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Supplementary Results and Methods - PDF file 255K, Additional information on results and methods

Supplementary Figure 1 - PDF file 147K, Additional evidence that miR-198 is regulated by MSLN in PC cells

Supplementary Figure 3 - PDF file 142K, miR-198 modulation in stable cell lines

Supplementary Figure 4 - PDF file 269K, Additional evidence that miR-198 targets the PBX-1/VCP axis

Supplementary Figure 2 - PDF file 207K, PBX-1 and VCP are predicted targets for miR-198

Supplementary Figure 6 - PDF file 388K, miR-198 modulation reduces the tumorigenic functions of mesothelin-overexpressing pancreatic cancer cells in vitro and in vivo

Purpose: The majority of pancreatic cancers overexpress mesothelin (MSLN), which contributes to enhanced proliferation, invasion, and migration. However, the MSLN regulatory network is still unclear. Here, we investigated the regulation of a panel of tumorigenic factors and explored the potential of MSLN-regulated miR-198 treatment in vivo.Experimental Design: The expression and functional regulation of the tumorigenic factors MSLN, NF-κB, and the homeobox transcription factors (TF) POU2F2 (OCT-2), Pre-B-cell leukemia homeobox factor 1 (PBX-1), valosin-containing protein (VCP), and miR-198 were studied in pancreatic cancer cell lines, patient tumor samples, and xenograft pancreatic cancer mouse models.Results: We found that miR-198 is downregulated in pancreatic cancer and is involved in an intricate reciprocal regulatory loop with MSLN, which represses miR-198 through NF-κB–mediated OCT-2 induction. Furthermore, miR-198 repression leads to overexpression of PBX-1 and VCP. The dysregulated PBX-1/VCP axis leads to increased tumorigenicity. Reconstitution of miR-198 in pancreatic cancer cells results in reduced tumor growth, metastasis, and increased survival through direct targeting MSLN, PBX-1, and VCP. Most interestingly, reduced levels of miR-198 in human tissue samples are associated with upregulation of these tumorigenic factors (MSLN, OCT-2, PBX-1, VCP) and predict poor survival. Reduced miR-198 expression links this tumor network signature and prognosticates poor patient outcome. High miR-198 disrupts the network and predicts better prognosis and increased survival.Conclusions: miR-198 acts as a central tumor suppressor and modulates the molecular makeup of a critical interactome in pancreatic cancer, indicating a potential prognostic marker signature and the therapeutic potential of attacking this tumorigenic network through a central vantage point. Clin Cancer Res; 19(21); 5901–13. ©2013 AACR.

Supplementary Figure 5 - PDF file 212K, Construct details for miR-198 target analysis of MSLN CDS

Supplementary Figure 7 - PDF file 166K, Real-time RT-PCR shows the mRNA levels of all the factors in the proposed network and Linear regression analyses depict the correlations between each factor and its counterparts

SummaryThe ampulla of Vater is a complex cellular environment from which adenocarcinomas arise to form a group of histopathologically heterogenous tumors. To evaluate the molecular features of these tumors, 98 ampullary adenocarcinomas were evaluated and compared to 44 distal bile duct and 18 duodenal adenocarcinomas. Genomic analyses revealed mutations in the WNT signaling pathway among half of the patients and in all three adenocarcinomas irrespective of their origin and histological morphology. These tumors were characterized by a high frequency of inactivating mutations of ELF3, a high rate of microsatellite instability, and common focal deletions and amplifications, suggesting common attributes in the molecular pathogenesis are at play in these tumors. The high frequency of WNT pathway activating mutation, coupled with small-molecule inhibitors of β-catenin in clinical trials, suggests future treatment decisions for these patients may be guided by genomic analysis.

BackgroundAlthough mortality following pancreaticoduodenectomy is decreasing, postoperative morbidity remains high. It was hypothesized that culture-directed treatment of bacteriobilia would decrease the incidence of infectious complications following pancreaticoduodenectomy.MethodsIn a retrospective study of 197 pancreaticoduodenectomy patients, those in the control group (n = 128, 2005–2009) were given perioperative prophylactic antibiotics, whereas those in the treatment group (n = 69, 2009–2011) were continued on antibiotics until intraoperative bile culture results became available. Patients with bacteriobilia received 10 days of antibiotic treatment, which was otherwise discontinued in patients without bacteriobilia. Various complication rates were compared using Fisher's exact test for categorical variables, Wilcoxon rank sum test for ordinal variables, and a two-sample t-test for continuous variables.ResultsDemographics, comorbidities, baseline clinical characteristics, and intraoperative and postoperative variables were similar between the two groups. There were higher incidences of elevated creatinine (19% versus 4%; P = 0.004) and preoperative hyperglycaemia (18% versus 7%; P = 0.053) in the control group. Fewer patients in the control group underwent preoperative biliary stenting (48% versus 67%; P = 0.017) and intraperitoneal drains were placed at the time of resection more frequently in the control group (85% versus 38%; P < 0.001). Bacteriobilia was found in 59% of patients. Treatment of bacteriobilia was associated with a decrease in the rate of postoperative wound infections (12% in the control group versus 3% in the treatment group; P = 0.036) and overall complication severity score (1 in the control group versus 0 in the treatment group; P = 0.027).ConclusionsProlonged antibiotic therapy for bacteriobilia may decrease postoperative wound infection rates after pancreaticoduodenectomy. A randomized prospective trial is warranted to provide evidence to further support this practice.

