Supplementary Figures 1-3. S1: Creation and characterization of EcSOD stably expressing PDA cell lines.; S2: Activity of EcSOD activity from stably expression PDA cell lines; S3: L-NNA decreases NOS activity in PDA cells.

Supplementary Table 1S from Progression of Barrett's Metaplasia to Adenocarcinoma Is Associated with the Suppression of the Transcriptional Programs of Epidermal Differentiation

Supplementary Table 1S from Progression of Barrett's Metaplasia to Adenocarcinoma Is Associated with the Suppression of the Transcriptional Programs of Epidermal Differentiation

Supplementary Table 2S from Progression of Barrett's Metaplasia to Adenocarcinoma Is Associated with the Suppression of the Transcriptional Programs of Epidermal Differentiation

Background Nicotinamide phosphoribosyltransferase (NAMPT) plays a key role in the biosynthesis of nicotinamide adenine dinucleotide (NAD(+)), which is a vital cofactor in redox reactions and a substrate for NAD(+) consuming enzymes including CD38, PARPs and sirtuins. NAMPT over-expression has been shown in various cancers and its inhibition decreases cancer cell growth, making it an attractive therapeutic target. Here we examine the NAMPT expression in a large cohort of resected stage I/II pancreatic ductal adenocarcinomas (PDAs) and correlate its expression with clinical outcomes and pathologic features. Methods A retrospective review of patients with PDAs was conducted at a single institution. Tissue microarrays (TMAs) containing primary PDAs and their metastatic lymph nodes (mLNs) were constructed and stained for NAMPT expression. Each TMA core was evaluated for staining intensity of cancer cells (0 = no staining, 1+ = weak, 2+ = moderate, 3+ = strong) and a mean score was calculated for each case with at least two evaluable cores. NAMPT expression was correlated with clinicopathological variables using chi-squared or Fisher's exact test, and t-tests for categorical and continuous variables, respectively. Survival probabilities were estimated and plotted using the Kaplan-Meier method. Cox proportional hazards regression was used to assess the effects of NAMPT staining values on recurrence-free survival (RFS) and overall survival (OS). This study was conducted under an approved IRB protocol. Results 173 primary PDAs had at least 2 TMA cores with identifiable cancer cells. The mean IHC score was 0.55 (range: 0 to 2.33). The mean IHC score of mLNs was 0.39 (range: 0-2), which was not significantly different from their primary tumors (mean IHC score = 0.47, P = 0.38). Sixty-four percent (111/173) of PDAs were positive for NAMPT staining. Stage II tumors were more likely to be positive (68% of 151 vs 41% of 22; P = 0.01). Non-obese non-diabetic patients were more likely to have NAMPT+ tumors (43.7% vs. 27.9%, P = 0.04). While RFS and OS were not statistically different between NAMPT+ vs. NAMPT-PDAs, patients with NAMPT-tumors tended to have a longer median OS (26.0 vs. 20.4 months, P = 0.34). Conclusion NAMPT expression was detected in 64% of stage I/II PDAs and up to 72% in non-obese non-diabetic patients. Frequency of NAMPT expression correlated with pathological stage, consistent with published literature regarding its role in cancer progression. While RFS and OS were not statistically significantly different, patients with NAMPT+ PDAs tended to have a shorter survival. Thus, NAMPT inhibition may prove beneficial in clinical trials.

