The aetiology for sporadic pancreatic adenocarcinoma is poorly characterized. Familial/hereditary causes account for about 10% cases and tobacco smoking is a well- established risk, however it is only responsible for about a third of cases. DNA repair mechanisms restore the genome damage caused by carcinogens including those derived from tobacco smoking. Increasing attention is being focused on single nucleotide polymorphisms, which exist amongst various physiological pathways including DNA repair mechanisms, and account for inter-individual variation in risk for cancer. This study is an effort to investigate the impact of family history of malignancy, tobacco smoking and selected genetic polymorphisms involved in DNA repair on pancreatic ductal adenocarcinoma development. A hospital-based case-control study of pancreatic adenocarcinoma cases and hospital- based controls was undertaken at the Freeman Hospital between 2005-2006. Pancreatic cancer cases were ascertained based on histology, cytology or a combination of clinical findings, tumour marker levels and progressive radiological changes. All participants were interviewed to establish a detailed clinical, family history and tobacco smoking (MONICA questionnaire) A sample of peripheral blood was obtained for genotyping of specific Base Excision repair genotypes – hOGG1, XRCC194, XRCC280, XRCC399 and APE148. Statistical analysis was performed on SPSS v16. Odds ratios (95% CI) were calculated for individual variables. Tobacco smoking was confirmed to be a risk factor for pancreatic cancer [OR (95% CI) on univariate: ever smoker [(present and past smokers) OR 3.01 (95% CI 1.73 to 5.24)] and multivariate analysis: present smokers [OR = 8.531 (3.198 to 22.759) and past smokers OR = 5.862 (2.223 to 15.460)]. Importantly a significantly decreased cumulative tobacco exposure was seen amongst pancreatic cancer cases with a family history of cancer [mean (SD): 30.00 (24.77) pack-years] as compared those who did not have such a history [44.69 (28.47) pack-years p=0/023]. No specific overall increased risk was associated with the individual base excision repair genotypes on both uni- variate and multi-variate analysis. Tobacco smoking is a risk factor and appears to play a more important role (for pancreatic cancer development) in the presence of a family history of cancer. Small risks associated with SNP’s are difficult to tease out from small studies like the current one. Larger multi institutional studies (as have been achieved for e.g. Lung Cancer) are required to confirm this latter finding and perform pooled analysis of data for specific sequence variants within a target biochemical pathway to uncover the risks associated with these genes.

Medulloblastoma (MB) is a paediatric tumour of the cerebellum which is responsible for 15-20% of all childhood brain tumours. Mortality due to this disease is high (~40%) and successful treatment is associated with significant neurological and cognitive consequences, making new therapies desirable. Disruption of the Sonic Hedgehog (SHH) signaling pathway, including mutations in PTCH1, define a major subset of human MB. Mice heterozygous for loss of function mutations in the Ptch1 ortholog develop MBs at low frequency. To identity genes that co-operate with Ptch1 in MB development, a Sleeping Beauty insertional mutagenesis screen in this murine model was performed. Mutagenesis significantly increased the frequency of MB formation in Ptch heterozygote mice from ~3% to ~25% after 8 months (p<0.0001). To identify the genes responsible for this enhanced tumour formation, Splinkerette-PCR and 454-FLX sequencing were applied to define SB insertion sites within 40 tumours. Statistical analysis of these data using Monte Carlo and Kernel Convolution methods identified 18 candidate medulloblastoma genes defined by common insertion sites (CISs), including 11 identified by both methods used. Many of these are genes known to be involved in neuronal development and cell fate determination. Subsequent ARACNe network analysis of the candidate genes in human gene expression datasets has established that seven (Nfia, Nfib, Tead1, Tgif2, Myt1l, Fgf13 and Crebbp) lie within a single network which is enriched for both neuronal genes and transcription factors, and includes genes known to interact with the SHH pathway. Bioinformatic analysis confirmed that the tumours generated from SB mutagenised mice were similar in their gene expression pattern to human SHH subgroup tumours, while microarray expression analysis of SB induced tumours identified Igf2, a gene previously implicated in MB development, as a key output of network activity. Finally, analysis of gene expression within granule neuron precursor cells (GNPCs), the cell of origin of SHH subgroup MB, identified Tgif2 and Myt1l as prime candidate genes for downstream functional analysis. Lentiviral based modulation of their gene expression was therefore attempted in primary GNPCs. shRNA based knock-down of Myt1l was achieved, and shown to be associated with significantly increased expression of both Gli1 (the downstream effector of the Shh pathway) and Math1 (a marker of undifferentiated neuronal cells), consistent with a reduction in MYT1L expression playing a role in human MB development. Further analysis of this gene, and others defined here, should both improve our understanding of MB and define potential targets for therapeutic intervention.

