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The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ 2 =5.4, P = 0.021). Guiding ctDNA detection in pre-operative ccfDNA based on mutations present in tumor DNA yielded a similar survival analysis. Detection of ctDNA in the post-operative ccfDNA with or without tumor-informed guidance was not associated with outcomes. Therefore, the detection of PDAC-derived ctDNA during a broad and untargeted survey of ccfDNA with NGS may be a valuable, non-invasive, prognostic biomarker to integrate into the clinical assessment and management of patients prior to surgery.

Background and AimsGenes associated with hereditary breast and ovarian cancer (HBOC) and colorectal cancer (CRC) susceptibility have been shown to play a role in pancreatic cancer susceptibility. Germline genetic testing of pancreatic cancer cases could be beneficial for at-risk relatives with pathogenic variants in established HBOC and CRC genes, but it is unclear what proportion of pancreatic cancer cases harbor pathogenic variants in these genes.Methods66 pancreatic cancer cases, unselected for family history and diagnosed at the Huntsman Cancer Hospital (HCH), were sequenced on a custom 34-gene panel including known HBOC and CRC genes. A second set of 156 unselected HCH pancreatic cancer cases were sequenced on an expanded 59-gene panel (n=95) or with a custom 14-gene clinical panel (n=61). Sequencing data from both sets of pancreatic cancer cases, the pancreatic cancer cases of the Cancer Genome Atlas (TCGA), and an unselected pancreatic cancer screen from the Mayo Clinic were combined in a meta-analysis to estimate the proportion of carriers with pathogenic and variants of uncertain significance.ResultsApproximately 8.9% of unselected pancreatic cancer cases at the HCH carried a variant with potential HBOC or CRC screening recommendations. A meta-analysis of unselected pancreatic cancer cases revealed that approximately 10.5% carry a pathogenic variant or HiP-VUS.ConclusionWith the inclusion of both HBOC and CRC susceptibility genes in a panel test, unselected pancreatic cancer cases have a high enough percentage of carriers to rationalize genetic testing for identification of variants that could be further used in cascade testing of healthy relatives to increase HBOC and CRC surveillance measures.

Many patients with stage I-II pancreatic adenocarcinoma do not undergo resection. We hypothesized that (1) clinical staging underestimates nodal involvement, causing stage IIB to have a greater percent of resected patients and (2) this stage-shift causes discrepancies in observed survival.The Surveillance, Epidemiology, and End Results (SEER) research database was used to evaluate cause-specific survival in patients with pancreatic adenocarcinoma from 2004-2012. Survival was compared using the log-rank test. Single-center data on 105 patients who underwent resection of pancreatic adenocarcinoma without neoadjuvant treatment were used to compare clinical and pathologic nodal staging.In SEER data, medium-term survival in stage IIB was superior to IB and IIA, with median cause-specific survival of 14, 9, and 11 months, respectively (P .001). Seventy-two percent of stage IIB patients underwent resection vs 28% in IB and 36% in IIA (P .001). In our institutional data, 12.4% of patients had clinical evidence of nodal involvement vs 69.5% by pathologic staging (P .001). Among clinical stage IA-IIA patients, 71.6% had nodal involvement by pathologic staging.Both SEER and institutional data support substantial underestimation of nodal involvement by clinical staging. This finding has implications in decisions regarding neoadjuvant therapy and analysis of outcomes in the absence of pathologic staging.

When epithelia become too crowded, some cells are extruded that later die. To extrude, a cell produces the lipid, Sphingosine 1-Phosphate (S1P), which activates S1P2 receptors in neighboring cells that seamlessly squeeze the cell out of the epithelium. Here, we find that extrusion defects can contribute to carcinogenesis and tumor progression. Tumors or epithelia lacking S1P2 cannot extrude cells apically and instead form apoptotic-resistant masses, possess poor barrier function, and shift extrusion basally beneath the epithelium, providing a potential mechanism for cell invasion. Exogenous S1P2 expression is sufficient to rescue apical extrusion, cell death, and reduce orthotopic pancreatic tumors and their metastases. Focal Adhesion Kinase (FAK) inhibitor can bypass extrusion defects and could, therefore, target pancreatic, lung, and colon tumors that lack S1P2 without affecting wild-type tissue. DOI: http://dx.doi.org/10.7554/eLife.04069.001, eLife digest Epithelial cells cover the surface of our bodies, line our lungs, stomach and intestines and serve as a protective layer around other organs. If too many epithelial cells die and are not replaced, this protective layer may erode and lead to organ damage. However, if too many new cells grow, tumors can form. One process that helps to maintain the right number of epithelial cells is called extrusion. When too many epithelial cells are present, the resulting overcrowding triggers this process to squeeze excess cells out of the layer and away from the organ. Usually, these cells quickly die. However, if the pathway that regulates this process—which involves a receptor protein called S1P2—is disturbed, the cells may instead be pushed into the space between the epithelial layer and the organ. When this happens, the cells are more likely to survive and may then form a tumor that invades the organ. Gu et al. interfered with extrusion by reducing the levels of the S1P2 receptor in layers of human epithelial cells grown in the laboratory. Fewer epithelial cells were squeezed out of these cell layers, making the layers up to three times as thick in places. Moreover, mutant zebrafish lacking the S1P2 receptor also accumulated epithelial masses throughout their bodies. Gu et al. found that disrupting the extrusion process made the cells resistant to chemotherapy, and that certain hard-to-treat human pancreatic, lung, and colon cancers had lower levels of the S1P2 receptors. Boosting the activity of S1P2 receptors helped to restore normal extrusion and reduced the size of pancreatic tumors in mice. Gu et al. then focused on an enzyme called Focal Adhesion Kinase that helps cells to survive. Treating zebrafish with a drug to block the activity of this enzyme left normal fish unharmed. However, in mutant fish with malfunctioning extrusion pathways, the drug rescued the number of cells that died, reduced the size and number of masses, and cured their leaky skin barrier. If further studies confirm the results, the drug may offer a new, less toxic, treatment for certain cancers that do not respond to currently available treatments. DOI: http://dx.doi.org/10.7554/eLife.04069.002

