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PDF file - 790K, S Table 1. Primary and secondary antibodies used in immunostaining and Western Blotting experiments S Table 2. Antibodies used for immunoprecipitation experiments S Table 3. Primers used in real time RT-PCR and PCR of the ChIP experiments S Figure 1: The expression pattern of Sirt1 and beta-catenin changes in Caerulein induced pancreatitis. S Figure 2: The expression pattern of Sirt1 and beta-catenin, but not Dbc1, changes in pancreatic duct ligation. S Figure 3: In an in vitro ADM model, Sirt1 undergoes transient changes in subcellular localisation without changes in expression levels. S Figure 4: Deletion of functional Sirt1 in Pdx1-Cre;Sirt1ex4lox/lox pancreas impacts on acinar cell differentiation during ADM. S Figure 5: Acinar cell differentiation is not altered during ADM in Sirt1 transgenic (SirtTg) pancreas. S Figure 6: Nicotinamide represses ADM of human cells in culture. S Figure 7: Sirt1 co-localizes and interacts with beta-Catenin in acinar cells. S Figure 8: Nicotinamide increases beta-catenin/Wnt signaling in ADM cultures. S Figure 9: Dbc1 expression is decreased in PDAC but not in chronic pancreatitis.

Cancer‐associated fibroblasts (CAFs) are the most abundant cell type in the tumor microenvironment and are responsible for producing the desmoplastic reaction that is a poor prognostic factor in ovarian cancer. Long non‐coding RNAs (lncRNAs) have been shown to play important roles in cancer. However, very little is known about the role of lncRNAs in the tumor microenvironment. We aimed to identify lncRNAs expressed in ovarian CAFs that were associated with patient survival and used computational approaches to predict their function. Increased expression of 9 lncRNAs and decreased expression of 1 lncRNA in ovarian CAFs were found to be associated with poorer overall survival. A “guilt‐by‐association” approach was used to predict the function of these lncRNAs. In particular, MIR155HG was predicted to play a role in immune response. Further investigation revealed high MIR155HG expression to be associated with higher infiltrates of immune cell subsets. In conclusion, these data indicate expression on several lncRNAs in CAFs are associated with patient survival and are likely to play an important role in regulating CAF function., This manuscript shows that long noncoding RNAs (lncRNAs) are heterogeneously expressed in ovarian cancer‐associated fibroblasts and expression differences in several lncRNAs are associated with differences in patient survival. Expression profiles were analyzed from 67 microdissected ovarian cancer‐associated fibroblast samples, one of the largest cohorts available. One lncRNA, MIR155HG, was associated with increases in tumor‐infiltrating lymphocytes.

Background: \ud Up to 60% of patients who undergo curative-intent pancreatic ductal adenocarcinoma (PDAC) resection experience disease recurrence within six months. We recently published a systematic review of prognostic immunohistochemical biomarkers in PDAC and shortlisted a panel of those reported with the highest level of evidence, including p53, p16, Ca-125, S100A4, FOXC1, EGFR, mesothelin, CD24 and UPAR. This study aims to discover and validate the prognostic significance of a combinatorial panel of tumor biomarkers in patients with resected PDAC.\ud \ud Methods: \ud Patients who underwent PDAC resection were included from a single institution discovery cohort and a multi-institutional validation cohort. Tumors in the discovery cohort were stained immunohistochemically for all nine shortlisted biomarkers. Biomarkers significantly associated with overall survival (OS) were reevaluated as a combinatorial panel in both discovery and validation cohorts for its prognostic significance.\ud \ud Results: \ud 224 and 191 patients were included in the discovery and validation cohorts, respectively. In both cohorts, S100A4, Ca-125 and mesothelin expression were associated with shorter OS. In both cohorts, the number of these biomarkers expressed was significantly associated with OS (discovery cohort 36.8 vs. 26.4 vs 16.3 vs 12.8 months, P

Objective We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. Background Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease. Method In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram. Results High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables. Conclusions Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.

Gliomas are the most common primary brain tumors in adults. IDH-mutated low-grade gliomas of astrocytic lineage (astrocytomas) have a relatively indolent clinical course; however, over time they progress to a higher grade. Molecular studies revealed recurrent tumors harbor more mutations upon malignant progression when treated with chemotherapy and/or radiotherapy compared to treatment-naïve tumors. However, genes which promote progression and recurrence in these tumors are unknown. This study aims to identify the transcriptional drivers of progression and recurrence of IDH-mutant gliomas. Longitudinal comparison of transcriptional profiles was performed on 6 matched pairs of primary lesions and their higher-grade recurrent lesion collected 1 to 8 years later. Three patients received no treatment and three patients received radiotherapy alone between their first and second surgeries. RNA was extracted from frozen specimens and following quality control, analyzed with the Illumina Next-Generation Sequencing platform (HiSeq HT paired end, TruSeq Kit). Comparison of RNA-seq results from primary and recurrent specimens identified 2042 differentially expressed genes (FC ≥2) for the no-treatment group and 2066 differentially expressed genes for the radiotherapy group. These genes include CADM2, SPP1, NDRG2 and CA9 which are associated with pro-tumor processes such as signal transduction, cell adhesion and treatment resistance. Validation is in progress. Identifying key genes driving glioma progression and recurrence will increase our understanding of the nature of these tumors and reveal core processes and potential novel targets that may be exploited for additional therapies for this deadly cancer.