Gastric adenocarcinoma most often presents at an advanced stage and overall five-year survival of ∼30%. Pharmacological ascorbate (high-dose IV ascorbate) has been proposed as a promising nontoxic adjuvant to standard radio-chemotherapies in several cancer types. In the current study, pharmacological ascorbate (0.5–2 mM) caused a dose-dependent decrease (70–85% at 2 mM) in clonogenic survival of gastric adenocarcinoma cells (AGS and MNK-45), but was relatively nontoxic to a small intestinal epithelial nonimmortalized human cell isolate (FHs 74 Int). The addition of pharmacological ascorbate (1 mM) to standard radio-chemotherapies [i.e., 5-FU (5 μM); cisplatin (0.5 μM); irinotecan (2.5 μM); carboplatin (5 μM); paclitaxel (2–4 nM); and X rays (1.8 Gy)] also potentiated gastric cancer clonogenic cell killing [additional decreases were noted with: ascorbate plus 5-FU/radiation (1%); ascorbate plus cisplatin/irinotecan (9–19%); and ascorbate plus paclitaxel/carboplatin (6–7%)]. The gastric cancer cell toxicity and chemosensitization seen with pharmacological ascorbate was dependent on H2O2 and the presence of catalytic metal ions. In addition, pharmacological ascorbate dosing resulted in a concentration-dependent decrease (64% at 20 mM, P ≤ 0.0001) in cancer cell invasion and migration that was inhibited by catalase. Finally, pharmacological ascorbate significantly increased the overall survival of mice with gastric cancer xenografts when used in combination with paclitaxel, carboplatin and radiation (P = 0.019). These results demonstrate that pharmacological ascorbate is selectively cytotoxic to gastric adenocarcinoma cells (relative to normal intestinal epithelial cells) by a mechanism involving H2O2 and redox active metal ions. Furthermore, pharmacological ascorbate significantly enhances gastric cancer xenograft responses to radio-chemotherapy as well as inhibiting tumor cell migration and invasiveness. Overall, these results support the hypothesis that pharmacological ascorbate can be used as an adjuvant with standard-of-care radio-chemotherapies for the treatment of gastric adenocarcinomas.

and a member of our Editorial Board, passed away on February 3, 2016, after a nearly 2-year battle with gastric cancer.Dr

Background Pancreatic ductal adenocarcinoma is an aggressive disease in which accurate staging is critical. Positron emission tomography has shown promise as a method of detecting metastatic disease in many cancers, but data supporting its use in pancreatic ductal adenocarcinoma is controversial. This study evaluated the impact of positron emission tomography on treatment in patients with pancreatic ductal adenocarcinoma. Methods A retrospective chart review identified patients with pancreatic ductal adenocarcinoma diagnosed between 2004-2012 who received positron emission tomography imaging as part of their disease assessment. The impact of positron emission tomography on therapy decisions was determined. Results Of the 62 patients evaluated, 7 (11.3%) had imaging prior to adjuvant therapy, 34 (54.8%) prior to neoadjuvant therapy, and 21 (33.9%) as part of initial staging. The median overall survival was 10.3 months (range: 1–31.6) and 14 patients (22.6%) underwent pancreatectomy. Positron emission tomography changed the treatment pathway in 6 patients (9.7%) including: 2/34 being staged prior to neoadjuvant therapy (5.9%) and 4/21 (19.0%) being evaluated with positron emission tomography as part of initial staging. There were 2 patients who had false positive findings resulting in unnecessary invasive testing. Conclusions In this study, Positron emission tomography imaging changed the treatment pathway in approximately 10% of patients with pancreatic ductal adenocarcinoma, primarily among patients with high risk clinical disease. The data suggests positron emission tomography imaging should be used selectively in patients with pancreatic ductal adenocarcinoma who have clinically advanced disease, where identification of distant disease would alter the patient’s treatment course.

Houwen, Frederick K. MD; O’Leary, Brianne R. PhD; Allen, Bryan G. MD, PhD; Keene, Jeffery L. PhD; Beardsley, Robert A. PhD; Spitz, Douglas R. PhD; Mezhir, James J. MD, FACS Author Information