OBJECTIVE: The goal of this investigation was to determine the role of calcitonin gene-related peptide (CGRP) in gastric mucosal resistance to ulceration. SUMMARY BACKGROUND DATA: CGRP is a 37-amino acid peptide found in the peripheral ends of afferent gastric neurons. CGRP is known to inhibit acid secretion, stimulate mucosal blood flow, and stimulate release of somatostatin. METHODS: The release of CGRP in response to intragastric and intra-arterial administration of capsaicin in the isolated, vascularly perfused rat stomach was measured by radioimmunoassay. The molecular forms of CGRP released were analyzed by gel filtration chromatography. The effect of intravenous CGRP or intragastric capsaicin on gastric ulceration induced by 100 mmol/L HCl and indomethacin was studied in intact and endogenous CGRP-depleted rats. RESULTS: Intra-arterial capsaicin (concentration range, 10(-7) to 10(-5) mol/L) stimulated a prompt and sustained release of immunoreactive CGRP, of which 84% coeluted with rat 1-37 CGRP I by gel filtration. Intragastric capsaicin (range, 10(-5) to 10(-4) mol/L) failed to release CGRP into the vascular perfusate. In intact rats, intragastric capsaicin (10(-6) mol/L) or intravenous CGRP I (10 micrograms/kg/hr) reduced the number and area of mucosal lesions caused by HCl and indomethacin compared with the findings in control rats. Rats depleted of endogenous CGRP were more susceptible to gastric ulceration than were normal rats. Intragastric capsaicin failed to protect the mucosa of CGRP-depleted rats, whereas exogenous intravenous CGRP was effective. CONCLUSIONS: These data support the hypothesis that CGRP released from gastric enteric neurons mediates gastric mucosal resistance to ulceration by noxious agents.

SummaryChronic pancreatitis is a well-known risk factor for pancreatic ductal adenocarcinoma (PDA) development in humans, and inflammation promotes PDA initiation and progression in mouse models of the disease. However, the mechanistic link between inflammatory damage and PDA initiation is unclear. Using a Kras-driven mouse model of PDA, we establish that the inflammatory mediator Stat3 is a critical component of spontaneous and pancreatitis-accelerated PDA precursor formation and supports cell proliferation, metaplasia-associated inflammation, and MMP7 expression during neoplastic development. Furthermore, we show that Stat3 signaling enforces MMP7 expression in PDA cells and that MMP7 deletion limits tumor size and metastasis in mice. Finally, we demonstrate that serum MMP7 level in human patients with PDA correlated with metastatic disease and survival.

hpb_177 352..358 Objective: Prognostic markers for pancreatic ductal adenocarcinoma (PDA) have failed to accurately predict patient prognosis. Recently, interest has developed in the accuracy of integrin-associated PINCH protein expression in human cancers as a predictive marker of tumour status. The goal of this study was to define the expression of PINCH protein in PDA. Methods: Human PDA samples and orthotopic tumours from a murine model were analysed by immunohistochemistry for PINCH expression. In the animal model, PINCH expression was compared between primary and metastatic tumours. In the human samples, PINCH expression was correlated with stage, nodal involvement, margin status and overall survival. Results: In the murine model, there was greater PINCH expression in metastatic tumours than in primary tumours. In the human PDA samples, greater staining for PINCH in the tumour cells was correlated with higher T status. Additionally, high PINCH expression in the stroma was associated with decreased overall survival. Conclusions: Findings of increased PINCH protein in more advanced stages of human PDA, as well as in metastatic tumours in the animal model, support the hypothesis that PINCH is an important controller of cell survival and migration. Additionally, the importance of the differential expression of PINCH in the human tumour and stroma warrants further evaluation.

