PDF file - 648K, Investigation of gene targets of miR-21 and miR-34a in human PDAC. A - C) Correlation plots of RT-PCR miR-21 expression (relative expression) versus cytoplasmic Bcl-2 protein expression (Spearman's correlation coefficient = 0.411, P = 0.003), cytoplasmic maspin expression ( = 0.653, P < 0.001), and cytoplasmic PTEN expression ( = 0.43, P = 0.004) (n = 43). IHC assessed using a semi-continuous histoscore. D) Kaplan-Meier analysis illustrating that patients with low PTEN have poorer outcome (n = 38, 13.0 months) compared to those with medium PTEN expression (n = 41, 16.7 months) and high PTEN expression (n = 38, 26.6 months) following pancreaticoduodenectomy (Log-Rank test, P = 0.01). The cohort was divided into tertiles according to the PTEN histoscore. E - F) Correlation plots of miR-34a expression with cytoplasmic Bcl-2 protein expression ( = -0.496, P = 0.001) and nuclear cyclin D1 protein expression ( = -0.293, P = 0.043) (n = 43). G) Correlation of nuclear p53 protein expression with miR-34a expression ( = 0.392, P = 0.01) (n = 43).

XLS file - 168K, A. Combination of predicted targets for miRNAs associated with favourable prognosis (miR-29c, miR-30d, miR-34a). B. Combination of predicted targets for miRNAs associated with poor prognosis (miR-21, miR-221, miR-224).

XLS file - 50K, Multivariate analysis of combined 72 patient cohort. A) All 72 patients. B) 38 patients with low miR-21 expression (less than median expression value). C) 34 patients with high miR-21 expression (greater than median expression).

XLS file - 47K, Conventional clinicopathological factors in PDAC cohort (n = 48) associated with survival following resection based on multivariate analysis.

PDF file - 209K, Top 25 miRNAs up-regulated and down-regulated in PDAC compared with normal pancreatic tissue, ranked by P-value.

PDF file - 274K, Differentially expressed microRNAs normal pancreas versus PDAC.

PDF file - 131K, Supplementary Results and Methods.

PDF file - 211K, Bioinformatics enrichment of miRNAs associated with poor outcome. A) Pathway enrichment of the targets of miR-21, miR-221, and miR-224 demonstrating union and intersections. B) Enrichment of TGF- and C) MAPKinase KEGG pathways for miR-21, miR-221, and miR-224 target expression.

PDF file - 243K, Supervised average linkage clustering with centred Pearson correlation using 48 PDACs and 10 normal pancreatic samples. miRNAs are in rows with the samples in columns. The non-tumor normal pancreatic samples are on the right highlighted in green, with the tumor samples highlighted in blue. The expression color bar is shown below the dendrogram. Red indicates a miRNA expression value higher than the average expression across all samples, while blue indicates a lower expression value.

XLS file - 433K, A. Gene expression according to miR-21 expression in 43 patients with PDAC. B. Gene expression according to miR-34a expression in 43 patients with PDAC.

PDF file - 204K, Bioinformatics enrichment of miRNAs associated with favourable outcome. A) Pathway enrichment of the targets of miR-29c, miR-30d, and miR-34a demonstrating union and intersections. B) Enrichment of focal adhesion and C) ECM-receptor interaction KEGG pathways for miR-29c, miR-30d, and miR-34a target expression.

XLS file - 48K, Conventional clinicopathological parameters in PDAC cohort (n = 48) univariately associated with survival following resection.

PDF file - 270K, Association of miRNA expression levels measured by RT-PCR with overall survival: univariate analysis.

PDF file - 189K, Multivariate Cox regression analysis of association of PTEN and clinicopathological factors and overall survival.

PDF file - 3.2MB, Immunohistochemical staining in PDAC. Immunohistochemistry staining for Bcl-2, maspin, PTEN, p53, cyclin D1 in human normal pancreatic ducts and pancreatic ductal adenocarcinoma with examples of expression of nil, low, moderate, and strong staining.

