Purpose Non-invasive biomarkers for early detection of pancreatic ductal adenocarcinoma (PDAC) are currently not available. Here, we aimed to identify a set of urine proteins able to distinguish patients with early stage PDAC from healthy individuals (H). Experimental design Proteomes of 18 urine samples from healthy controls, chronic pancreatitis and PDAC patients (six/group) were assayed using GeLC/MS/MS analysis. The selected biomarkers were subsequently validated using ELISA assays using multiple logistic regression applied to a training dataset in a multicentre cohort comprising 488 urine samples. Results LYVE-1, REG1A and TFF1 were selected as candidate biomarkers. When comparing PDAC (n=192) to healthy (n=87) urines, the resulting areas under the receiver operating characteristic curves (AUCs) of the panel were 0.89 (95%CI 0.84-0.94) in the training (70% of the data), and 0.92 (95%CI 0.86-0.98) in the validation (30% of the data) datasets. When comparing PDAC stage I-II (n=71) to healthy urines, the panel achieved AUCs of 0.90 (95%CI 0.84-0.96) and 0.93 (95%CI 0.84-1.00) in the training and validation datasets, respectively. In PDAC stage I-II and healthy samples with matching plasma CA19.9 the panel achieved a higher AUC of 0.97 (95%CI 0.94-0.99) than CA19.9 (AUC=0.88, 95%CI 0.81-0.95, p=0.005). Adding plasma CA19.9 to the panel increased the AUC from 0.97 (95%CI 0.94-0.99) to 0.99 (95%CI 0.97-1.00, p=0.04) but did not improve the comparison of stage I-IIA PDAC (n=17) to healthy urine. Conclusion We have established a novel, three-protein biomarker panel that is able to detect patients with early stage pancreatic cancer in urine specimens.

Despite a wealth of genomic information, a comprehensive alternative splicing (AS) analysis of pancreatic ductal adenocarcinoma (PDAC) has not been performed yet. In the present study, we assessed whole exome-based transcriptome and AS profiles of 43 pancreas tissues using Affymetrix exon array. The AS analysis of PDAC indicated on average two AS probe-sets (ranging from 1–28) in 1,354 significantly identified protein-coding genes, with skipped exon and alternative first exon being the most frequently utilised. In addition to overrepresented extracellular matrix (ECM)-receptor interaction and focal adhesion that were also seen in transcriptome differential expression (DE) analysis, Fc gamma receptor-mediated phagocytosis and axon guidance AS genes were also highly represented. Of note, the highest numbers of AS probe-sets were found in collagen genes, which encode the characteristically abundant stroma seen in PDAC. We also describe a set of 37 ‘hypersensitive’ genes which were frequently targeted by somatic mutations, copy number alterations, DE and AS, indicating their propensity for multidimensional regulation. We provide the most comprehensive overview of the AS landscape in PDAC with underlying changes in the spliceosomal machinery. We also collate a set of AS and DE genes encoding cell surface proteins, which present promising diagnostic and therapeutic targets in PDAC.

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses.

The mechanisms of initiation of pancreatic ductal adenocarcinoma (PDAC) are still largely unknown. In the present study, we analysed the role of anterior gradient-2 (AGR2) in the earliest stages of pancreatic neoplasia. Immunohistochemical analysis of chronic pancreatitis (CP) and peritumoral areas in PDAC tissues showed that AGR2 was present in tubular complexes (TC) and early pancreatic intraepithelial neoplasia (PanINs). Moreover, AGR2 was also found in discrete subpopulations of non-transformed cells neighbouring these pre-neoplastic lesions. In primary cells derived from human patient-derived xenograft (PDX) model, flow-cytometry revealed that AGR2 was overexpressed in pancreatic cancer stem cells (CSC) compared with non-stem cancer cells. In LSL-KrasG12D;Pdx1-Cre (KC) mouse model Agr2 induction preceded the formation of pre-neoplastic lesions and their development was largely inhibited by Agr2 deletion in engineered LSL-KrasG12D;Pdx1-Cre; Agr2−/− mice. In vitro, AGR2 expression was stimulated by tunicamycin-induced endoplasmic reticulum (ER) stress in both KRAS wild-type normal pancreas cells, as well as in KRAS mutated pancreatic cancer cells and was essential for ER homoeostasis. The unfolded protein response proteins GRP78, ATF6 and XBP1s were found expressed in CP and PDAC peritumoral tissues, but in contrast to AGR2, their expression was switched off during TC and PanIN formation. Real-time PCR and ELISA analyses showed that ER stress induced a pro-inflammatory phenotype in pancreatic normal, cancer and stellate cells. Moreover, AGR2 expression was inducible by paracrine transfer of ER stress and pro-inflammation between different pancreatic cell types. Our findings demonstrate that AGR2 induced in ER-stressed and inflammatory pre-neoplastic pancreas is a potential marker of cancer progenitor cells with an important functional role in PDAC initiation.