BackgroundThe American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) was started in 2004. Presently, 58% of the 198 hospitals participating in ACS-NSQIP are academic or teaching hospitals. In 2008, ACS-NSQIP initiated a number of changes and made risk-adjusted data available for use by participating hospitals. This analysis explores the ACS-NSQIP database for utility in developing hepato-pancreato-biliary (HPB) surgery-specific outcomes (HPB-NSQIP).MethodsThe ACS-NSQIP Participant Use File was queried for patient demographics and outcomes for 49 HPB operations from 1 January 2005 through 31 December 2007. The procedures included six hepatic, 16 pancreatic and 23 complex biliary operations. Four laparoscopic or open cholecystectomy operations were also studied. Risk-adjusted probabilities for morbidity and mortality were compared with observed rates for each operation.ResultsDuring this 36-month period, data were accumulated on 9723 patients who underwent major HPB surgery, as well as on 44189 who received cholecystectomies. The major HPB operations included 2847 hepatic (29%), 5074 pancreatic (52%) and 1802 complex biliary (19%) procedures. Patients undergoing hepatic resections were more likely to have metastatic disease (42%) and recent chemotherapy (7%), whereas those undergoing complex biliary procedures were more likely to have significant weight loss (20%), diabetes (13%) and ascites (5%). Morbidity was high for hepatic, pancreatic and complex biliary operations (20.1%, 32.4% and 21.2%, respectively), whereas mortality was low (2.3%, 2.7% and 2.7%, respectively). Compared with laparoscopic cholecystectomy, the open operation was associated with higher rates of morbidity (19.2% vs. 6.0%) and mortality (2.5% vs. 0.3%). The ratios between observed and expected morbidity and mortality rates were

Background Gastric ileus is an unsolved clinical problem and current treatment is limited to supportive measures. Models of ileus using anesthetized animals, muscle strips or isolated smooth muscle cells do not adequately reproduce the clinical situation. Thus, previous studies using these techniques have not led to a clear understanding of the pathophysiology of ileus. The feasibility of using food intake and fecal output as simple, clinically relevant endpoints for monitoring ileus in a conscious mouse model was evaluated by assessing the severity and time course of various insults known to cause ileus. Methods Delayed food intake and fecal output associated with ileus was monitored after intraperitoneal injection of endotoxin, laparotomy with bowel manipulation, thermal injury or cerulein induced acute pancreatitis. The correlation of decreased food intake after endotoxin injection with gastric ileus was validated by measuring gastric emptying. The effect of endotoxin on general activity level and feeding behavior was also determined. Small bowel transit was measured using a phenol red marker. Results Each insult resulted in a transient and comparable decrease in food intake and fecal output consistent with the clinical picture of ileus. The endpoints were highly sensitive to small changes in low doses of endotoxin, the extent of bowel manipulation, and cerulein dose. The delay in food intake directly correlated with delayed gastric emptying. Changes in general activity and feeding behavior were insufficient to explain decreased food intake. Intestinal transit remained unchanged at the times measured. Conclusion Food intake and fecal output are sensitive markers of gastric dysfunction in four experimental models of ileus. In the mouse, delayed gastric emptying appears to be the major cause of the anorexic effect associated with ileus. Gastric dysfunction is more important than small bowel dysfunction in this model. Recovery of stomach function appears to be simultaneous to colonic recovery.

Background: Early detection screening of asymptomatic populations for low prevalence cancers requires a highly specific test in order to limit the cost and anxiety produced by falsely positive identifications. Most solid cancers are a heterogeneous collection of diseases as they develop from various combinations of genetic lesions and epigenetic modifications. Therefore, it is unlikely that a single test will discriminate all cases of any particular cancer type. We propose a novel, intuitive biomarker panel design that accommodates disease heterogeneity by allowing for diverse biomarker selection that increases diagnostic accuracy. Methods: Using characteristics of nine pancreatic ductal adenocarcinoma (PDAC) biomarkers measured in human sera, we modeled the behavior of biomarker panels consisting of a sum of indicator variables representing a subset of biomarkers within a larger biomarker data set. We then chose a cutoff for the sum to force specificity to be high and delineated the number of biomarkers required for adequate sensitivity of PDAC in our panel design. Results: The model shows that a panel consisting of 40 non-correlated biomarkers characterized individually by 32% sensitivity at 95% specificity would require any 7 biomarkers to be above their respective thresholds and would result in a panel specificity and sensitivity of 99% each. Conclusions: A highly accurate blood-based diagnostic panel can be developed from a reasonable number of individual serum biomarkers that are relatively weak classifiers when used singly. A panel constructed as described is advantageous in that a high level of specificity can be forced, accomplishing a prerequisite for screening asymptomatic populations for low-prevalence cancers.