There has been recent evidence supporting post-pancreatectomy pancreatitis as a factor in the development of postoperative pancreatic fistula (POPF). The aims of this study were to evaluate: (i) the correlation of the acinar cell density at the pancreatic resection margin with the intra-operative amylase concentration (IOAC) of peri-pancreatic fluid, postoperative pancreatitis, and POPF; and (ii) the association between postoperative pancreatitis on the first postoperative day and POPF.Consecutive patients who underwent pancreatic resection between June 2016 and July 2017 were included for analysis. Fluid for IOAC was collected, and amylase concentration was determined in drain fluid on postoperative days 1, 3, and 5. Serum amylase and lipase and urinary trypsinogen-2 concentrations were determined on the first postoperative day. Histology slides of the pancreatic resection margin were scored for acinar cell density.Sixty-one patients were included in the analysis. Acinar cell density significantly correlated with IOAC (r = 0.566, p 0.001), and was significantly associated with postoperative pancreatitis (p 0.001), and POPF (p = 0.003). Postoperative pancreatitis was significantly associated with the development of POPF (OR 17.81, 95%CI 2.17-145.9, p = 0.001).The development of POPF may involve a complex interaction between acinar cell density, immediate leakage of pancreatic fluid, and postoperative pancreatitis.

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy in women, with high-grade serous ovarian cancer (HGSOC) the most common and aggressive subtype. The tumor microenvironment is acknowledged to play a vital role in the growth and metastasis of many solid tumors, including ovarian cancer, and as such represents an attractive new therapeutic target. In ovarian cancer, patients with a higher proportion of desmoplasia have a poorer survival. Cancer-associated fibroblasts (CAFs) represent the most abundant cell type in the tumor stroma and are responsible for producing the desmoplastic reaction that is a poor prognostic factor in HGSOC. Genetic aberrations in ovarian CAFs are extremely rare, raising the possibility of alternative mechanisms that regulate gene expression in CAFs, such as regulation by long non-coding RNAs (lncRNAs). LncRNAs are transcripts that do not encode for protein, but have been shown to play important roles in several diseases, including cancer. However, very little is known about the role of lncRNAs in the tumor microenvironment. OBJECTIVES: To identify lncRNAs whose expression levels in CAFs are associated with patient survival and use computational approaches to predict their function. METHODS: CAFs were laser capture microdissected from 67 advanced stage HGSOCs. RNA was extracted from the microdissected samples and expression analyzed using Affymetrix U133 Plus 2.0 Arrays. Probes identified as lncRNAs were used in this analysis. Samples were normalized and background corrected using the robust multiarray average (RMA) method and expression values were log2 transformed. Expression levels of each lncRNA were clustered into low and high expression groups. Kaplan Meier /log-rank analysis was used to assess the association between expression levels of each lncRNA and the patients' overall survival. Multivariate cox regression analysis was used to determine if differential expression of lncRNAs were independent predictors of survival. A network based ‘guilt-by-association' approach was used to predict the function of lncRNAs associated with patient survival. RESULTS: Increased expression of 9 lncRNAs including DANCR, MALAT1 and NEAT1 and decreased expression of 1 lncRNA in ovarian CAFs were found to be associated with poorer overall survival by the log-rank test. Expression profiles of 5 lncRNAs as well as response to chemotherapy and debulking status were significant predictors of survival by univariate cox proportional hazards analysis. To adjust for existing collinearity of the 10 lncRNAs, the first principal component of these lncRNAs (capturing 98% of variations), as well as response to chemotherapy and debulking status were incorporated into a multivariate model. The first principal component (HR=0.74, P=0.0001163) and response to chemotherapy (HR=0.22, P=0.000168) were found to be independent predictors of survival. Functional enrichment analysis revealed these lncRNAs are likely to play a role in metabolism, autophagy or immune response. CONCLUSIONS: We have identified several lncRNAs whose expression levels in CAFs are associated with survival of HGSOC patients, raising the likelihood that they play an important role in the tumor-promoting functions of CAFs. A further understanding of the role of lncRNAs in CAFs may be useful when designing novel therapies that target the tumor microenvironment. Citation Format: Emily K. Colvin, Viive M. Howell, Samuel C. Mok, Goli Samimi and Fatemeh Vafaee. EXPRESSION OF LNCRNAS IN OVARIAN CANCER-ASSOCIATED FIBROBLASTS IS ASSOCIATED WITH PATIENT SURVIVAL [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-067.

Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.

Gliomas are the most common primary brain tumors in adults. Driver mutations in Isocitrate Dehydrogenase 1 and 2 (IDH1/2) result in overproduction of an oncometabolite, 2-hydroxy-keto glutartate with subsequent downstream Tp53 mutations or loss of 1p19q. IDH-mutated low grade gliomas have a relatively indolent clinical course; however over time they migrate to a higher grade. Initial standard of care involves surgery with the addition of radiation and alkylating chemotherapy for more aggressive lesions. Molecular studies of recurrent lesions have documented numerous DNA mutations consistent with alkylating agent damage in those treated with chemotherapy. To ascertain the natural history of progressive IDH mutated 1p19q intact tumors this study compared differences in the proteomic profiles of 6 matched pairs of primary and recurrent lesions from patients who had NOT received chemotherapy. Three patients presented with grade 2 lesions and following resection were observed. Time to recurrence (TTR) was 38, 30 and 14 months respectively. Three patients presented with grade 3 lesions and received radiotherapy alone following surgery. Their TTR was 99, 52 and 71 months. Protein was extracted from frozen specimens and following quality control analyzed by SWATH-MS (on a Sciex TripleToF 6600). Overall, 1781 proteins were identified by SWATH-MS analysis. Of those proteins, 40 were significantly differentially expressed (FC ≥2-fold; p ≤ 0.05) between primary and recurrent specimens. In both datasets, these key proteins were associated with activation of the Acute Phase Response, Cell Death and Survival and Cellular Compromise, Development and Movement. Proteins significantly differentially expressed included PTPRZ1, GLS, IGFBP7 and LUM, which are involved in tumor cell proliferation and migration, angiogenesis and chemotherapy resistance. The identification of differentially expressed proteins will improve our understanding of the mechanisms driving recurrence and progression of gliomas and may also reveal potential novel targets for additional therapies for this deadly cancer.