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide (O2•-), which may contribute to the aggressive nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of O2•- to hydrogen peroxide (H2O2) in the extracellular environment. By limiting O2•- concentration and producing H2O2, EcSOD is thought to regulate the redox state of the tumor microenvironment that could contribute to pancreatic cancer progression. The current work tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the balance between H2O2 and ONOO- formation. Methods: EcSOD and 3-nitrotyrosine (3-NT), a marker of protein nitration by ONOO-, were evaluated in specimens from patients with PDA. EcSOD expression was correlated with clinicopathologic and treatment-related variables and survival in a tissue microarray (TMA) of 114 patients with Stage I-IV PDA. EcSOD was constitutively overexpressed in two primary PDA cell lines and clonogenic survival and doubling time were measured. A Matrigel invasion assay was used to determine the impact of EcSOD on PDA tumor cell invasive capacity. We also utilized and GC4419, a selective SOD mimic currently in clinical development. GC4419 dismutates O2•- but does not react with H2O2 under physiologic conditions. PDA cells stably expressing EcSOD and luciferase were used to create hind leg xenografts in athymic nude mice to assess the impact of EcSOD on tumor growth and then via peritoneal injection to determine the impact on peritoneal metastasis formation using bioluminescence imaging (BLI). Results: EcSOD expression was reduced in PDA compared to normal pancreas in 11/16 specimens evaluated (69%). A decrease in expression was also seen in areas of premalignant ductal epithelium (PanIN-3) relative to adjacent pancreas. In normal pancreas tissue, there was no evidence of immunoreactive 3-NT; however, 11/16 PDA specimens (69%) had robust levels of immunoreactive 3-NT. In the TMA, EcSOD expression was absent in 41/114 of the PDA biopsies (36%). The median survival of patients with intact EcSOD expression was 11.0 months vs. 6.5 months for patients with loss of EcSOD expression. Compared to patients with intact EcSOD, patients with loss of EcSOD more often had advanced stage disease and were less likely to be treated with surgery. In an adjusted proportional hazard model of more than 10 clinical variables, the HR for death with loss of EcSOD was 1.63 (95% CI=1.02-2.58, p=0.04). EcSOD overexpression significantly reduced clonogenic survival, doubling time, and tumor cell invasion relative to controls. GC4419 also significantly reduced PDA invasiveness in the Matrigel assay. PDA cells stably expressing EcSOD showed decreased growth and doubling time in hind limb xenografts and significantly less peritoneal metastasis using BLI. Conclusions: Loss of EcSOD expression and oxidative stress as indicated by the presence of 3-NT is a common finding in PDA. Loss of EcSOD expression correlates with negative prognostic factors and shorter survival in patients with PDA. Overexpression of EcSOD resulted in reduced PDA growth, invasion, and peritoneal metastasis. The selective SOD mimic GC4419 also reduced invasion of PDA tumor cells. Together these findings support the hypothesis that modulating O2•- in the PDA microenvironment alters parameters of progression, oxidative damage, and may represent a target for limiting the spread of PDA. (Supported by The American Surgical Association Foundation Fellowship (JJM) and the American Cancer Society (JJM). Citation Format: James J. Mezhir, Brianne R. O'Leary, Andrew M. Bellizzi, Sean Altekruse, Charles F. Lynch, Brenda Y. Hernandez, Wendy Cozen, Michael D. Henry, Jeffrey Keene, Robert A. Beardsley, Douglas R. Spitz, Frederick E. Domann. The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B52.

BACKGROUND AND OBJECTIVE:Pancreatic resection is the standard treatment option for patients with stage I/II pancreatic ductal adenocarcinoma (PDA), yet many studies demonstrate low rates of resection. The objective of this study was to evaluate whether increasing resection rates would result in an increase in average survival in patients with stage I/II PDA.METHODS:SEER (Surveillance, Epidemiology, and End Results) data were analyzed for patients with stage I/II pancreatic head cancers treated from 2004 to 2009. Pancreatectomy rates were examined within Health Service Areas (HSAs) across 18 SEER regions. An instrumental variable analysis was performed, using HSA rates as an instrument, to determine the impact of increasing resection rates on survival.RESULTS:Pancreatectomy was performed in 4322 of 8323 patients evaluated with stage I/II PDA (overall resection rate = 51.9%). The resection rate across HSAs ranged from an average of 38.6% (lowest quintile) to 67.3% (highest quintile). Median survival was improved in HSAs with higher resection rates. Instrumental variable analysis revealed that, for patients whose treatment choices were influenced by rates of resection in their geographic region, pancreatectomy was associated with a statistically significant increase in overall survival.CONCLUSIONS:When controlling for confounders using instrumental variable analysis, pancreatectomy is associated with a statistically significant increase in survival for patients with resectable PDA. On the basis of these results, if resection rates were to increase in select patients, then average survival would also be expected to increase. It is important that this information be provided to physicians and patients so that they can properly weigh the risks and advantages of pancreatectomy as treatment of PDA.

