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Background: Serine hydrolases (SHs) are among the largest classes of enzymes in human and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the pro-drug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. Methods: CES2 expression in PDAC and paired non-tumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. Results: Significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared to paired non-tumor tissue (P < .0001), with 48/118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in PDAC cell lines was inversely correlated with irinotecan IC50 values (P = .021), while no molecule involved in irinotecan metabolism yielded a significant correlation between its expression and sensitivity to the drug. Remarkably, we recently found that high CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (P = .024). Conclusion: Our findings suggest that CES2 expression and activity, by mediating the intra-tumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy. Citation Format: Michela Capello, Minhee Lee, Hong Wang, Ingrid Babel, Matthew H. Katz, Jason B. Fleming, Anirban Maitra, Huamin Wang, Weihua Tian, Ayumu Taguchi, Samir M. Hanash. Identification of Carboxylesterase-2 as a determinant of response to irinotecan and neoadjuvant FOLFIRINOX therapy in pancreatic ductal adenocarcinoma. [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 LB-121. doi:10.1158/1538-7445.AM2015-LB-121

Pancreatic ductal adenocarcinoma (PDA) is one of the most difficult cancer to treat, both for lack of effective screening method and for resistance phenomenon. At present surgical resection is the only potentially curative option, but the absence of early symptoms or clinical-pathological markers results in diagnosis at a late, inoperable stage in mostly of cases. Once diagnosed, a number of chemotherapy, radiation and combination therapy regimens are usually used to treat patients, but responses remain poor. However, chemotherapy is able to enhance tumor immunogenicity. Thus more immunogenic neo-antigens can be induced by chemotherapy and targeted by passive or active immunotherapy. We have previously identified a number of Tumor Associated Antigens (TAA) that are recognized by autoantibodies in PDAC patients. One of these, alpha-enolase, was found to have good antigen capability and immunization with DNA coding for alpha-enolase effectively enhanced the survival of genetically engineered mice that develop spontaneously PDAC. To discover neo-antigens that might be selected for immunotherapy, we have analysed antibody response in PDAC patients’ sera before and after chemotherapy. The reactivity of PDAC patients’ sera obtained before and after two rounds of chemotherapy was analysed on 2-dimensional gel electrophoresis proteome map of CF-PAC pancreatic cancer cell line and the antigens recognized by autoantibodies were identified by mass spectrometry. A number of antigens, mainly metabolic enzymes, proteins involved in transcription and structural proteins, were recognized by autoantibody only after one or two rounds of chemotherapy. The identification of common and individual antigens from chemotherapy treated-patients formed the platform for ongoing immunological studies aimed to assess the ability of these neo-antigens, compared to that identified before chemotherapy treatment, to induce specific helper and cytotoxic response to PDAC. A stronger and sustained immune response correlated to survival is expected to be induced by neo-antigens. Validation of these neo-antigens will allow to develop specific immunotherapy combined with chemotherapy. Citation Format: Giorgia Mandili, Emanuela Mazza, Michela Capello, Daniele Giordano, Paola Cappello, Francesco Novelli. Identification of neoantigens recognized by autoantibodies in chemotherapy-treated pancreatic cancer patients. [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 1329. doi:10.1158/1538-7445.AM2015-1329