BRCA1 mutations are associated with ovarian cancer. Previous studies reported that murine granulosa cell (GC) Brca1 loss caused ovarian-uterine tumors resembling serous cystadenomas, but the pathogenesis of these tumors may have been confounded by ectopic Brca1 expression and altered estrous cycling. We have used Tg.AMH.Cre conferring proven ovarian and GC-specific Cre activity to selectively target Brca1 disruption, denoted Brca1 GC−/−. Furthermore, ovary-specific Brca1 GC−/− was combined with global Trp53 haploinsufficiency (Trp53 +/−) and transgenic follicle-stimulating hormone (Tg.FSH) overexpression as a multi-hit strategy to investigate additional genetic and hormonal ovarian tumorigenesis mechanisms. However, 12-month-old Brca1 GC−/− mice had no detectable ovarian or uterine tumors. Brca1 GC−/− mice had significantly increased ovary weights, follicles exhibiting more pyknotic granulosa cells, and fewer corpora lutea with regular estrous cycling compared to controls. Isolated Brca1 GC−/− mutation lengthened the estrous cycle and proestrus stage; however, ovarian cystadenomas were not observed, even when Brca1 GC−/− was combined with Trp53 +/− and overexpressed Tg.FSH. Our Brca1 GC−/− models reveal that specific intra-follicular Brca1 loss alone, or combined with cancer-promoting genetic (Trp53 loss) and endocrine (high serum FSH) changes, was not sufficient to cause ovarian tumors. Our findings show that the ovary is remarkably resistant to oncogenesis, and support the emerging view of an extragonadal, multi-hit origin for ovarian tumorigenesis.

Cancer-associated fibroblasts (CAFs) contribute to the poor prognosis of ovarian cancer. Unlike in tumour cells, DNA mutations are rare in CAFs, raising the likelihood of other mechanisms that regulate gene expression such as long non-coding RNAs (lncRNAs). We aimed to identify lncRNAs that contribute to the tumour-promoting phenotype of CAFs. RNA expression from 67 ovarian CAF samples and 10 normal ovarian fibroblast (NOF) samples were analysed to identify differentially expressed lncRNAs and a functional network was constructed to predict those CAF-specific lncRNAs involved in metastasis. Of the 1,970 lncRNAs available for analysis on the gene expression array used, 39 unique lncRNAs were identified as differentially expressed in CAFs versus NOFs. The predictive power of differentially expressed lncRNAs in distinguishing CAFs from NOFs were assessed using multiple multivariate models. Interrogation of known transcription factor-lncRNA interactions, transcription factor-gene interactions and construction of a context-specific interaction network identified multiple lncRNAs predicted to play a role in metastasis. We have identified novel lncRNAs in ovarian cancer that are differentially expressed in CAFs compared to NOFs and are predicted to contribute to the metastasis-promoting phenotype of CAFs.

background: Adjuvant chemotherapy improves survival for patients with resected pancreatic cancer. Elderly patients are under-represented in Phase III clinical trials, and as a consequence the efficacy of adjuvant therapy in older patients with pancreatic cancer is not clear. We aimed to assess the use and efficacy of adjuvant chemotherapy in older patients with pancreatic cancer.\ud methods: We assessed a community cohort of 439 patients with a diagnosis of pancreatic ductal adenocarcinoma who underwent operative resection in centres associated with the Australian Pancreatic Cancer Genome Initiative.\ud results: The median age of the cohort was 67 years. Overall only 47% of all patients received adjuvant therapy. Patients who received adjuvant chemotherapy were predominantly younger, had later stage disease, more lymph node involvement and more evidence of perineural invasion than the group that did not receive adjuvant treatment. Overall, adjuvant chemotherapy was associated with prolonged survival (median 22.1 vs 15.8 months; P\ud conclusion: Patients aged greater than or equal to70 are less likely to receive adjuvant therapy although it is associated with improved outcome. Increased use of adjuvant therapy in older individuals is encouraged as they constitute a large proportion of patients with pancreatic cancer.

The SV40 viral oncogene has been used since the 1970s as a reliable and reproducible method to generate transgenic mouse models. This seminal discovery has taught us an immense amount about how tumorigenesis occurs, and its success has led to the evolution of many mouse models of cancer. Despite the development of more modern and targeted approaches for developing genetically engineered mouse models of cancer, SV40-induced mouse models still remain frequently used today. This review discusses a number of cancer types in which SV40 mouse models of cancer have been developed and highlights their relevance and importance to preclinical research.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis.

Background: Current staging methods for pancreatic cancer (PC) are inadequate, and biomarkers to aid clinical decision making are lacking. Despite the availability of the serum marker carbohydrate antigen 19.9 (CA19.9) for over two decades, its precise role in the management of PC is yet to be defined, and as a consequence, it is not widely used.\ud \ud Methods: We assessed the relationship between perioperative serum CA19.9 levels, survival and adjuvant chemotherapeutic responsiveness in a cohort of 260 patients who underwent operative resection for PC.\ud \ud Results By specifically assessing the subgroup of patients with detectable CA19.9, we identified potential utility at key clinical decision points. Low postoperative CA19.9 at 3 months (median survival 25.6 vs 14.8 months, P = 0.0052) and before adjuvant chemotherapy were independent prognostic factors. Patients with postoperative CA 19.9 levels >90 U/ml did not benefit from adjuvant chemotherapy (P = 0.7194) compared with those with a CA19.9 of ≤90 U/ml median 26.0 vs 16.7 months, P = 0.0108). Normalization of CA19.9 within 6 months of resection was also an independent favorable prognostic factor (median 29.9 vs 14.8 months, P = 0.0004) and normal perioperative CA19.9 levels identified a good prognostic group, which was associated with a 5-year survival of 42%.\ud \ud Conclusions: Perioperative serum CA19.9 measurements are informative in patients with detectable CA19.9 (defined by serum levels of of >5 U/ml) and have potential clinical utility in predicting outcome and response to adjuvant chemotherapy. Future clinical trials should prioritize incorporation of CA19.9 measurement at key decision points to prospectively validate these findings and facilitate implementation.