Transforming growth factor β-activated kinase 1 (TAK1) is critical for survival of many KRAS mutated colorectal cancer cells, and TAK1 inhibition with 5Z-7-oxozeaenol has been associated with oxidative stress leading to tumor cell killing. When SW 620 and HCT 116 human colon cancer cells were treated with 5 µM 5Z-7-oxozeaenol, cell viability, growth, and clonogenic survival were significantly decreased. Consistent with TAK1 inhibition being causally related to thiol-mediated oxidative stress, 10 mM N-acetylcysteine (NAC) partially reversed the growth inhibitory effects of 5Z-7-oxozeaenol. In addition, 5Z-7-oxozeaenol also increased steady-state levels of H2DCFDA oxidation as well as increased levels of total glutathione (GSH) and glutathione disulfide (GSSG). Interestingly, depletion of GSH using buthionine sulfoximine did not significantly potentiate 5Z-7-oxozeaenol toxicity in either cell line. In contrast, pre-treatment of cells with auranofin (Au) to inhibit thioredoxin reductase activity significantly increased levels of oxidized thioredoxin as well as sensitized cells to 5Z-7-oxozeaenol-induced growth inhibition and clonogenic cell killing. These results were confirmed in SW 620 murine xenografts, where treatment with 5Z-7-oxozeaenol or with Au plus 5Z-7-oxozeaenol significantly inhibited growth, with Au plus 5Z-7-oxozeaenol trending toward greater growth inhibition compared to 5Z-7-oxozeaenol alone. These results support the hypothesis that thiol-mediated oxidative stress is causally related to TAK1-induced colon cancer cell killing. In addition, these results support the hypothesis that thioredoxin metabolism is a critical target for enhancing colon cancer cell killing via TAK1 inhibition and could represent an effective therapeutic strategy in patients with these highly resistant tumors.

Introduction: The mainstay of treatment for pancreatic and gastric cancers is surgical resection. Unfortunately many of these patients present with locally advanced, unresectable or distant disease and therefore medical management may be of more benefit. Accurate staging of patients with pancreatic and gastric cancer is essential in determining the best treatment strategy. Despite preoperative imaging there remains a group of patients that have clinically occult metastatic disease. Positive peritoneal cytology is a poor prognostic indicator for survival in both gastric and pancreatic cancer. Surgical resection may not be of benefit in those with positive peritoneal cytology. At our institution, a diagnostic laparoscopy with peritoneal washings is performed prior to surgical resection. We performed a retrospective review to evaluate the accuracy of immediate peritoneal washing interpretation in both gastric cancer and pancreatic cancer. Results: There were 51 patients that underwent immediate peritoneal washing interpretations. There were 5 patients with gastric adenocarcinoma, 2 patients with cholangiocarcinoma, and 44 patients with pancreatic adenocarcinoma. Four of the patients had positive cytology for tumor cells with immediate interpretation, and 47 patients had cytology negative for tumor cells with immediate interpretation. There was only one patient with negative cytology on immediate interpretation with cytology positive for malignant cells on final pathologic results. There were many RBCs noted within the peritoneal fluid specimen, which is probably why the cancer cells were missed. Immediate peritoneal cytology results had a 100% positive predictive value, and a 97% negative predictive value. There were no false positive results. Discussion: Positive peritoneal cytology is considered a poor prognostic factor for survival in both gastric and pancreatic cancer. In pancreatic and gastric cancer, previous studies have shown resection in the presence of metastatic disease does not improve survival. Diagnostic laparoscopy has been used as an adjunct to help stage patients before proceeding with a radical resection that has an associated significant morbidity andmortality rate is futile and potentially harmful, delaying or obviating systemic therapy that may be of more benefit to the patient. Our results show that utilizing immediate interpretation of peritoneal cytology is reliable and accurate. There was only one patient with a false negative result and there were no false positive results. Therefore, prior to surgical resection immediate interpretation can be utilized. Conclusion: Diagnostic laparoscopy with peritoneal washings and immediate interpretation of cytology can be used prior to proceeding with surgical resection. A futile operation can potentially be avoided with the use of immediate interpretation of the peritoneal fluid.