No description available.

No description available.

No description available.

Although many of the symptoms of cancer are well documented, they are also very common in benign conditions. It is therefore difficult for a General Practitioner (GP) to identify which patients, presenting with symptoms, warrant further investigation and the level of cancer risk these features represent. Two case-control studies examined all cases of pancreatic (3,647) and oesophagogastric (7.657) cancers arising in the Clinical Practice Research Datalink (CPRD) (previously known as the General Practice Research Database (GPRD» United Kingdom (UK) from 2000-2009. Each case was matched with up to five age, sex and practice matched controls, randomly generated by CPRD. The entire primary care records for one year before the diagnosis of the cancer for all cases and controls were searched to identity features from medical text books, literature, cancer charities and websites. Conditional logistic regression analysis reported all features independently associated with each cancer. Likelihood ratios (LRs) and positive predictive values (PPVs) for these variables were calculated for patients consulting in primary care. Nine features were independently associated with pancreatic cancer. PPVs of individual symptoms and pairs of symptoms were calculated to identify patients with apparently low risk symptoms, but whose total risk warranted further investigation. 1.n the oesophago-gastric study, sixteen features were independently associated with cancer, including a relatively new finding of an association with thrombocytosis which has not been previously reported in association with oesophago-gastric cancer. Although the results largely support national referral guidelines for suspected cancer, they provide a robust evidence base for many of the recommendations. They should also inform commissioning policy for upper gastro-intestinal (GI) endoscopy and help in the selection of patients for endoscopic investigation if the UK's poor survival record is to be improved.

Aims of the investigation The aims of this investigation were to detect pancreatic cancer earlier using both genetic and proteomic approaches. The aims of the genetic approach were to use Amplification Refractory Mutation System (ARMS) to detect KRAS mutations in blood serum and plasma, pancreatic juice and tissue from patients with pancreatic cancer, chronic pancreatitis and other benign disease controls. The aims of the proteomic approach were to use Isobaric Tags for Relative and Absolute Quantification (iTRAQ) to identify and quantify the proteins present in blood serum from patients with 'early' and 'later' stage pancreatic cancer, chronic pancreatitis and healthy controls. In this thesis 'early' stage pancreatic cancer was defined as patients with lymph node negative status while 'later' stage pancreatic cancer was defined as patients with lymph node positive status. Results achieved DNA extraction from blood serum and plasma, pancreatic juice and tissue was successful. DNA concentrations were higher in pancreatic juice compared with serum and plasma. DNA concentrations were higher in serum compared with plasma. KRAS mutations were detected in 71.7 % of pancreatic cancer patients in pancreatic juice samples, 51.9 % in serum samples, 45.1 % in plasma samples and 79 % in pancreatic tissue samples. KRAS mutations were detected in 42.9 % of chronic pancreatitis patients in pancreatic juice samples, 43.8 % in serum samples and 20 % in plasma samples. A total of 254 proteins were identified and 234 proteins were quantified using iTRAQ in serum from early and later stage pancreatic cancer patients, chronic pancreatitis patients and healthy controls. Of these proteins, 48 showed a greater than 3-fold change between the early stage pancreatic cancer group and controls. Of these proteins, three were selected for further analysis: von Willebrand Factor, Collagen I and Collagen Ill. Further validation using Western blotting and ELlSA verified the iTRAQ result for von Willebrand Factor that this protein was elevated in the early stage pancreatic cancer group compared with controls.

Introduction: Pancreatic cancer is a malignancy with a very poor prognosis, and therefore insights into the derangement in cell signalling pathways which result in cancer development may provide important information in understanding the disease process, and in developing future therapeutic strategies. The aim of this study was to examine the expression and potential importance of several intracellular signalling proteins. Particular attention was given to Notch3. Methods: An immunohistochemical study was performed on 23 samples of human pancreatic adenocarcinorna and 12 samples of normal pancreas, to assess expression of Notch3, cyclin D1, pAkt, STAT3 and pSTAT3, β-catenin, and pin1. The degree of staining, and intracellular locations of the proteins were assessed. Immunohistochemical data were correlated with clinical parameters, including tumour resectability, size, presence of metastases, and plasma levels of CA19-9. In vitro studies were also performed on two pancreatic cell lines. Basal levels of protein expression were assessed by western blotting. The effect of Notch inhibition by γ-secretase inhibitors and by transfecting cells with Notch3 siRNA was also examined. Cell survival studies were performed using the Annexin V apoptosis assay. Results: Pin1 was not overexpressed in pancreatic adenocarcinoma. However, Notch3 was significantly overexpressed in the cytoplasm of 73.9% of tumours. Nuclear expression, never observed in normal ductal tissue, was noted in 43.5% of cases. No tumour expressing nuclear Notch3 was surgically resectable, and there was a significant correlation between nuclear Notch3 staining and increasing levels of CA19-9. There were also significant correlations between expression and intracellular location of Notch3 and each of STAT3, pSTAT3, and pAkt, but not cyclin D1. Notch3 was expressed in untreated ASPC1 cells, but not in PANC1 cells. Its levels could be reduced using either γ-secretase inhibitors or siRNA. Notch inhibition also resulted in a fall in levels of pAkt, but initial experiments did not demonstrate an effect upon cell survival. Conclusion: The presence of Notch3 in tumour nuclei is likely to represent functional activation of the protein, and is clearly linked to more aggressive tumours. In the future, targeting the Notch pathway may prove to be of therapeutic benefit.