Objective Pancreatic cancer is a leading cause of cancer-related death in the Western world. Current chemotherapy regimens have modest survival benefit. Thus, novel, effective therapies are required for treatment of this disease.Design Activating KRAS mutation almost always drives pancreatic tumour initiation, however, deregulation of other potentially druggable pathways promotes tumour progression. PTEN loss leads to acceleration of Kras(G12D)-driven pancreatic ductal adenocarcinoma (PDAC) in mice and these tumours have high levels of mammalian target of rapamycin (mTOR) signalling. To test whether these KRAS PTEN pancreatic tumours show mTOR dependence, we compared response to mTOR inhibition in this model, to the response in another established model of pancreatic cancer, KRAS P53. We also assessed whether there was a subset of pancreatic cancer patients who may respond to mTOR inhibition.Results We found that tumours in KRAS PTEN mice exhibit a remarkable dependence on mTOR signalling. In these tumours, mTOR inhibition leads to proliferative arrest and even tumour regression. Further, we could measure response using clinically applicable positron emission tomography imaging. Importantly, pancreatic tumours driven by activated KRAS and mutant p53 did not respond to treatment. In human tumours, approximately 20% of cases demonstrated low PTEN expression and a gene expression signature that overlaps with murine KRAS PTEN tumours.Conclusions KRAS PTEN tumours are uniquely responsive to mTOR inhibition. Targeted anti-mTOR therapies may offer clinical benefit in subsets of human PDAC selected based on genotype, that are dependent on mTOR signalling. Thus, the genetic signatures of human tumours could be used to direct pancreatic cancer treatment in the future.

s / Pancreatology 13 (2013) e1–e20 e8 University College London, Institute of Liver and Digestive Health, Royal Free Hospital Guy's and St Thomas’ NHS Foundation Trust, London, Department of Gastroenterology King's College London and King's Health Partners, Division of Women's Health University College London, Department of Pathology University College London, HPB & Liver Transplant Unit, Royal Free Hospital Category: Benign and Inflammatory Background and aims: Some 35% of women of reproductive age are obese. Emerging evidence suggests that maternal obesity(MO) may predispose offspring to an increased risk of obesity and its related disorders, including Non-Alcoholic Fatty Pancreas Disease(NAFPD), a newly described disease entity which may link obesity and pancreatic adenocarcinoma. We have investigated the impact of MO on the pathogenesis of NAFPD using a pathophysiologically relevant model and mechanistically explored a role for altered expression of circadian genes(CG). Methods: Female C57BL6 mice were fed standard or obesogenic diet(OD) for 6 weeks prior to and throughout pregnancy and lactation. Litters were standardised to a minimum of 6 pups and offspring then weaned onto either standard or OD to produce 4 groups before sacrifice at 6 months. We assessed biochemical, histological and pro-inflammatory/profibrogenic markers associated with the NAFPD phenotype and the expression of CG in the pancreas. Results and Interpretation: Offspring exposed to MO and OD had increased bodyweights (p<0.001), pancreas tissue triglycerides (p<0.001) alongside an increased expression of TNF-a (p<0.0001), IL-6 (p<0.001), aSMA (p<0.0001), TGF-b (p<0.001) and collagen (p<0.0001) when compared to controls. Additionally, cosinor analysis of CG demonstrated a phase shift in CLOCK (p<0.01), REV-ERB-a (p<0.05) and PER2(p<0.05). 2-way ANOVA further demonstrated CLOCK (p< 0.005), PER1 (p< 0.005) and PER2 (p< 0.05) to display a significant interaction between MO and post-weaning diet. Summary: Our results show that MO and a post-natal OD interact to induce offspring NAFPD. The mechanisms through which MO programs offspring NAFPD may be through a perturbation of CG. Take-home message: Maternal obesity in conjunction with a postweaning obesogenic diet may interact to induce offspring NAFPD, potentially through pertubation of core circadian genes. Abstract previously presented? no (Although it is due for poster presentation at UEGW in October) Any disclosures? no ()