Deregulated expression of zinc transporters was linked to several cancers. However, the detailed expression profile of all human zinc transporters in normal human organs and in human cancer, especially in pancreatic cancer is not available. The objectives of this study are to investigate the complete expression patterns of 14 ZIP and 10 ZnT transporters in a large number of normal human organs and in human pancreatic cancer tissues and cell lines. We examined the expression patterns of ZIP and ZnT transporters in 22 different human organs and tissues, 11 pairs of clinical human pancreatic cancer specimens and surrounding normal/benign tissues, as well as 10 established human pancreatic cancer cell lines plus normal human pancreatic ductal epithelium (HPDE) cells, using real time RT-PCR and immunohistochemistry. The results indicate that human zinc transporters have tissue specific expression patterns, and may play different roles in different organs or tissues. Almost all the ZIPs except for ZIP4, and most ZnTs were down-regulated in human pancreatic cancer tissues compared to the surrounding benign tissues. The expression patterns of individual ZIPs and ZnTs are similar among different pancreatic cancer lines. Those results and our previous studies suggest that ZIP4 is the only zinc transporter that is significantly up-regulated in human pancreatic cancer and might be the major zinc transporter that plays an important role in pancreatic cancer growth. ZIP4 might serve as a novel molecular target for pancreatic cancer diagnosis and therapy.

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 Experience with the Whipple procedure has been associated with improved outcomes, but the learning curve for this complex procedure is not well defined. Methods Outcomes with 162 consecutive Whipple procedures during the 1st 11.5 years of practice was documented in a prospective database. A period of low (≤11/y) and high (≥23/y) case volume was compared using the Wilcoxon rank-sum test and Fisher exact test. Results With low case volume, blood loss was higher (800 vs 400 mL, P = .001), more patients were transfused (44% vs 18%, P = .027), there were more complications (58% vs 46%, P = .0337), and a longer length of stay (10 vs 7 days, P = .006). There was only 1 mortality (.7%). Conclusions Frequent repetition of the Whipple procedure is associated with an improvement in quantifiable quality benchmarks, and improvement continues with extensive experience. However, with proper training and the right environment, this procedure can be performed during the learning curve with acceptable outcomes.

Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.

BackgroundMost surgeons routinely place a nasogastric tube at the time of a pancreatic resection. The goal of the present study was to evaluate the outcome when a pancreatic resection is performed without routine post-operative nasogastric suction.MethodsOne hundred consecutive patients underwent a pancreatic resection (64a pancreaticoduodenectomy, 98% pylorus sparing and 36a distal pancreatectomy). In the first cohort (50 patients), a nasogastric tube was routinely placed at the time of surgery and in the second cohort (50 patients) the nasogastric was removed in the operating room. Outcomes for these two cohorts were recorded in a prospective database and compared using the χ2 or Fisher's exact test and Wilcoxon's rank-sum test.ResultsDemographical, surgical and pathological details were similar between the two cohorts. A post-operative complication occurred in 22 (44%) in each group (P= 1.000). There were no statistically significant differences in the frequency or severity of complications, or length of stay between groups. The spectrum of complications experienced by the two cohorts was similar including complications that could potentially be related to the use of nasogastric suction such as delayed gastric emptying, anastomotic leak, wound dehiscence and pneumonia. There was no difference between the two groups in the number of patients who required post-operative nasogastric tube placement (or replacement) [2 (4%) vs. 4 (8%), P= 0.678].ConclusionIt may be safe to place a nasogastric tube post-operatively in a minority of patients after a pancreatic resection and spare the majority the discomfort associated with routine post-operative nasogastric suction.