Pancreatic (PC) is the eleventh most common malignancy in the UK but it has the poorest prognosis of all human adenocarcinoma. The autopsy, epidemiological and clinical studies have consistently identified PC as one of the most highly angiogenic and invasive malignancies, with the greatest prevalence and incidence of thrombo-embolism (TE). The incidence of TE in PC has been reported as high as 57%. Tissue factor (TF) bearing microparticles (MP) have recently been shown to promote thrombosis. The biological link between cancer haemostasis, cell invasion and angiogenesis remains unclear. These three indices may be driven by PC cells directly, be a reflection of the individual tumour stromal microenvironment and/or a result of the inflammatory response of the host. The hypothesis of the thesis is that factors directly attributed to the cancer promote the observed pathophysiology and that the removal of the tumour should result in reversal of these abnormalities. Flow cytometry was used for the evaluation of MP in plasma and quantification of surface-expressed TF, VEGFR-1 and-2 and EGFR. Cellular TF activity and pro-coagulant activity (prothrombin time) of PC patients were measured using a coagulometer. Matrigel Invasion Chambers and Boyden chambers with collagen IV were used to measure cellular invasion. A two dimensional angiogenesis assay was used to evaluate tubule formation in vitro in response to PC sera. Relative levels of protein expression of 55 angiogenic markers in the sera of PC patients were evaluated using a human angiogenesis array kit. Enzyme-linked immunosorbent assay was undertaken on VEGF, TF, TFPI, Leptin and annexin autoantibodies using sera or plasma from PC patients as appropriate. Immunohistochemical analysis of key markers of angiogenesis and thrombosis was also undertaken on resected PC samples. The in vitro optimisation experiments revealed that the cell invasion was significantly correlated with TF antigen expression and activity on PC cell lines (MIA-PaCa-2, AsPC-1 and CFPAC1) and that blocking TF on these cells decreased cell invasion. In the same manner neutralising soluble TF in PC serum samples also significantly decreased cell invasion, as did spiking of the serum with low molecular weight heparin. Analysis of sera from patients showed that TF bearing MP, pro-coagulant activity, cell invasion and angiogenesis (total length and number of capillaries) of PC cases were significantly higher than the control. Furthermore, the post-operative median number of TF bearing MP, procoagulant activity, cell invasion and angiogenesis (total length and total number of capillaries) were all significantly lower compared with pre-operative samples. Out of 55 angiogenic markers studied in 6 PC patients, pre- and post-operatively there was a significant decrease of angiopoietin-1, angiostatin/plasminogen, PDGF-AA, PDGF-AB/PDGF-BB and VEGF post-operatively. This result was supported by ELISA analysis of 29 samples and 14 controls that also showed significantly higher levels of VEGF in pancreatic cancer sera versus control groups, and that there was a significant decrease observed post-operatively only in the cancer patients. Furthermore both angiogenesis array and ELISA showed increased leptin levels post-operatively. Immunohistochemical analysis of the pancreatic tissue sections revealed that TF was expressed on 62 % of PC samples. There was significant correlation between TF expression on the tissue and procoagulant activity. Also, there was a significant correlation between tissue-expressed TF and in vitro angiogenesis, i.e. total length and number of capillaries. Furthermore, there was a significant correlation between TF expression on tissue with intratumoural microvascular density (MVD) and tumour-expressed with VEGFR 2. As expected, high levels of MVD correlated with high levels of tissue-expressed VEGF. Finally serum from patients who showed a high level of tissue-expressed VEGF also induced the greatest level of in vitro angiogenesis, i.e. number of capillaries. In summary, it was shown that TF expression on cell lines was significantly correlated with TF activity and cell invasion, and that TF expression in plasma and on tissue from PC patients was significantly correlated with procoagulant activity, cell invasion and angiogenesis. PC tissue-expressed VEGF was significantly associated with the angiogenic activity of PC sera and tissue MVD. Thus, the pathophysiology represented by a high procoagulant state, elevated cell invasion and angiogenic properties seen in PC patient sera appears to be driven by the malignant cells, as removal of the tumour causes a return towards the normal state.

No description available.