Background: Although resectable pancreatic cancers are associated with better survival, only about 10% of pancreatic patients present with localized disease. Imaging modalities, notably endoscopic ultrasound and Magnetic resonance cholangiopancreatography, allow detection of early stage pancreatic cancer or small pancreatic cysts. However, these imaging modalities are not suitable for screening in terms of throughput, cost effectiveness, or invasiveness. Blood-based biomarkers could be ideal for screening of early stage pancreatic cancer. The performance of CA19-9 as a pancreatic cancer biomarker is limited and moreover CA 19-9 is not detectable in 5-10% of subjects with fucosyltransferase deficiency. As a result there is a need for additional markers that complement CA 19-9 for reliable detection of early stage pancreatic cancer. We have previously identified and obtained initial validation data for a set of blood based biomarkers which distinguished cases from control in the pre-diagnostic setting (Faca et al PLoS Medicine, 2008). We have undertaken further validation of this marker panel augmented with novel candidates including autoantibodies to tumor antigens identified by recombinant protein array. Methods: The selected biomarker candidates as well as CA19-9 were further assayed in a training set, consisting of plasmas from 138 pancreatic cancer patients and 81 controls (52 healthy subjects and 29 subjects with chronic pancreatitis) resulting in a combination rule which was tested in an independent cohort consisting of plasmas from 42 early stage pancreatic cancer patients, 50 healthy controls, 29 subjects with chronic pancreatitis, and 14 subjects with benign pancreatic cysts. Results: A logistic regression model was built in the training set using 5 markers. The AUCs of the model and CA19-9 alone were 0.80 (95% CI = 0.71-0.89) and 0.76 (95% CI = 0.67-0.86) (P value for difference in AUC = 0.014) in the training set. In the test set, the AUCs of the model in a comparison of early stage pancreatic cancer vs. (1) healthy controls, (2) subjects with chronic pancreatitis, and (3) subjects with benign pancreatic cysts were 0.91, 0.85, and 0.94 respectively. The marker panel substantially improved the negative predictive values (NPV) at 98% sensitivity in comparison of early stage pancreatic cancer vs. (1) healthy controls and (2) subjects with chronic pancreatitis (NPVs of the panel = (1) 0.96, (2) 0.95, and (3) 0.88; NPVs of CA19-9 alone = (1) 0.83, (2) 0.83, and (3) 0.88). Stratification based on CA19-9 positivity (cut off = 37 units/ml) yielded AUCs of (1) 0.79, (2) 0.72, and (3) 0.90, and NPVs were (1) 0.99, (2) 0.98, and (3) 0.95 in CA19-9 negative subjects. Conclusions: We have validated a marker combination with a potential to differentiate early stage pancreatic cancer from healthy controls, subjects with chronic pancreatitis and subjects with pancreatic cysts. Citation Format: Ayumu Taguchi, Michela Capello, Yang Zhao, Ingrid Babel, Gary Goodman, Margaret A. Tempero, Matthew A. Firpo, Matthew H. Katz, Ziding Feng, Samir Hanash. Development and validation of diagnostic biomarker model for detection of early stage pancreatic cancer. [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 B20.

Background Pancreatic Ductal Adenocarcinoma (PDAC) is a highly aggressive malignancy with only a 5% 5-year survival rate. Reliable biomarkers for early detection are still lacking. The goals of this study were (a) to identify early humoral responses in genetically engineered mice (GEM) spontaneously developing PDAC; and (b) to test their diagnostic/predictive value in newly diagnosed PDAC patients and in prediagnostic sera. Methods and results The serum reactivity of GEM from inception to invasive cancer, and in resectable or advanced human PDAC was tested by two-dimensional electrophoresis Western blot against proteins from murine and human PDAC cell lines, respectively. A common mouse-to-human autoantibody signature, directed against six antigens identified by MALDI-TOF mass spectrometry, was determined. Of the six antigens, Ezrin displayed the highest frequency of autoantibodies in GEM with early disease and in PDAC patients with resectable disease. The diagnostic value of Ezrin-autoantibodies to discriminate PDAC from controls was further shown by ELISA and ROC analyses (P Conclusions Autoantibodies against Ezrin are induced early in PDAC and their combination with other serological markers may provide a predictive and diagnostic signature.

Aberrant metabolism together with invasion and metastasis are hallmarks of cancer. This is particularly true for pancreatic ductal adenocarcinoma (PDAC), characterized by rapid progression, invasiveness and resistance to treatments. We have previously described α-enolase (ENOA) as a PDAC-associated antigen. It is a moonlighting protein that works both as a key metabolic enzyme and as a membrane plasminogen receptor. In order to clarify its multifunctional role in pancreatic tumorigenesis we investigated the effect of ENOA knockdown in PDAC cells. Protein expression alterations following ENOA knockdown in the human PDAC cell line CFPAC-1 were revealed by LC-MS/MS analysis. On the basis of a spectra count label-free quantitation approach a large number of proteins mainly involved in cell adhesion, metabolism and proliferation were found to be differentially expressed in ENOA silenced cells compared to the control. After ENOA silencing, PDAC cells displayed a delay in proliferation and decreased survival and colony formation capabilities, even if the pyruvate production was not affected. The growth inhibition was partially due to an increased concentration of intracellular reactive oxygen species (ROS) mainly generated through the sorbitol and NADPH oxidase pathways. Moreover in ENOA silenced cells, the in vitro plasminogen-driven invasion was abolished and the number of lung tumor masses was significantly reduced in SCID-beige mice injected with ENOA silenced cells compared to mice injected with control cells. These effects are under further confirmation in other PDAC cell lines. All together, these findings propose ENOA as a promising target for developing new therapies in pancreatic cancer management. Citation Format: Michela Capello, Moitza Principe, Michelle Samuel Chattaragada, Chiara Riganti, Weidong Zhou, Sammy Ferri-Borgogno, Simona Rolla, Lance Liotta, Emanuel Petricoin, Paola Cappello, Francesco Novelli. Can the moonlighting glycolytic enzyme α-enolase be a therapeutic target in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1889. doi:10.1158/1538-7445.AM2013-1889