Recent evidence suggests that the quantity of intra-abdominal fat may be a more important predictor of postoperative complications than body mass index (BMI). We hypothesized that increased intra-abdominal fat would be associated with longer operations, increased blood loss, more complications, and prolonged length of stay after pancreatic resection.Retrospective cohort study. Intra-abdominal fat was quantified using CT imaging, and patients were divided into three groups (low, moderate, high). Unconditional multiple logistic regression was used to evaluate the relationship between obesity measures and complications.Between 2002 and 2010, 255 patients underwent pancreaticoduodenectomy or distal pancreatectomy, and 201 had preoperative CT imaging available for review. Operative time was significantly prolonged in patients with high quantities of intra-abdominal fat compared with those with low fat quantity (median 438 versus 354 min, P0.05), while BMI was not associated with changes in duration of surgery. Neither obesity defined by BMI (OR 0.90, 95% CI 0.36-2.21) nor visceral fat (OR 1.20, 95% CI 0.46-3.16) significantly predicted risk of complications. Median length of stay was similar in patients who were obese by BMI (7 versus 7.5 d) or amount of intra-abdominal fat (7 d).Intra-abdominal fat was a better predictor than BMI for determining length of procedure. However, in contrast to previous studies evaluating abdominal surgery, neither BMI nor intra-abdominal fat significantly predicted risk of complication or length of hospital stay. Further research is needed to determine the best measure to assist in risk prediction of obese patients undergoing pancreatic surgery.

BackgroundPost-operative pancreatic fistula (POPF) is a common and potentially devastating complication of pancreas resection. Management of this complication is important to the pancreas surgeon.ObjectiveThe aim of the present study was to evaluate whether drain data accurately predicts clinically significant POPF.MethodsA prospectively maintained database with daily drain amylase concentrations and output volumes from 177 consecutive pancreatic resections was analysed. Drain data, demographic and operative data were correlated with POPF (ISGPF Grade: A – clinically silent, B – clinically evident, C – severe) to determine predictive factors.ResultsTwenty-six (46.4%) out of 56 patients who underwent distal pancreatectomy and 52 (43.0%) out of 121 patients who underwent a Whipple procedure developed a POPF (Grade A-C). POPFs were classified as A (24, 42.9%) and C (2, 3.6%) after distal pancreatectomy whereas they were graded as A (35, 28.9%), B (15, 12.4%) and C (2, 1.7%) after Whipple procedures. Drain data analysis was limited to Whipple procedures because only two patients developed a clinically significant leak after distal pancreatectomy. The daily total drain output did not differ between patients with a clinical leak (Grades B/C) and patients without a clinical leak (no leak and Grade A) on post-operative day (POD) 1 to 7. Although the median amylase concentration was significantly higher in patients with a clinical leak on POD 1–6, there was no day that amylase concentration predicted a clinical leak better than simply classifying all patients as ‘no leak’ (maximum accuracy = 86.1% on POD 1, expected accuracy by chance = 85.6%, kappa = 10.2%).ConclusionDrain amylase data in the early post-operative period are not a sensitive or specific predictor of which patients will develop clinically significant POPF after pancreas resection.

Background RECQL is a DNA helicase involved in DNA mismatch repair. The RECQL polymorphism, 3′ untranslated region (UTR) A159C, was previously associated with overall survival of patients with resectable pancreatic adenocarcinoma treated with neoadjuvant chemoradiation. In the present study, we examined RECQL for somatic mutations and other polymorphisms and compared these findings with the outcome in patients who received adjuvant or neoadjuvant chemoradiation. We hypothesized that RECQL (i) would be mutated in cancer, (ii) would have polymorphisms linked to the 3′UTR A159C and that either or both events would affect function. We also hypothesized that (iii) these changes would be associated with survival in both cohorts of patients. Material and methods We sequenced RECQL ' s 15 exons and surrounding sequences in paired blood and tumour DNA of 39 patients. The 3′UTR A159C genotype was determined in blood DNA samples from 176 patients with resectable pancreatic adenocarcinoma treated with adjuvant (53) or neoadjuvant (123) chemoradiation. Survival was calculated using the Kaplan–Meier method, with log rank comparisons between groups. The relative impact of genotype on time to overall survival was performed using the Cox proportional hazards model. Results Somatic mutations were found in UTRs and intronic regions but not in exonic coding regions of the RECQL gene. Two single nucleotide polymorphisms (SNPs), located in introns 2 and 11, were found to be part of the same haplotype block as the RECQL A159C SNP and showed a similar association with overall survival. No short-term difference in survival between treatment strategies was found. We identified a subgroup of patients responsive to neoadjuvant therapy in which the 159 A allele conferred strikingly improved long-term survival. Discussion The RECQL 3′UTR A159C SNP is not linked with other functional SNPs within RECQL but may function as a site for regulatory molecules. The mechanism of action needs to be clarified further.