Epithelial ovarian cancer is the fifth leading cause of cancer related deaths in women and the most lethal gynecological malignancy. Extracellular matrix (ECM) is an integral component of both the normal and tumor microenvironment. ECM composition varies between tissues and is crucial for maintaining normal function and homeostasis. Dysregulation and aberrant deposition or loss of ECM components is implicated in ovarian cancer progression. The mechanisms by which tumor cells induce ECM remodelling to promote a malignant phenotype are yet to be elucidated. A thorough understanding of the role of the ECM in ovarian cancer is needed for the development of effective biomarkers and new therapies.

Background and Aim: Clinicopathological data regarding pancreatic solid pseudopapil- lary tumors (SPT) in a multiethnic country are limited. The aim of the present study was to characterize pancreatic SPT in Australia. Methods: Clinicopathological features, treatment, immunohistochemical findings and outcome data of 34 patients (79% Caucasian, 12%Asian, 6% South Pacific Islander and 3% African) with pancreatic SPT were reviewed. Results: The most presenting complaint was abdominal pain. Median diameter of tumors was 60 mm (range: 20-220); predominantly located in the pancreatic tail (tail : body : head = 23:3:8). All tumors were resected and patients underwent surgery, including a liver resection for metastasis, all patients were alive after a median follow up of 70 months (IQR: 48-178). Two patients underwent repeated surgery for local recurrences with liver metastases after 8 and 18 months, which were successfully managed by surgical resection. Completeness of excision, perineural spread, vascular space invasion, mitotic rate and cellular atypia did not predict recurrence. In all cases, there was aberrant nuclear staining of beta-catenin and a loss of membranous expression of E-cadherin with aberrant nuclear localization of the cytoplasmic domain. Most pancreatic SPT were also strongly positive for CD10 (96%), progesterone receptor (79%), cytokeratin (28%), synapthophysin (26%) and chromogranin (15%). Conclusions: Pancreatic SPT occur in all races and are uniformly indolent. Given com- plete resection of a pancreatic SPT is usually curative and recurrences can be treated with re-operation, correct diagnosis is important.

BACKGROUND: Gliomas are the most common primary brain tumours in adults. IDH-mutated low-grade gliomas of astrocytic lineage (astrocytomas) have a relatively indolent clinical course; however, over time they progress to a higher grade. Molecular studies revealed recurrent tumours harbour more mutations upon malignant progression when treated with chemotherapy and/or radiotherapy compared to treatment-naïve tumours, however, genes which promote progression and recurrence in these tumours are unknown. This study aims to ascertain the natural history and treatment-induced effects of progressive IDH-mutated astrocytomas using whole transcriptome profiling. MATERIAL AND METHODS: This study compared differences in the whole transcriptome profiles of 6 matched pairs of primary lesions with their higher-grade recurrent lesion. Tumour specimens from three patients who presented with grade 2 lesions and received no treatment following resection were collected. Tumours from another three patients who presented with grade 3 lesions and received radiotherapy alone following surgery were also collected. The recurrent high grade tumour of each patient that was collected from the second surgery was also analysed. RNA was extracted from frozen specimens and following quality control, analysed with the Illumina Next-Generation Sequencing platform (HiSeq HT paired end, TruSeq Kit). RESULTS: Comparison of primary and recurrent specimens identified 2042 differentially expressed genes (Fold Change ≥2) for the no-treatment group and 2066 differentially expressed genes for the radiotherapy group. Several differentially expressed gene families, including HLAs corresponding to MHC class II, cytokine families (IL, CXC, CC, TNF) and metalloproteinase families (ADAM, MMP), were associated with immune response and modification of the tumour microenvironment. The identification of these key gene families suggests the importance of the immune response and the tumour microenvironment to promote glioma progression. CONCLUSION: The identification of differentially expressed genes will improve our understanding of the mechanisms driving recurrence and progression of gliomas and may also reveal potential novel targets for additional therapies for this deadly cancer.

Microbiol. 2007; 114: 267–74. 12 Torok VA, Ophel-Keller K, Loo M, Hughes RJ. Application of methods for identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl. Environ. Microbiol. 2008; 74: 783–91. 13 Kamil R, Geier MS, Butler RN, Howarth GS. Lactobacillus rhamnosus GG exacerbates intestinal ulceration in a model of indomethacin-induced enteropathy. Dig. Dis. Sci. 2007; 52: 1247–52. 14 Guarner F. Prebiotics in inflammatory bowel diseases. Br. J. Nutr. 2007; 98 (Suppl. 1): S85–9. 15 Geier MS, Butler RN, Giffard PM, Howarth GS. Prebiotic and synbiotic fructooligosaccharide administration fails to reduce the severity of experimental colitis in rats. Dis Colon Rectum 2007; 50: 1061–9. 16 Rachmilewitz D, Karmeli F, Takabayashi K et al. Immunostimulatory DNA ameliorates experimental and spontaneous murine colitis. Gastroenterology 2002; 122: 1428–41.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-β, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Transplantation of insulin-producing cells offers a promising therapy to treat diabetes. However, due to the limited number of donor islet cells available, researchers are looking for different sources of pancreatic islet progenitor or stem cells. A stem cell with extensive proliferative ability may provide a valuable source of islet progenitor cells. Several studies have demonstrated that a progenitor/stem-cell population can be expanded in vitro to generate large numbers of islet progenitor cells. However, efficient and directed differentiation of these cells to an endocrine pancreatic lineage has been difficult to achieve. We discuss here various pancreatic islet stem cells that we and others have obtained from embryonic, fetal or adult human tissues. We review the progress that has been achieved with pancreatic islet progenitor cell differentiation in the last 2 decades and discuss how close we are to translate this research to the clinics.