Prostatic differentiation is modelled through enrichment for stem-like populations through a combination of putative stem-cell markers. However, in vitro cultures demonstrate a phenotypic drift that abrogates normal physiology. Induced Pluripotent Stem Cell (iPSC)- reprogramming allows for any somatic cell to be transformed into an embryonic-stem-celllike state although molecular properties as well as differentiation abilities are limited by the primary tissue type of origin. This project describes the derivation of Prostate-iPSC (ProiPSC) from the prostate of an individual in his sixth decade. Prostate cells were reprogrammed through use of a specific Cre-Recombinase/LoxP polycistronic transduction protocol. Resultant iPS clones (14 cell lines) were checked for identical DNA fingerprinting with the parent fibroblasts and then tested for pluripotency and exogene silencing. Morphologically the Pro-iPSC are identical to human embryonic stem cells. Normal karyotyping was confirmed following which Pro-iPSC were immunostained for a panel of 6 pluripotent markers including nuclear-transcription factors Oct4 and NANOG. Messenger RNA studies confirmed a gene-expression profile that was similar to embryonic-stem cells. These Pro-iPSC are able to differentiate into all the three germ layers (embryoid body and teratoma formation) and demonstrate in vitro differentiation along a prostate-specific lineage when treated with specific differentiation media. Preliminary tissue recombination grafts of Pro-iPSC with the urogenital messenchyme have further demonstrated in vivo differentiation of these cells along a specific urological route. In conclusion a novel iPSC model has been established whereby aged prostatic fibroblasts have been progressively de-differentiated into a primitive embryonic state - this model demonstrates crucial events in prostate embryology which in turn allows the scrutiny of some complex signalling pathways as well as molecular mechanisms behind prostate carcinogenesis.

Literature Review : Contemporary qualitative evidence exploring the adjustment experiences of young people treated for a brain tumour was synthesised. A systematic search of the literature was undertaken and eight papers met the criteria for inclusion in the review. A thematic synthesis of the findings revealed two superordinate themes: (a) no longer playing well; and (b) mastering the game of life. The findings from the review demonstrated the range and extent of difficulties experienced by survivors. However, the survivors showed resilience in adapting to the effects of their illness and developed a number of effective coping strategies. The findings also suggested that some survivors may experience post-traumatic growth. The review highlighted the need for multidisciplinary support and liaison between healthcare services and schools. Further implications for clinical practice and directions for future research are considered. Research Report : Young people’s experiences of reintegrating into school following treatment for a brain tumour were explored using an Interpretative Phenomenological Analysis (IPA) approach. Four girls (ages 14 to 16) were interviewed and the transcripts were analysed. Three superordinate themes emerged across the cases, relating to experiences of identity, relationships and reengaging with schoolwork. The subthemes retained the complexity of how these overarching themes were experienced differently for the individuals. The present study provided insight into the lived experiences of these young people as they returned to school following treatment for a brain tumour. The study highlighted the challenging nature of this experience, and implications for future research and clinical practice are discussed. Critical Appraisal : The processes involved in conducting a research project are discussed reflectively in the critical appraisal section. This includes personal reflections, a critique of the research, and a consideration of future research opportunities.

Background: Electrolytic ablation (EA) is a technique of liver ablation that produces extreme pH changes in the local microenvironment. An ex-vivo perfused liver model compared EA vs. radiofrequency ablation (RFA) and analysed biochemical, immunological and histologic parameters. Methods: Seventeen pigs were perfused extracorporeally with normothermic autologous blood, five of which underwent RFA and five EA after one hour from reperfusion. Arterial, venous blood samples and histologic specimen were collected hourly and analysed for 1) arterial blood gases content, 2) biochemical parameters, 3) cytokines, 4) and tissue modifications. Results: No significant differences were registered among techniques for biochemical and immunologic parameters investigated. EA created a smaller transitional zone of ablation compared to RFA and histologically a particular pattern of changes in which the coagulative necrosis and hemorrhages affected mainly the peripheral area of the lobule, while the sinusoidal dilatations the centrilobular area. No significant changes were found for the apoptosis and the regeneration activity. Conclusions: Although results of the histological changes are interesting, the technical complexity of the ex-vivo model increased the number of parameters to monitor, especially concerning the liver viability and the administration of external substances to maintain a physiologic environment. Furthermore, the high portal vein pressures used and long warm ischemia times registered could have biased results. For the purpose of this study an in-vivo model would have been more appropriate.