With recent advances in DNA sequencing technology, medicine is entering an era in which a personalized genomic approach to diagnosis and treatment of disease is feasible. However, discovering the role of altered DNA sequences in various disease states will be a challenging task. The genomic approach offers great promise for diseases, such as pancreatic cancer, in which the effect of current diagnostic and treatment modalities is disappointing. To facilitate the characterization of the genome of pancreatic cancer, high-quality and well-annotated tissue repositories are needed. This article summarizes the basic principles that guide the creation of such a repository, including sample processing and preservation techniques, sample size and composition, and collection of clinical data elements.

Background. Cystic lesions of the pancreas are being identified more frequently. Deciding which asymptomatic lesions can be safely followed with serial imaging and which require resection due to malignant potential is an increasingly common question. Current clinical practice is to rely on characteristics of the lesions on CT scan, and additional information from endoscopic ultrasound with fine-needle aspiration (EUS-FNA) and cyst fluid analysis or endoscopic retrograde pancreatography (ERCP) to assess malignant potential. Hypothesis. The malignant potential of pancreatic cystic lesions cannot be accurately predicted by CT scan. Methods. CT scans from 48 patients with cystic lesions of the pancreas were stripped of patient identifiers and retrospectively presented to two expert radiologists. The radiologists recorded specific characteristics of the lesions thought to be important in the differential diagnosis and their opinion of the likely diagnosis. Diagnostic accuracy was assessed by comparing the radiologists’ diagnoses to the final pathologic diagnosis after resection. To determine if clinical history, EUS-FNA or ERCP findings improved diagnostic accuracy, medical records were retrospectively reviewed and scored as either supporting or not supporting malignant potential of the lesion. Results. Specific diagnoses based on CT findings alone were correct in an average of 39% of the cases. Even when diagnoses were dichotomized as benign (43%) or potentially malignant (57%, papillary mucinous neoplasms, mucinous cystic neoplasms, cancer), determinations based on CT alone were accurate in an average of 61% of cases. Accuracy rates were 60.4 and 62.5% for the two radiologists, although there was only fair agreement between them (Kappa=0.28, 95% CI=(0.01–0.55), p=0.05). When all clinical information available was considered together as a single dichotomous indicator of malignant potential, the indicator was accurate in 90% of the cases (Kappa=0.73, 95% CI=(0.51–0.95, p

Background Accurate preoperative staging is essential in pancreatic cancer to select the 15% of patients who can benefit from surgery and avoid surgery in the 85% with advanced disease. With improvements in computed tomography (CT) scanning, the value of routine laparoscopy for preoperative staging of pancreatic cancer has been questioned because it changes the preoperative plan in less than 20% of unselected cases. Methods We retrospectively reviewed our experience with preoperative staging in 88 consecutive patients with pancreatic cancer. All patients had preoperative CT scans, and selective criteria were used to determine which patients would also undergo preoperative staging laparoscopy. Patients were categorized preoperatively as resectable or not resectable (locally advanced or metastatic). Medical records, operative, and pathology reports were reviewed to determine the accuracy of preoperative predictions. Results Thirty patients were deemed resectable based on CT alone and 27 (90%) were resected (25 R0, 2 R1). Two (7%) had metastatic disease discovered at laparotomy and one (3%) had a R2 resection. Only 19 patients (39%) of 49 patients deemed resectable by CT met our selective criteria for preoperative staging laparoscopy. Laparoscopy changed the treatment plan in 11 (58%) of these patients. Eight were still deemed resectable after staging laparoscopy and 7 (88%) were resected (6 R0, 1 R1). One patient (12%) had metastatic disease diagnosed at laparotomy. If selective staging laparoscopy were eliminated from our algorithm, 49 patients would have been deemed potentially resectable based on CT alone, 34 (69%) would have been found to be resectable at laparotomy (31 R0, 3 R1), and 15 (31%) would have been found to be unresectable at laparotomy (positive predictive value of 69%). The addition of selective staging laparoscopy avoided unnecessary laparotomy in 11 patients and increased the positive predictive value to (34/38) 89%. Conclusion Selective use of laparoscopy increases the positive predictive value of preoperative staging in pancreatic cancer and avoids unnecessary laparoscopy in the majority of patients.