Nature 543, 65–71 (2017); doi:10.1038/nature21063 It has been brought to our attention that in Fig. 2d of this Article, an incorrect Sanger trace was used to represent the breakpoint of the EWSR1 and FLI1 type 2 fusion. This was due to an error during manuscript preparation, when we inadvertently inserted the electrophoretic trace referring to EWSR1 splicing variants.

The ability to accurately model human cancer in mice enables in vivo examination of the biological mechanisms related to cancer initiation and progression as well as preclinical testing of new anticancer treatments and potential targets. The emergence of the genetically engineered Sleeping Beauty system of insertional mutagenesis has led to the development of a new generation of genetic mouse models of cancer and identification of novel cancer-causing genes. This chapter reviews the published cancer models of Sleeping Beauty and strategies using available strains to generate several models of cancer.

Despite incremental advances in the diagnosis and treatment for pancreatic cancer (PC), the 5‑year survival rate remains

Ovarian cancer is the leading cause of death in women with gynecological malignancy and improvements in current treatments are needed. As with many other solid cancers, the ovarian tumor microenvironment is emerging as a key player in tumor progression and a potential therapeutic target. The tumor microenvironment contains several non-malignant cell types that are known to contribute to tumor progression and metastasis. Included in this population of non-malignant cells are several different types of immune cells, of which tumor-associated macrophages (TAMs) are the most abundant. An increasing amount of evidence is emerging to suggest that TAMs display a unique activation profile in ovarian tumors and are able to create an immunosuppressive microenvironment, allowing tumors to evade immune detection and promoting tumor progression. Therefore, an increased understanding of how these immune cells interact with tumor cells and the microenvironment will greatly benefit the development of more effective immunotherapies to treat ovarian cancer. This review focuses on the role of TAMs in the ovarian tumor microenvironment and how they promote tumor progression.

The ability to accurately model human cancer in mice enables in vivo examination of the biological mechanisms related to cancer initiation and progression as well as preclinical testing of new anticancer treatments and potential targets. The emergence of the genetically engineered Sleeping Beauty system of insertional mutagenesis has led to the development of a new generation of genetic mouse models of cancer and identifi cation of novel cancer-causing genes. This chapter reviews the published cancer models of Sleeping Beauty and strategies using available strains to generate several models of cancer.

The exocrine pancreas can undergo acinar-to-ductal metaplasia (ADM), as in the case of pancreatitis where precursor lesions of pancreatic ductal adenocarcinoma (PDAC) can arise. The NAD+-dependent protein deacetylase Sirtuin-1 (Sirt1) has been implicated in carcinogenesis with dual roles depending on its subcellular localization. In this study, we examined the expression and the role of Sirt1 in different stages of pancreatic carcinogenesis, i.e. ADM models and established PDAC. In addition, we analyzed the expression of KIAA1967, a key mediator of Sirt1 function, along with potential Sirt1 downstream targets. Sirt1 was co-expressed with KIAA1967 in the nuclei of normal pancreatic acinar cells. In ADM, Sirt1 underwent a transient nuclear-to-cytoplasmic shuttling. Experiments where during ADM, we enforced repression of Sirt1 shuttling, inhibition of Sirt1 activity or modulation of its expression, all underscore that the temporary decrease of nuclear and increase of cytoplasmic Sirt1 stimulate ADM. Our results further underscore that important transcriptional regulators of acinar differentiation, that is, Pancreatic transcription factor-1a and β-catenin can be deacetylated by Sirt1. Inhibition of Sirt1 is effective in suppression of ADM and in reducing cell viability in established PDAC tumors. KIAA1967 expression is differentially downregulated in PDAC and impacts on the sensitivity of PDAC cells to the Sirt1/2 inhibitor Tenovin-6. In PDAC, acetylation of β-catenin is not affected, unlike p53, a well-characterized Sirt1-regulated protein in tumor cells. Our results reveal that Sirt1 is an important regulator and potential therapeutic target in pancreatic carcinogenesis. Cancer Res; 73(7); 2357–67. ©2012 AACR.