Resveratrol, a polyphenol present in red wine and peanuts exerts an array of beneficial effects, including anticancer activity and calorie restriction (CR) mimicry. Work from our laboratory has shown for the first time that low doses of resveratrol, comparable to amounts contained in a glass of red wine significantly reduced adenoma development in ApcMin/+ mice when given with a high fat diet. The protective effect was more pronounced than that caused by higher amounts, equivalent to doses used in clinical trials. The CR activity of resveratrol is mediated via the energy regulator AMP-activated kinase (AMPK). This study aims to investigate whether AMPK signalling contributes to the chemopreventive effect of resveratrol. The extent of AMPK activation in vivo correlated with the degree of adenoma inhibition; AMPK expression and phosphorylation was evident in mucosa of resveratrol treated mice, but not in control animals, with highest levels in those that ingested the low dose. This chronic resveratrol treatment was also associated with increased expression of p21, a biomarker of senescence, particularly at the low dose. Moreover, we identify a bell-shaped dose-response to resveratrol: In Apc10.1 cells resveratrol caused significant increases in AMPK phosphorylation at 0.01, 0.1 and 1μM, but not at 10μM, consistent with a bell-shaped dose-response. Activity was accompanied by significantly reduced phosphorylation of mTOR, which is regulated by AMPK signalling, plus its downstream targets p70S6K and 4E-BP1. Furthermore, these concentrations also increased autophagy and senescence, as measured by LC3-II and p21 expression, respectively. Modulation of cellular energy balance by increasing the AMP/ATP and/or ADP/ATP ratios and ability of resveratrol to produce ROS are most likely to be two of the mechanisms responsible for resveratrol stimulating AMPK activation in Apc10.1cells. The novel finding that low amounts of resveratrol are more efficient than higher does will influence future attempts towards clinical implementation of resveratrol for cancer chemoprevention.

Tumour growth is dependent on angiogenesis. Angiogenesis often predicts a poorer prognosis in cancer patients and is also associated with the increased development of tumour metastases. Angiogenesis is stimulated by vascular endothelial growth factor, which increases vascular permeability, induces endothelial cell proliferation and promotes endothelial cell survival, which, are pivotal in tumourgenesis. As such angiogenesis and vascular endothelial growth factor inhibitors are now commonplace in the armoury of anti-cancer treatments. In vitro, in-vivo and epidemiological studies have demonstrated that omega-3 fatty acids, principally eicosapentaenoic acid and docosahexaenoic acid have profound anti-inflammatory, anti-apoptotic, anti-proliferative and anti-angiogenic properties. Anti-angiogenic actions include a reduction vascular endothelial growth factor concentration and alteration of vascular endothelial growth factor receptor signalling response. Unfortunately only negligible amounts of EPA and DHA can be produced by human metabolism. Omega-3 fatty acids are however synthesised in abundance by algae and plankton and as a consequence fish are the main dietary source of essential omega-3 fatty acids. However even with a balanced diet rich in fish, human plasma concentrations are minimal. In contrast parenteral administration of a fish-oil lipid emulsion leads to a significant and rapid increase in eicosapentaenoic acid and docosahexaenoic acid concentrations in plasma, and platelet and leukocyte membrane phospholipids within hours. There is a large body of work in the scientific literature that supports the theory that omega-3 fish oils have marked anti-angiogenic properties and that plasma concentrations of omega-3 fatty acids can be quickly and safely manipulated with intravenous infusion. I present the findings from a randomised controlled pilot study in patients with hepatic colorectal metastases to assess the response of biomarkers of angiogenesis to intravenous administration of omega-3 fatty acids compared to controls.

Colorectal carcinoma is the third commonest cancer in the UK and is the second commonest cause of cancer related death. There is a need for efficacious chemotherapeutic and chemopreventative agents against colorectal carcinogenesis. Indole-3-carbinol and its acid condensation product, 3,3 diindolylmethane (DIM), exhibit anti-tumourigenic activity in preclinical models. This study investigated their therapeutic potential in colorectal carcinoma. A panel of four colorectal carcinoma cell lines (HT29, HCT116, SW480 and SW620) was used to compare effects of DIM and I3C on cell viability, induction of apoptosis and cell cycle arrest. Treatment with DIM or I3C resulted in dose-dependent reductions in cell viability. Cell lines were sensitive to 25–75 μM DIM (GI[subscript 50] = 45 μM), but not to 200 μM I3C. DIM (40 μM) induced apoptosis in all cell lines. This was associated with downregulation of survivin and appeared to be independent of p53. Furthermore it induced G[subscript 1] arrest in HCT116, SW480 and SW620 and G[subscript 2]/M arrest in HT29. DIM induced activatory phosphorylation of Chk2 in HT29 but not in HCT116. DIM downregulated cell cycle proteins: PLK1, cdc25C, cdc2 and cyclins B1, D1 and E. Since all colon cancer cell lines expressed significant levels of Src, their response to Src inhibition was investigated. Colon cancer cells were modestly sensitive to AZD0530 (AstraZeneca), but combination with indoles reduced viability to a greater extent than single agent treatment. Combining 40 μM DIM with 1 μM AZD0530 was particularly effective, reducing viability by half after 48 hours on average. Decreases were sustained and progressive over extended timepoints. Attempts were made to establish primary cultures from resected hepatic metastases however issues were identified with cell death, fibroblast overgrowth and slow cell turnover. Potential changes to address these were identified. These data suggest chemotherapeutic potential of DIM in colon cancer, particularly in combination with Src inhibition.