Fatty Acid Synthase (FASN), a key enzyme of de novo lipogenesis, is upregulated in many cancers including colorectal cancer (CRC); increased FASN expression is associated with poor prognosis. Potent FASN inhibitors developed by 3-V Biosciences demonstrate anti-tumor activity in vitro and in vivo and a favorable tolerability profile in a Phase I clinical trial in solid tumor patients. However, CRC characteristics associated with responsiveness to FASN inhibition are not fully understood. The purpose of our study was: (i) to determine the effect of FASN inhibition on tumor growth in CRC patient-derived xenografts (PDXs); (ii) to identify potential biomarkers associated with CRC responsiveness to FASN inhibition; and (iii) to explore new combination strategies with FASN inhibitors. METHODS. Tumor growth was assessed in 9 PDXs established in NSG mice using freshly resected specimens. Once the xenografts grew to ~100 mm3, mice were randomized into two groups (n=5) to receive either vehicle or TVB-3664 or four groups (n=10) for TVB-3664 treatment in combination with either MK2206 or Chloroquine (CQ). Tumor volume and animal weights were measured weekly. Western blot analysis and immunohistochemistry staining were used to identify FASN-mediated changes in signaling pathways. Changes in metabolites and lipids were analyzed by nuclear magnetic resonance and mass spectrometry in plasma and tumor tissues. Next Generation Sequencing was used to assess the mutation profile of 198 oncogenes in patient tumors and PDXs. RESULTS. PDXs showed a wide range of sensitivity to FASN inhibition: TVB-3664 treatment attained significant response (reduced tumor volume) in 3 PDXs, significant response followed by developed resistance in one PDX, and no response in 5 PDXs. Activation of Akt and AMPK pathways was associated with resistance to FASN inhibition and combination of TVB-3664 with either MK2206 or CQ led to a significant reduction in tumor volume as compared to either drug alone. Moreover, TVB-3664 treatment significantly decreased the total palmitate level in plasma and the levels of triglycerides, diglycerides, phosphatidylserines, phosphatidylethanolamines, and phosphatidylcholines in tumor tissues. Furthermore, a significant decrease in the levels of AXP-1, AXP-2 and myo-Inositol-2 was observed in tumors responsive to FASN inhibition. CONCLUSIONS. Our studies demonstrate that TVB-3664 shows anti-tumor activity in CRC. Importantly, our results suggest that activation of Akt and autophagy are major mechanisms of resistance to FASN inhibition and demonstrate that combine inhibition of these pathways and FASN may be a new therapeutic approach in CRC. Ongoing studies of correlation between mutation and metabolic profiles of tumors and tumor response to FASN inhibition aim to identify a subset of CRC patients that are likely to respond to FASN-targeted therapy. Citation Format: Yekaterina Y. Zaytseva, Piotr G. Rychahou, Anh-Thu Le, Robert M. Flight, Timothy L. Scott, Jennifer W. Harris, Sally Hodges, Brent J. Hallahan, Dana L. Napier, Jinpeng Liu, Chi Wang, Manjula Sunkara, Andrew Morris, Ji Tae Kim, Sivakumaran Theru Arumugam, Andrew Lane, Teresa W. Fan, Hunter Moseley, Tianyan Gao, Eun Y. Lee, Heidi L. Weiss, Timothy S. Heuer, George Kemble, B Mark Evers. Activation of Akt pathway and autophagy promotes resistance to FASN inhibition in colorectal cancer patient-derived xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 452. doi:10.1158/1538-7445.AM2017-452

Pancreatic ductal adenocarcinoma (PDAC) is difficult to detect early and is often resistant to standard chemotherapeutic options, contributing to extremely poor disease outcomes. Members of the nuclear receptor superfamily carry out essential biological functions such as hormone signaling and are successfully targeted in the treatment of endocrine-related malignancies. Liver X receptors (LXRs) are nuclear receptors that regulate cholesterol homeostasis, lipid metabolism, and inflammation, and LXR agonists have been developed to regulate LXR function in these processes. Intriguingly, these compounds also exhibit antiproliferative activity in diverse types of cancer cells. In this study, LXR agonist treatments disrupted proliferation, cell-cycle progression, and colony-formation of PDAC cells. At the molecular level, treatments downregulated expression of proteins involved in cell cycle progression and growth factor signaling. Microarray experiments further revealed changes in expression profiles of multiple gene networks involved in biological processes and pathways essential for cell growth and proliferation following LXR activation. These results establish the antiproliferative effects of LXR agonists and potential mechanisms of action in PDAC cells and provide evidence for their potential application in the prevention and treatment of PDAC.