Background: Pancreatic cancer is the 5th leading cause of cancer death in both males and females. In recent years, a wealth of gene and protein expression studies have been published broadening our understanding of pancreatic cancer biology. Due to the explosive growth in publicly available data from multiple different sources it is becoming increasingly difficult for individual researchers to integrate these into their current research programmes. The Pancreatic Expression database, a generic web-based system, is aiming to close this gap by providing the research community with an open access tool, not only to mine currently available pancreatic cancer data sets but also to include their own data in the database.

Pancreatic ductal adenocarcinoma (PDAC) accounts for over 213 000 deaths worldwide each year, largely due to late diagnosis. One of the risk factors for the development of PDAC is chronic pancreatitis (CP); the intense desmoplastic reaction makes differentiation between the two conditions extremely difficult. In order to identify biomarkers for noninvasive diagnosis, we performed 2-D DIGE analysis of urine samples from healthy individuals and patients with PDAC and CP. Despite considerable intersample heterogeneity, a total of 127 statistically valid (p<0.05), differentially expressed protein spots were detected, 101 of which were identified using MALDI-TOF MS. A number of these, including annexin A2, gelsolin and CD59 have already been associated with PDAC, however, their validation using immunoblotting proved challenging. This is probably due to extensive PTMs and processing thus indicating the need for raising specific antibodies for urinary proteins. Despite this, our study clearly demonstrates that urine is a valid source of noninvasive biomarkers in patients with pancreatic diseases.

Perineural invasion ( PNI ) is a common and characteristic feature of pancreatic ductal adenocarcinoma ( PDAC ) that is associated with poor prognosis, tumor recurrence, and generation of pain. However, the molecular alterations in cancer cells and nerves within PNI have not previously been comprehensively analyzed. Here, we describe our proteomic analysis of the molecular changes underlying neuro‐epithelial interactions in PNI using liquid chromatography–mass spectrometry ( LC ‐ MS / MS ) in microdissected PNI and non‐ PNI cancer, as well as in invaded and noninvaded nerves from formalin‐fixed, paraffin‐embedded PDAC tissues. In addition, an in vitro model of PNI was developed using a co‐culture system comprising PDAC cell lines and PC 12 cells as the neuronal element. The overall proteomic profiles of PNI and non‐ PNI cancer appeared largely similar. In contrast, upon invasion by cancer cells, nerves demonstrated widespread plasticity with a pattern consistent with neuronal injury. The up‐regulation of SCG 2 (secretogranin II ) and neurosecretory protein VGF (nonacronymic) in invaded nerves in PDAC tissues was further validated using immunohistochemistry. The tested PDAC cell lines were found to be able to induce neuronal plasticity in PC 12 cells in our in vitro established co‐culture model. Changes in expression levels of VGF , as well as of two additional proteins previously reported to be overexpressed in PNI , Nestin and Neuromodulin ( GAP 43), closely recapitulated our proteomic findings in PDAC tissues. Furthermore, induction of VGF , while not necessary for PC 12 survival, mediated neurite extension induced by PDAC cell lines. In summary, here we report the proteomic alterations underlying PNI in PDAC and confirm that PDAC cells are able to induce neuronal plasticity. In addition, we describe a novel, simple, and easily adaptable co‐culture model for in vitro study of neuro‐epithelial interactions.

Anterior gradient 2 (AGR2), a protein disulfide isomerase, shows two subcellular localizations: intracellular (iAGR2) and extracellular (eAGR2). In healthy cells that express AGR2, the predominant form is iAGR2, which resides in the endoplasmic reticulum. In contrast, cancer cells secrete and express eAGR2 on the cell surface. We wanted to test if AGR2 is a cancer-specific tumor-associated antigen. We utilized two AGR2 antibodies, P3A5 and P1G4, for in vivo tumor localization and tumor growth inhibition. The monoclonal antibodies recognized both human AGR2 and mouse Agr2. Biodistribution experiments using a syngeneic mouse model showed high uptake of P3A5 AGR2 antibody in xenografted eAgr2 + pancreatic tumors, with limited uptake in normal tissues. In implanted human patient-derived eAGR2 + pancreatic cancer xenografts, tumor growth inhibition was evaluated with antibodies and Gemcitabine (Gem). Inhibition was more potent by P1G4 + Gem combination than Gem alone or P3A5 + Gem. We converted these two antibodies to human:mouse chimeric forms: the constructed P3A5 and P1G4 chimeric mV L hC κ and mV H hC γ (γ1, γ2, γ4) genes were inserted in a single mammalian expression plasmid vector, and transfected into human 293F cells. Expressed human:mouse chimeric IgG1, IgG2 and IgG4 antibodies retained AGR2 binding. Increase in IgG yield by transfected cells could be obtained with serial transfection of vectors with different drug resistance. These chimeric antibodies, when incubated with human blood, effectively lysed eAGR2 + PC3 prostate cancer cells. We have, thus, produced humanized anti-AGR2 antibodies that, after further testing, might be suitable for treatment against a variety of eAGR2 + solid tumors.