Purpose Individuals with adenocarcinoma of the ampulla of Vater demonstrate a broad range of outcomes, presumably because these cancers may arise from any one of the three epithelia that converge at that location. This variability poses challenges for clinical decision making and the development of novel therapeutic strategies. Patients and Methods We assessed the potential clinical utility of histomolecular phenotypes defined using a combination of histopathology and protein expression (CDX2 and MUC1) in 208 patients from three independent cohorts who underwent surgical resection for adenocarcinoma of the ampulla of Vater. Results Histologic subtype and CDX2 and MUC1 expression were significant prognostic variables. Patients with a histomolecular pancreaticobiliary phenotype (CDX2 negative, MUC1 positive) segregated into a poor prognostic group in the training (hazard ratio [HR], 3.34; 95% CI, 1.69 to 6.62; P < .001) and both validation cohorts (HR, 5.65; 95% CI, 2.77 to 11.5; P < .001 and HR, 2.78; 95% CI, 1.25 to 7.17; P = .0119) compared with histomolecular nonpancreaticobiliary carcinomas. Further stratification by lymph node (LN) status defined three clinically relevant subgroups: one, patients with histomolecular nonpancreaticobiliary (intestinal) carcinoma without LN metastases who had an excellent prognosis; two, those with histomolecular pancreaticobiliary carcinoma with LN metastases who had a poor outcome; and three, the remainder of patients (nonpancreaticobiliary, LN positive or pancreaticobiliary, LN negative) who had an intermediate outcome. Conclusion Histopathologic and molecular criteria combine to define clinically relevant histomolecular phenotypes of adenocarcinoma of the ampulla of Vater and potentially represent distinct diseases with significant implications for current therapeutic strategies, the ability to interpret past clinical trials, and future trial design.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy in women. Despite advances in our understanding of the molecular biology of EOC, patient survival has not significantly improved in decades and new treatments are needed. There is increased interest in therapeutic targeting of the tumor microenvironment (TME). The lysyl oxidase (LOX) family of enzymes are involved in extracellular matrix remodelling through crosslinking of collagen and elastin. They have been shown to play an important role in the TME of other cancers and are regulated by hypoxia, however little is known about their role in EOC. Therefore we aimed to investigate the role of LOX and LOXL2 in EOC using in vitro models. Tumor epithelial (n = 2) and matched cancer-associated fibroblast cell lines (n = 2) derived from an SV40-induced mouse model of EOC were generated. MTT assays were performed on all cell lines to assess the effect of LOX and LOXL2 inhibition on cell viability. The effect of LOX and LOXL2 inhibition on cancer cell migration and invasion towards cancer-associated fibroblast conditioned media was also investigated using transwell assays. RNA and protein were isolated and analysed from cells cultured in normoxic and hypoxic conditions to assess whether LOX and LOXL2 gene and protein expression is regulated by hypoxia. Inhibition of LOX and LOXL2 did not affect cell viability. LOX and LOXL2 inhibition significantly reduced the migration and invasion of EOC cells towards cancer-associated fibroblast conditioned media in vitro (p This study demonstrates a role for LOX and LOXL2 in promoting migration and invasion of EOC cells in vitro. The upregulation of LOX and LOXL2 in hypoxia suggests a role in facilitating tumor growth and progression within hypoxic tumors. These results provide support for the utility of LOX and LOXL2 inhibition as promising and novel therapeutic strategies in EOC. Citation Format: Angela Cho, Amanda L. Hudson, Samuel Yuen, Nham Tran, Viive M. Howell, Emily K. Colvin. LOX and LOXL2 inhibition as a treatment for ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 764.

SummaryColony-forming units – fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

Background and Aims:\ud Chronic pancreatitis and pancreatic cancer are characterised by extensive stellate cell mediated fibrosis, and current therapeutic development includes targeting pancreatic cancer stroma and tumor-host interactions. Recent evidence has suggested that circulating bone marrow derived stem cells (BMDC) contribute to solid organs. We aimed to define the role of circulating haematopoietic cells in the normal and diseased pancreas.\ud \ud Methods:\ud Whole bone marrow was harvested from male β-actin-EGFP donor mice and transplanted into irradiated female recipient C57/BL6 mice. Chronic pancreatitis was induced with repeat injections of caerulein, while carcinogenesis was induced with an intrapancreatic injection of dimethylbenzanthracene (DMBA). Phenotype of engrafted donor-derived cells within the pancreas was assessed by immunohistochemistry, immunofluorescence and in situ hybridisation.\ud \ud Results:\ud GFP positive cells were visible in the exocrine pancreatic epithelia from 3 months post transplantation. These exhibited acinar morphology and were positive for amylase and peanut agglutinin. Mice administered caerulein developed chronic pancreatitis while DMBA mice exhibited precursor lesions and pancreatic cancer. No acinar cells were identified to be donor-derived upon cessation of cerulein treatment, however rare occurrences of bone marrow-derived acinar cells were observed during pancreatic regeneration. Increased recruitment of BMDC was observed within the desmoplastic stroma, contributing to the activated pancreatic stellate cell (PaSC) population in both diseases. Expression of stellate cell markers CELSR3, PBX1 and GFAP was observed in BMD cancer-associated PaSCs, however cancer-associated, but not pancreatitis-associated BMD PaSCs, expressed the cancer PaSC specific marker CELSR3.\ud \ud Conclusions:\ud This study demonstrates that BMDC can incorporate into the pancreas and adopt the differentiated state of the exocrine compartment. BMDC that contribute to the activated PaSC population in chronic pancreatitis and pancreatic cancer have different phenotypes, and may play important roles in these diseases. Further, bone marrow transplantation may provide a useful model for the study of tumor-host interactions in cancer and pancreatitis.

Clinicopathological data regarding pancreatic solid pseudopapillary tumors (SPT) in a multiethnic country are limited. The aim of the present study was to characterize pancreatic SPT in Australia.Clinicopathological features, treatment, immunohistochemical findings and outcome data of 34 patients (79% Caucasian, 12% Asian, 6% South Pacific Islander and 3% African) with pancreatic SPT were reviewed.The most presenting complaint was abdominal pain. Median diameter of tumors was 60 mm (range: 20-220); predominantly located in the pancreatic tail (tail : body : head = 23:3:8). All tumors were resected and patients underwent surgery, including a liver resection for metastasis, all patients were alive after a median follow up of 70 months (IQR: 48-178). Two patients underwent repeated surgery for local recurrences with liver metastases after 8 and 18 months, which were successfully managed by surgical resection. Completeness of excision, perineural spread, vascular space invasion, mitotic rate and cellular atypia did not predict recurrence. In all cases, there was aberrant nuclear staining of beta-catenin and a loss of membranous expression of E-cadherin with aberrant nuclear localization of the cytoplasmic domain. Most pancreatic SPT were also strongly positive for CD10 (96%), progesterone receptor (79%), cytokeratin (28%), synapthophysin (26%) and chromogranin (15%).Pancreatic SPT occur in all races and are uniformly indolent. Given complete resection of a pancreatic SPT is usually curative and recurrences can be treated with re-operation, correct diagnosis is important.