Objective To test by randomized prospective multicenter trial the hypothesis that pancreaticoduodenectomy (PD) without the use of intraperitoneal drainage does not increase the frequency or severity of complications. Background Some surgeons have abandoned the use of drains placed during pancreas resection. Methods We randomized 137 patients to PD with (n = 68, drain group) and without (n = 69, no-drain group) the use of intraperitoneal drainage and compared the safety of this approach and spectrum of complications between the 2 groups. Results There were no differences between drain and no-drain cohorts in demographics, comorbidities, pathology, pancreatic duct size, pancreas texture, baseline quality of life, or operative technique. PD without intraperitoneal drainage was associated with an increase in the number of complications per patient [1 (0-2) vs 2 (1-4), P = 0.029]; an increase in the number of patients who had at least 1 ≥grade 2 complication [35 (52%) vs 47 (68%), P = 0.047]; and a higher average complication severity [2 (0-2) vs 2 (1-3), P = 0.027]. PD without intraperitoneal drainage was associated with a higher incidence of gastroparesis, intra-abdominal fluid collection, intra-abdominal abscess (10% vs 25%, P = 0.027), severe (≥grade 2) diarrhea, need for a postoperative percutaneous drain, and a prolonged length of stay. The Data Safety Monitoring Board stopped the study early because of an increase in mortality from 3% to 12% in the patients undergoing PD without intraperitoneal drainage. Conclusions This study provides level 1 data, suggesting that elimination of intraperitoneal drainage in all cases of PD increases the frequency and severity of complications.

Purpose: The majority of pancreatic cancers overexpress mesothelin (MSLN), which contributes to enhanced proliferation, invasion, and migration. However, the MSLN regulatory network is still unclear. Here, we investigated the regulation of a panel of tumorigenic factors and explored the potential of MSLN-regulated miR-198 treatment in vivo. Experimental Design: The expression and functional regulation of the tumorigenic factors MSLN, NF-κB, and the homeobox transcription factors (TF) POU2F2 (OCT-2), Pre-B-cell leukemia homeobox factor 1 (PBX-1), valosin-containing protein (VCP), and miR-198 were studied in pancreatic cancer cell lines, patient tumor samples, and xenograft pancreatic cancer mouse models. Results: We found that miR-198 is downregulated in pancreatic cancer and is involved in an intricate reciprocal regulatory loop with MSLN, which represses miR-198 through NF-κB–mediated OCT-2 induction. Furthermore, miR-198 repression leads to overexpression of PBX-1 and VCP. The dysregulated PBX-1/VCP axis leads to increased tumorigenicity. Reconstitution of miR-198 in pancreatic cancer cells results in reduced tumor growth, metastasis, and increased survival through direct targeting MSLN, PBX-1, and VCP. Most interestingly, reduced levels of miR-198 in human tissue samples are associated with upregulation of these tumorigenic factors (MSLN, OCT-2, PBX-1, VCP) and predict poor survival. Reduced miR-198 expression links this tumor network signature and prognosticates poor patient outcome. High miR-198 disrupts the network and predicts better prognosis and increased survival. Conclusions: miR-198 acts as a central tumor suppressor and modulates the molecular makeup of a critical interactome in pancreatic cancer, indicating a potential prognostic marker signature and the therapeutic potential of attacking this tumorigenic network through a central vantage point. Clin Cancer Res; 19(21); 5901–13. ©2013 AACR.

Changes in the intracellular levels of the essential micronutrient zinc have been implicated in multiple diseases including pancreatic cancer; however, the molecular mechanism is poorly understood. Here, we report a novel mechanism where increased zinc mediated by the zinc importer ZIP4 transcriptionally induces miR-373 in pancreatic cancer to promote tumour growth. Reporter, expression and chromatin immunoprecipitation assays demonstrate that ZIP4 activates the zinc-dependent transcription factor CREB and requires this transcription factor to increase miR-373 expression through the regulation of its promoter. miR-373 induction is necessary for efficient ZIP4-dependent enhancement of cell proliferation, invasion, and tumour growth. Further analysis of miR-373 in vivo oncogenic function reveals that it is mediated through its negative regulation of TP53INP1, LATS2 and CD44. These results define a novel ZIP4-CREB-miR-373 signalling axis promoting pancreatic cancer growth, providing mechanistic insights explaining in part how a zinc transporter functions in cancer cells and may have broader implications as inappropriate regulation of intracellular zinc levels plays an important role in many other diseases.