Pancreatic Ductal Adenocarcinoma (PDAC) is one of the five leading causes of cancer-related deaths in the West, and this, largely, is due to metastatic disease. In order to better understand PDAC metastatic spread and identify novel therapeutic targets, we analysed the proteome of primary tumours and matched lymph node (LN) metastases. As frozen specimens of metastatic lesions are scarce, we examined formalin-fixed paraffin-embedded (FFPE) tissues. Whilst such tissue is in routine diagnostic use, the cross-linkages induced by fixation have, in the past, precluded proteomic investigation for research purposes. Recent technological advances have, however, overcome this technical limitation. Using laser capture microdissection (P.A.L.M system), we isolated malignant epithelia from seven FFPE primary PDAC tumours and matched LN metastases. Following dissection, samples were analysed in duplicate using Multidimensional Protein Identification Technology (MudPIT); this resulted in the identification of 1504 proteins, 854 of which were common to all samples analysed. Comparison of the obtained proteins with data from previous proteomics studies on pancreatic tissue, pancreatic juice, serum and urine resulted in a less than 30 % overlap, indicating that our study has expanded the current database of proteins expressed in this malignancy substantially. Statistical analysis further showed that 115/854 proteins (13.5%) were significantly differentially expressed (g-value ≥ 3.8). Two proteins, S100P and 14-3-3 sigma, with highly significant g-values were confirmed to be significantly differentially expressed (S100P: p = 0.05 and 14-3-3 sigma: p < 0.001) 4 in a larger series of 55 cases of matched primary PDAC and LN metastases using immunohistochemistry. We chose to investigate further the roles of S100P in lymphatic invasion in vitro and in vivo. By co-culturing a Panc1 S100P-overexpressing clone (S5L), or a vector control clone (V3L), with human dermal lymphatic endothelial cells (HDLEC), we were able to show that different receptors mediate S5L adhesion to resting and activated HDLEC as opposed to V3L; and that the presence of S5L cells in these co-cultures significantly increased permeability at one (p = 0.02), four (p = 0.002) and eight (p = 0.007) hours post-seeding, and significantly increased translymphatic endothelial migration at 72 hours (p = 0.006). Using the V3L and S5L cell lines, which were transduced to express luciferase, we also created an orthotopic mouse model of PDAC, as well as experimental metastatic mouse models, in CD1 nude mice. These models were used to evaluate the effects of S100P on primary tumour growth, metastasis and site-specific growth. S100P was only found to significantly increase primary tumour growth in this model (n = 10 animals/group), both by bioluminescence (p = 0.002) and tumour weight (p = 0.01). No metastases (spontaneous and/or experimental) were seen however. Thus, this model can be used to evaluate the anti-tumour efficacy of novel therapies to S100P in the future.

Activating mutations of the kras proto-oncogene are found in more than 90% of human pancreatic ductal adenocarcinoma (PDAC) and can result in increased activity of the NF-κB pathway, leading to constitutive production of proinflammatory cytokines such as TNF-α. Pancreatic cancer progression occurs through a series of pre-invasive lesions, pancreatic intraepithelial neoplasias (PanIN lesions), which progress into invasive carcinoma. The aim of this thesis is to understand the autocrine role of TNF-α produced by premalignant epithelial cells in pancreatic tumour progression. This cytokine has already been shown to be involved in the progression of cancer. The major hypothesis therefore tested was that TNF-α secreted by pre-malignant epithelial cells promotes the early stages of pancreatic carcinogenesis by sustaining an inflamed microenvironment. In the spontaneous kras+/LSL-G12D; pdx1-cre mouse model of pancreatic cancer, concomitant genetic deletion of the TNF-α/IKK2 pathway substantially delayed pancreatic cancer progression and resulted in downregulation of the classical Notch target genes hes1 and hey1. Cell lines from the different PanIN bearing mice were established and used to dissect the cooperation between TNF-α/IKK2 and Notch signalling during PanIN progression. Optimal expression of Notch target genes was induced upon TNF-α stimulation of the canonical NF-κB signalling pathway, in cooperation with basal Notch signals. Mechanistically, TNF-α stimulation resulted in phosphorylation of histone H3 at the hes1 promoter and this signal was lost upon ikk2 genetic deletion. HES1 suppressed the expression of pparg, which encodes for the anti-inflammatory nuclear receptor PPAR-γ. Thus, crosstalk between TNF-α/IKK2 and Notch sustained an intrinsic inflammatory profile of the transformed cells. The treatment of PanIN bearing mice with rosiglitazone, a PPAR-γ agonist, also delayed PanIN progression. A malignant cell-autonomous, low-grade inflammatory process was shown to operate from the very early stages of kras-driven pancreatic carcinogenesis, which may cooperate with the Notch signalling pathway to promote pancreatic cancer progression.