PurposeCurrent adjuvant therapies for pancreatic cancer (PC) are inconsistently used and only modestly effective. Because a high proportion of patients who undergo resection for PC likely harbor occult metastatic disease, any adjuvant trials assessing therapies such as radiotherapy directed at locoregional disease are significantly underpowered. Stratification based on the probability (and volume) of residual locoregional disease could play an important role in the design of future clinical trials assessing adjuvant radiotherapy.Patients and MethodsWe assessed the relationships between margin involvement, the proximity to operative resection margins and outcome in a cohort of 365 patients who underwent operative resection for PC.ResultsMicroscopic involvement of a resection margin by tumor was associated with a poor prognosis. Stratifying the minimum clearance of resection margins by 0.5-mm increments demonstrated that although median survival was no different to clear margins based on these definitions, it was not until the resection margin was clear by more than 1.5 mm that optimal long-term survival was achieved.ConclusionThese data demonstrate that a margin clearance of more than 1.5 mm is important for long-term survival in a subgroup of patients. More aggressive therapeutic approaches that target locoregional disease such as radiotherapy may be beneficial in patients with close surgical margins. Stratification of patients for entry onto future clinical trials based on this criterion may identify those patients who benefit from adjuvant radiotherapy.

BackgroundMorphologically similar cancers display heterogeneous patterns of molecular aberrations and follow substantially different clinical courses. This diversity has become the basis for the definition of molecular phenotypes, with significant implications for therapy. Microarray or proteomic expression profiling is conventionally employed to identify disease-associated genes, however, traditional approaches for the analysis of profiling experiments may miss molecular aberrations which define biologically relevant subtypes.Methodology/principal findingsHere we present Messina, a method that can identify those genes that only sometimes show aberrant expression in cancer. We demonstrate with simulated data that Messina is highly sensitive and specific when used to identify genes which are aberrantly expressed in only a proportion of cancers, and compare Messina to contemporary analysis techniques. We illustrate Messina by using it to detect the aberrant expression of a gene that may play an important role in pancreatic cancer.Conclusions/significanceMessina allows the detection of genes with profiles typical of markers of molecular subtype, and complements existing methods to assist the identification of such markers. Messina is applicable to any global expression profiling data, and to allow its easy application has been packaged into a freely-available stand-alone software package.

Background & Aims: Current methods of preoperative staging and predicting outcome following pancreatectomy for pancreatic cancer (PC) are inadequate. We evaluated the utility of multiple biomarkers from distinct biologic pathways as potential predictive markers of response to pancreatectomy and patient survival. Methods: We assessed the relationship of candidate biomarkers known, or suspected, to be aberrantly expressed in PC, with disease-specific survival and response to therapy in a cohort of 601 patients. Results: Of the 17 candidate biomarkers examined, only elevated expression of S100A2 was an independent predictor of survival in both the training (n = 162) and validation sets (n = 439; hazard ratio [HR], 2.19; 95% confidence interval [CI]: 1.48-3.25; P < .0001) when assessed in a multivariate model with clinical variables. Patients with high S100A2 expressing tumors had no survival benefit with pancreatectomy compared with those with locally advanced disease, whereas those without high S100A2 expression had a survival advantage of 10.6 months (19.4 vs 8.8 months, respectively) and a HR of 3.23 (95% CI: 2.39-4.33; P < .0001). Of significance, patients with S100A2-negative tumors had a significant survival benefit from pancreatectomy even in the presence of involved surgical margins (median, 15.7 months; P = .0007) or lymph node metastases (median, 17.4 months; P = .0002). Conclusions: S100A2 expression is a good predictor of response to pancreatectomy for PC and suggests that high S100A2 expression may be a marker of a metastatic phenotype. Prospective measurement of S100A2 expression in diagnostic biopsy samples has potential clinical utility as a predictive marker of response to pancreatectomy and other therapies that target locoregional disease. © 2009 AGA Institute.

Identification of a biomarker of prognosis and response to therapy that can be assessed preoperatively would significantly improve overall outcomes for patients with pancreatic cancer. In this study, patients whose tumours exhibited high LMO4 expression had a significant survival advantage following operative resection, whereas the survival of those patients whose tumours had low or no LMO4 expression was not significantly different when resection was compared with operative biopsy alone.

OBJECTIVE—The use of human fetal pancreatic tissue may provide a potential source of transplantable β-cells as a therapy for type 1 diabetes. Human fetal pancreas has a remarkable capacity to grow and differentiate in vivo and has been shown to reverse diabetes in rodents. However, it is known that human fetal pancreas obtained from the second trimester of gestation is immunogenic and is rejected after transplantation. Tissue obtained from earlier stages might prove to be immune privileged, as has been shown for other tissues.RESEARCH DESIGN AND METHODS—In this study, we determined the immunogenicity of human fetal pancreatic tissue obtained from the first trimester of gestation in a humanized mouse model. A microarray study of immunoregulatory gene expression in first- and second-trimester human fetal pancreas was also undertaken.RESULTS—The analysis of transplanted human fetal pancreata revealed a significantly decreased immunogenicity of the first-trimester tissue. The first-trimester grafts showed only limited cellular infiltration and contained numerous insulin-positive cells, whereas second-trimester tissue was completely infiltrated and rejected. Furthermore an analysis of immunoregulatory genes expressed in first- and second-trimester human fetal pancreas by microarray demonstrated the upregulation of several key immunoregulatory genes in the second-trimester tissue. This might account for the reduced immunogenicity of the younger tissue.CONCLUSIONS—Our results provide the first indication that the use of first-trimester human fetal pancreas for transplantation might increase the survival of the grafts and might decrease the requirement for immunosuppressive drugs.