Perioperative transfusion of packed red blood cells (PRBC) has been associated with negative side effects. We hypothesized that a majority of transfusions in our series of patients who underwent pancreaticoduodenectomy (PD) were unnecessary. A retrospective analysis was performed to determine whether transfusions were indicated based on pre-determined criteria, and the impact of perioperative transfusions on postoperative outcomes was assessed.Our prospectively maintained database was queried for patients who underwent PD between 2004 and 2011. 200 patients were divided into Cohort 1 (no transfusion) and Cohort 2 (transfusion). Rates of various graded 90-day postoperative complications were compared. Categorical values were compared according to the Common Terminology Criteria for Adverse Events. All cases involving intraoperative blood transfusion were reviewed for associated blood loss, intraoperative vital signs, urine output, hemoglobin values, and presence or absence of EKG changes to determine whether the transfusion was indicated based on these criteria.There were 164 patients (82%) in Cohort 1 (no transfusion) and 36 patients (18%) in Cohort 2 (transfused). Both groups had similar demographics. Patients in Cohort 2 had lesser median preoperative values of hemoglobin (12.3 vs 13.1, P = .002), a greater incidence of vein resection (33% vs. 16%, P = .021), longer operative times (518 vs 440 minutes, P.0001), a greater estimated blood loss (850 vs. 300 mL, P.001), and greater intraoperative fluid resuscitation (6,550 vs. 5,300 mL, P = .002). Ninety-day mortality was similar between the 2 groups (3% vs 1%, P = .328). Patients in Cohort 2 (transfused) had increased rates of delayed gastric emptying (36% vs. 20%, P = .031), wound infection (28% vs. 7%, P = .031), pulmonary complications (6% vs. 0%, P = .032), and urinary retention (6% vs. 0%, P = .032). A greater incidence of any complication of grade II severity (67% vs. 35%, P = .0005) or grade III severity (36% vs. 17%, P = .010) was also noted in Cohort 2. Of the 33 intraoperative transfusions, 15 (46%) did not meet any of the predetermined criteria: intraoperative hypotension (90/60 mmHg), tachycardia (110 beats per minute), low urine output (10 mL/hour), decreased oxygen saturation (95%), excessive blood loss (1,000 mL), EKG changes, and low hemoglobin (7.0 g/dL).Perioperative transfusions among patients with PD were associated with increased rates of various postoperative complications. A substantive portion (∼46%) of perioperative transfusions in this patient population did not meet predetermined criteria, indicating a potential opportunity for improved blood product use. Further prospective studies are required to determine whether the implementation of these criteria may a positive impact on perioperative outcomes.

Ampullary adenocarcinomas are located and expand out of the ampulla of Vater. Their exact cellular origin is often hard to determine due to the diverse cellular constitution of the ampulla itself. The ampulla is the site where the bile and the pancreatic ducts unite and then open by a common orifice into the duodenum creating a diversified environment from which the adenocarcinoma can arise. We hypothesize that the cellular origin of ampullary adenocarcinomas might be elucidated with genetic profiling. DNA was isolated from 6 ampullary adenocarcinomas classified as periampullary by the pathologist and from the matched patient blood. Coding sequences of the tumor and normal matched samples were enriched by capture on a Nimblegen system and sequenced on a SOLiD platform. All mutations identified were validated on a 454 platform. Sixteen ductal pancreatic adenocarcinomas were studied in parallel and their genetic profile was compared with the ampullary adenocarcinoma profile. We identified a total of 492 and 458 somatic events in the ductal and ampullary tumor groups, respectively. Whereas the mutation distribution profile was similar in the 2 tumor types, their mutation spectrum was different with the T-to-G transitions being up to 6 fold more common in the ampullary than in the ductal adenocarcinomas suggesting that the development of each of these tumors was initiated by different mechanisms. The ampullary and the ductal adenocarcinoma shared several mutated genes and pathways defined by point mutations such as KRAS signaling, apoptosis, DNA repair, MAPK Wnt signaling, focal adhesion, and calcium signaling pathways and the axon guidance gene group. However, 4 of the periampullary adenocarcinoma had a distinct mutation landscape with mutations found in the significant mutated TCGA gene list of colon cancer and/or in the Catalog of Somatic Mutations in Cancer (COSMIC) for gastric cancer. In conclusion, distinct mutation spectrum and mutation profile can be identified in ampullary tumors that might help in the determination of their cellular origin. Citation Format: Marie-Claude Gingras, David A. Wheeler, Donna M. Muzny, Sally E. Hodges, William E. Fisher, Richard A. Gibbs. Can mutation profile be of help to define the cellular origin of ampullary adenocarcinoma? [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 A14.

ObjectiveConventional chemotherapy and radiotherapy produce marginal survival benefits in pancreatic cancer, underscoring the need for novel therapies. The aim of this study is to develop an adoptive Tcell transfer approach to target tumours expressing prostate stem cell antigen (PSCA), a tumour-associated antigen that is frequently expressed by pancreatic cancer cells.MethodsExpression of PSCA on cell lines and primary tumour samples was confirmed by immunohistochemistry. Healthy donor- and patient-derived Tcells were isolated, activated in vitro using CD3/CD28, and transduced with a retroviral vector encoding a chimeric antigen receptor (CAR) targeting PSCA. The ability of these cells to kill tumour cells was analysed by chromium-51 (Cr51) release.ResultsProstate stem cell antigen was expressed on >70% of the primary tumour samples screened. Activated, CAR-modified Tcells could be readily generated in clinically relevant numbers and were specifically able to kill PSCA-expressing pancreatic cancer cell lines with no non-specific killing of PSCA-negative target cells, thus indicating the potential efficacy and safety of this approach.ConclusionsProstate stem cell antigen is frequently expressed on pancreatic cancer cells and can be targeted for immune-mediated destruction using CAR-modified, adoptively transferred Tcells. The safety and efficacy of this approach indicate that it deserves further study and may represent a promising novel treatment for patients with pancreatic cancer.