Enolase is one of the most abundantly expressed cytosolic proteins in many organisms, and it is a key glycolytic enzyme that catalyzes the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Recently, accumulating evidence revealed that, in addition to its innate glycolytic function, the well characterized old enzyme enolase is a multifunctional protein and plays an important role in several biological and pathophysiological processes: 1) by using an alternative start codon, the α-enolase mRNA can be translated into a 37 kDa protein which lacks the first 96 amino acid residues. This protein is localized in the nucleus and can bind to the c-myc P2 promoter and negatively regulate transcription of the proto-oncogene; 2) α-enolase has been detected on the surface of hematopoietic cells such as monocytes, T cells and B cells, neuronal cells and endothelial cells as a strong plasminogen-binding receptor, modulating the pericellular and intravascular fibrinolytic system. The expression of α-enolase on the surface of a variety of eukaryotic cells has been found to be dependent on the pathophysiological conditions of these cells, however, how α-enolase is displayed on the cell surface remains unknown; 3) increased expression of enolase has been reported to correlated with progression of tumors, such as neuroendocrine tumors, neuroblastoma and lung cancers, and enolase has been considered to be a diagnostic marker for many tumors. Our previous studies demonstrated that α-enolase is up-regulated at both the mRNA and protein levels in pancreatic ductal adenocarcinoma (PDAC). PDAC is the fourth leading cause of cancer-related deaths in Western countries. The mean life expectancy is 15-18 months for patients with local and regional disease, and only 3-6 months for those with metastatic disease. In this present work, we further characterized the α-enolase from PDAC by reversed-phase liquid chromatography nanospray tandem mass spectrometry (LC-MS/MS) analysis and an LTQ-Orbitrap instrument, and identified multiple posttranslational modifications of α-enolase, such as phosphorylation, acetylation, and methylation. The mass spectrometer LTQ-Orbitrap provides high accuracy mass measurement that is essential for the validation of modified peptide identifications and the reduction of false positive identifications. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5575.

We have demonstrated that sera from pancreatic ductal adenocarcinoma (PDAC) patients contain IgG autoantibodies to α-enolase, a glycolytic enzyme that also works as surface plasminogen receptor. As this implies that α-enolase elicits a B-cell dependent humoral response in vivo in PDAC patients, its ability to induce a T cell mediated response to PDAC was investigated. Here we show that α-enolase-pulsed dendritic cells were capable to specifically stimulate healthy autologous T cells to proliferate, secrete IFN-γ and lyse PDAC cells. In vivo, α-enolase-specific T cells completely inhibited the growth of PDAC cells in immunodeficient mice. In PDAC patients, and in particular in those with circulating IgG autoantibodies to α-enolase, the existence of memory T cells to α-enolase was also demonstrated. As a whole, these results indicate that α-enolase elicits a PDAC-specific humoral and cellular responses. The sum of its features makes α-enolase a promising candidate for new immunotherapeutic strategies in the cure of PDAC that lacks until now of efficacious therapies to associate the conventional ones.

The frequency of gastroduodenal lesions has been investigated in 142 patients with liver cirrhosis of various degrees of severity and in 63 patients with mild liver disease (controls) in whom liver biopsy excluded nodular regeneration. Cirrhotic patients were subdivided in three groups according to the Pugh modification of the Child-Turcotte criteria. Although the frequency of peptic ulcer was not different, gastroduodenal erosions were observed more frequently in cirrhotics than in controls (29.6% vs 11.1%, P less than 0.01). The occurrence of erosions was related to the severity of the disease: in Child A and B patients their frequency was 21 and 26% respectively, but rose to 48.4 (15 of 31 vs 7 of 63 in controls, P less than 0.001) in the Child C group. Both mild and severe gastroduodenitis occurred more frequently, although not significantly, in patients with liver cirrhosis. All together one or more endoscopic lesions were observed in almost 60% of cirrhotics but only in 25.4% of controls (P less than 0.001). In conclusion, our data do not show an increased prevalence of peptic ulcer in cirrhotic patients; in contrast, liver cirrhosis is significantly associated with the endoscopic finding of gastroduodenal erosions, especially in the more advanced stages of the disease. These findings would suggest a cautious use, in cirrhotic patients, of drugs which may damage the gastroduodenal mucosa; moreover, long-term administration of antacids or of other drugs with a protective effect on gastroduodenal mucosa might be taken into consideration for Child C patients.