Pancreatic cancer is a leading cause of cancer-related death in the Western world and, in most patients, current chemotherapies have only slight survival benefit. Evaluation of targeted therapies however, is a relatively recent development. Therefore, we have attempted to model genetic subsets of the disease in the mouse. These models can be used to test targeted therapies in the most appropriate genetic backgrounds. For example, we have shown previously that mutant p53 drives metastasis, and that the Src inhibitor Dasatinib inhibits mutant p53-mediated pancreatic cancer metastasis in vivo. We have now assessed the efficacy of further targeted therapies in this model, in particular those that target the tumor microenvironment, and found that tumor growth and metastasis can be suppressed in certain cases. We also previously showed that loss of Pten leads to acceleration of PDAC in the mouse, and that in the human disease those patients with high levels of active mTOR represent a discrete cluster in which, in contrast with the majority of patients, mTOR inhibitors should be effective. We now show, in a mouse model driven by KrasG12D and Pten deficiency, that mTOR inhibition significantly enhances survival of animals presenting with advanced PDAC and causes regression of tumors in several cases. In tumors driven by KrasG12D and Lkb1 deficiency we also observed some enhancement of survival. In tumors driven by KrasG12D and mutant p53, however, mTOR inhibition had little survival benefit, suggesting that its chemotherapeutic effect is specifically due to the reliance on mTOR signaling. The genetic signatures of tumors could be used to direct therapy in the future. Clearly, promising treatments may work only in certain patient groups, or only as part of combinatorial approaches. Thus, careful consideration should be taken before selecting models to test new agents. Citation Format: Jennifer P. Morton, Douglas C. Morran, Saadia A. Karim, Nigel B. Jamieson, TR Jeffry Evans, Owen J. Sansom. The use of mouse models of pancreatic cancer to evaluate sensitivity to targeted therapies. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B31.

Abstract Purpose: MicroRNAs (miRNA) have potential as diagnostic and prognostic biomarkers and as therapeutic targets in cancer. We sought to establish the relationship between miRNA expression and clinicopathologic parameters, including prognosis, in pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Global miRNA microarray expression profiling of prospectively collected fresh-frozen PDAC tissue was done on an initial test cohort of 48 patients, who had undergone pancreaticoduodenectomy between 2003 and 2008 at a single institution. We evaluated association with tumor stage, lymph node status, and site of recurrence, in addition to overall survival, using Cox regression multivariate analysis. Validation of selected potentially prognostic miRNAs was done in a separate cohort of 24 patients. Results: miRNA profiling identified expression signatures associated with PDAC, lymph node involvement, high tumor grade, and 20 miRNAs were associated with overall survival. In the initial cohort of 48 PDAC patients, high expression of miR-21 (HR = 3.22, 95% CI: 1.21–8.58) and reduced expression of miR-34a (HR = 0.15, 95% CI: 0.06–0.37) and miR-30d (HR = 0.30, 95% CI: 0.12–0.79) were associated with poor overall survival following resection independent of clinical covariates. In a further validation set of 24 patients, miR-21 and miR-34a expression again significantly correlated with overall survival (P = 0.031 and P = 0.001). Conclusion: Expression patterns of miRNAs are significantly altered in PDAC. Aberrant expression of a number of miRNAs was independently associated with reduced survival, including overexpression of miR-21 and underexpression of miR-34a. Summary: miRNA expression profiles for resected PDAC were examined to identify potentially prognostic miRNAs. miRNA microarray analysis identified statistically unique profiles, which could discriminate PDAC from paired nonmalignant pancreatic tissues as well as molecular signatures that differ according to pathologic features. miRNA expression profiles correlated with overall survival of PDAC following resection, indicating that miRNAs provide prognostic utility. Clin Cancer Res; 18(2); 534–45. ©2011 AACR.