Body mass index (BMI) has proven unreliable in predicting survival following pancreaticoduodenectomy for cancer. While measures of intra-abdominal fat correlate with medical and postoperative complications of obesity, the impact of intra-abdominal fat on pancreatic cancer survival is uncertain. We hypothesized that the quantity of intra-abdominal fat would predict survival following resection of pancreatic cancer. Preoperative CT imaging was used to measure intra-abdominal fat. Cox regression analyses were used to identify independent predictors of survival. Sixty-one patients from 2000–2009 underwent pancreaticoduodenectomy for exocrine pancreatic adenocarcinoma. After adjusting for age and perineural invasion status, preoperative BMI did not predict overall survival (p

Despite numerous modifications of surgical technique, pancreatic fistula remains a serious problem and occurs in about 10% of patients following pancreas resection. BioGlue is a new sealant that creates a flexible mechanical seal within minutes independent of the body’s clotting mechanism. Application of BioGlue sealant will reduce the incidence of pancreatic fistula following pancreas resection. A retrospective cohort study was performed with 64 patients undergoing pancreas resection. BioGlue sealant was applied to the pancreatic anastomosis (Whipple) or resection margin (distal pancreatectomy) in 32 cases. Factors that could affect the rate of postoperative pancreatic fistula were recorded. Pancreatic fistula was defined as greater than 50 ml of drain output with an amylase content greater than three times normal serum value after postoperative day 10. To improve the sensitivity of our study, we also examined pancreatic fistula with a strict definition of any drain output on or after postoperative day 3 with a high amylase content and graded the fistulas in terms of clinical severity. Grade A leaks were defined as subclinical. Grade B leaks required some response such as making the patient nil per os, parenteral nutrition, octreotide, antibiotics, or a prolonged hospital stay. Grade C leaks were defined as serious and life threatening. They were associated with hemorrhage, sepsis, resulted in deterioration of other organ systems, and mandated intensive care. Comparisons between the two groups were made using the chi-square test or Fisher’s exact test for categorical variables and by the Wilcoxon rank-sum test for continuous variables. P values of 0.05 or less were deemed statistically significant. There were no differences between the patients who received BioGlue and the control cohort in terms of comorbid conditions, tumor location, texture of the pancreas, size of the pancreatic duct, or surgical technique. By the common definition, pancreatic fistula occurred in 6% (control) vs. 22% (BioGlue). By the strict definition, a fistula occurred in 41% (control) vs. 60% (BioGlue). In the control group, ten were subclinical (grade A) and two were clinically apparent leaks (grade B). In the BioGlue group, seven were subclinical (grade A), five were clinically apparent (grade B), and three were severe (grade C). There were no statistically significant differences in the incidence or severity grades of postoperative pancreatic fistulas between the two groups. Application of BioGlue sealant probably does not reduce the incidence of pancreatic fistula following pancreas resection.

The onset and progression of cancer is driven by extensive rearrangement and mutation of the genome. The ever-expanding sequencing capacity ushered in by the second generation of DNA sequencers has enabled complete base-level description of the cancer genome and is therefore deepening our understanding of the etiology of the disease. We have combined a variety of approaches centered on next generation sequencing technologies aimed at comprehensive characterization of the cancer genome to define the key molecular lesions that drive tumor initiation and progression. These include targeted exonic sequencing to define coding sequence mutation, whole genome shotgun sequencing to define structural rearrangements, and cDNA sequencing to define alteration in expression levels, aberrant splicing and fusion transcripts. We have applied these approaches to pancreatic adenocarcinoma, hepatocellualr carcinoma and colorectal carcinoma. Results of genome sequencing have yielded an unprecedented picture of the rearrangements and mutations from analysis of read sequence and read coverage across the chromosomes. Evident in both the cancers is a dramatic reduction in SNP variation in the tumor genome compared to the germline from the same individual owing to extensive loss of heterozygosity. Superimposed on this are a few thousand mutations, some of which drive the tumor phenotype. The corresponding set of analog data generated by SNP-array microarray platforms provide validation of the central results from sequencing which leads to a new appreciation of the relationship between genetic change and the malignant phenotype. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2218.