Curcumin inhibits the proliferation of chemotherapy-resistant cancer stem-like cells in cell lines but whether this contributes to its chemopreventive activity is unknown. This study aims to determine whether curcumin modulates the growth and expansion of colorectal stem-like cells in primary adenoma and carcinoma tissues, and in vivo using a patient-derived xenograft, then to elucidate a possible mechanism of action. Colorectal tissue obtained post-operatively (normal n=32, adenoma n=6, carcinoma n=40) was FACS profiled for markers of stem-like cells, aldehyde dehydrogenase (ALDH) activity and CD133 expression. The percentage of cells with ALDH[superscript high] activity was 11.8±1.8, 4.6±0.7 and 2.8±0.4 in adenoma, normal and carcinoma tissues, respectively. Equivalent values for CD133 expression were 1.2±0.6, 0.5±0.2 and 7.7±1.8%. To assess in vitro activity, single cells from adenomas and carcinomas (three patients each, in triplicate) were cultured as spheroids with clinically achievable curcumin concentrations. Curcumin significantly reduced adenoma and carcinoma sphere number, compared to controls for all patient samples, with a U-shaped dose-response in >50% of cases. Sphere size was also impaired at concentrations >1μM. Curcumin (0.2%) consumption in NOD/SCID mice injected (s.c) with cancer stem-like cells was associated with significant delay in time to palpable tumours, increased survival and reduced rate of tumour growth. There was also a ~60% reduction in proportion of ALDH[superscript high] cells in tumours from curcumin treated mice compared to controls (p<0.05). Curcumin (0.1, 1μM) treatment of Caco2 cells caused a significant decrease (p<0.01) in Nanog expression, an embryonic stem cell transcription factor, in cancer stem-like cells specifically. A protein pull-down assay confirmed the interaction between curcumin and Nanog. Curcumin reduced (p<0.01) Nanog phosphorylation in cancer stem-like cells which may destabilise the protein, leading to reduced levels. These results indicate that clinically achievable concentrations of curcumin target stem-like cells in colorectal adenomas and carcinomas, which may contribute to anti-cancer efficacy in humans.

Men with localised prostate cancer can now expect to live longer with the disease and the side-effects of treatment than a decade ago. These side effects include urinary, bowel and sexual dysfunction which can affect their quality of life. Aim: There is a lack of information on how radiotherapy with neo-adjuvant hormone treatment affects men's quality of life over one year post-radiotherapy and how they cope with the disease and side-effects during this time. This study aims to address these gaps. Method: One hundred and forty-nine men who were about to undergo radiotherapy and neo-adjuvant hormone treatment for localised prostate cancer participated in this longitudinal study. They completed the EORTC QLQ C-30 & PR25 and the Brief Cope scale at four times (prior to radiotherapy, at four to six weeks post-radiotherapy; at six months and one year post-radiotherapy). The response rate was over 90% at each time-point. Results: Men reported the biggest decline in all aspects of health related quality of life at 4-6 weeks after radiotherapy, generally followed by improvements in health between six months and a year after treatment. Those who were experiencing late urinary side-effects, one year after radiotherapy had poor urinary function prior to radiotherapy. Some men who reported acute bowel effects at 4-6 weeks after radiotherapy developed late effects. Coping and HRQoL in men receiving prostate cancer treatment were influenced by the individual's life circumstances inclusive of age, spousal support and their perceived HRQoL as well as time (pre and post treatment). Conclusion: Psycho-social interventions in follow-up care need to be developed and these need to be flexible enough to take account of the individual physical and psycho-social needs of men with prostate cancer pre and post-radiotherapy. They are particularly vulnerable in the immediate post-radiotherapy period. Men with little social support may also be most in need of help.