Ovarian cancer is the most lethal gynecological malignancy in women. Expression profiling of the high-grade serous subtype has revealed that patients exhibiting a “stromal expression signature” demonstrate the poorest prognosis1. However, genetic aberrations in ovarian cancer-associated fibroblasts (CAFs) are extremely rare2, raising the possibility that alternative mechanisms that regulate gene expression, such as long non-coding RNAs (lncRNAs), are present in CAFs. LncRNAs are polyadenylated RNA transcripts that do not encode for protein, but have been shown to correlate with cancer progression and outcome. Therefore, our aim was to investigate the presence of differentially expressed lncRNAs in ovarian CAFs versus normal ovarian fibroblasts. CAFs were laser-capture microdissected from 51 advanced-stage high-grade serous ovarian cancers and 10 normal ovaries removed from women for non-cancer reasons. RNA was extracted from the microdissected samples and expression analyzed using Affymetrix U133 Plus 2.0 Arrays. Probes previously identified as lncRNAs were used in this analysis3. Samples were normalized and background corrected using the robust multi-arrray average (RMA) method and expression values were log2 transformed. Differentially expressed probes between cancer versus normal samples were calculated using linear models for microarray data (limma) package from Bioconductor and a moderated t-statistic used to assess significance. Based on a significance cutoff of fold-change > 2 and a p-value < 0.05, 54 lncRNAs were identified as differentially expressed in cancer versus normal fibroblasts. These included lncRNAs previously implicated in ovarian cancer such as XIST and H19, the p53-regulated lncRNA TUG1, as well as several lncRNAs that are known to play a role in other cancer types such as NEAT1, GAS5 and CASC2. In addition, 16 lncRNAs were differentially expressed between patients with short (15 months) overall survival. These included MALAT1, MEG3, TUG1, XIST and CRNDE. In summary, we have identified lncRNAs that are differentially expressed in ovarian CAFs compared to normal ovarian fibroblasts, as well as lncRNAs that are differentially expressed based on survival. Given that CAFs are known to promote tumor progression and metastasis, these lncRNAs present suitable candidates for their role in the tumor-promoting function of CAFs. References 1. Tothill, R.W. et al. Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res 14, 5198-208 (2008). 2. Qiu, W. et al. No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas. Nat Genet 40, 650-5 (2008). 3. Zhang, X. et al. Long non-coding RNA expression profiles predict clinical phenotypes in glioma. Neurobiol Dis 48, 1-8 (2012). Citation Format: Emily K. Colvin, Fatemeh Vafaee, Samuel C. Mok, Viive M. Howell, Goli Samimi. Differential expression of long non-coding RNAs in ovarian cancer-associated fibroblasts versus normal ovarian fibroblasts. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2885. doi:10.1158/1538-7445.AM2015-2885

Background: The goal of this project is to increase our understanding of the molecular aetiology of ovarian cancer by utilizing Sleeping Beauty (SB) insertional mutagenesis. Methods: Breeding colonies of the following genetically engineered mice were established and cross bred: homozygous floxed SB (STOCK Rosa26-LsL-D-SB11;T2/Onc2,TG6113), homozygous floxed Brca1 knock-out (C57BL/6.Brca1tm2Brn) and Tp53 mutant (C57BL/6-Trp53tm1Tyj/J). CRE recombinase packaged into an adenoviral vector (AdCreM2, MicroBix Biosystems Inc, CA) was surgically injected under the ovarian bursal membrane of mature female mice to delete Brca1 and activate SB mutagenesis in the ovarian surface epithelium. Mice were monitored and sacrificed at ethically defined endpoints or a maximum of 15 months post-surgery. Tumors were assessed for SB transposase activity by immunohistochemical staining. DNA was extracted from paraffin embedded sections of ovarian tumors and underwent high-throughput sequencing for T2/Onc2 insertion sites (Illumina, University of Iowa). Genes with insertion sites were interrogated against SEOC data from The Cancer Genome Atlas (TCGA) to identify genes also altered in the human malignancy. Functional annotation clustering was performed using DAVID (Database for Annotation, Visualization and Integrated Discovery, v6.7). Results: Ovarian tumors were observed at low penetrance starting at 30 weeks post-surgery in SBflox/+Tp53mut/+ mice (6%, 3/48) and SBflox/+Brca1flox/flox p53mut/+ mice (8%, 4/50). No ovarian tumors were observed in SBflox/+Brca1flox/flox mice (n=38) or in SBflox/+Brca1flox/+ mice (n=26). Sequencing of the insertion sites identified a number of genes of which 67 were altered in 10 - 30% of cases in the TCGA SEOC dataset (N = 316). This gene-set was enriched for kinases (P = 0.003, False discovery rate (FDR) = 3%) including Fgfr2, Dyrk1a and Gsk3b, and small GTPase regulators (P = 0.01, FDR = 11%) including Smap2, Trio and Dock10. Other genes of interest included tumor suppressor genes Arid1b, Cdh4 and Wwox and the E3 ubiquitin ligase Ube3a. In the TCGA dataset, decreased progression free survival was associated with high expression of GSK3B, TRIO and UBE3A and low expression of WWOX. Conclusions: This screen identified a number of novel potential driver genes of ovarian cancer. In addition genes previously associated with ovarian cancer were also identified, providing proof of principle for this approach. Investigation of these novel genes may lead to further insights into the pathogenesis of ovarian cancer. This work was supported by Cancer Institute NSW, Cancer Council NSW and The Northern Translational Cancer Research Unit, NSW, Australia. Citation Format: Emily K. Colvin, Emily Fuller, Jizhou Cheng, Anthony Gill, Deborah J. Marsh, Viive M. Howell. A mutagenesis screen identifies tumor suppressors and kinases as potential driver genes of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A10.