Notch and Kras in pancreatic cancer: At the crossroads of mutation,differentiation and signaling

Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.     

Genetic and epigenetic alterations in pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers worldwide. Despite significant progresses in the last decades, the origin of this cancer remains unclear and no efficient therapy exists. PDAC does not arise de novo: three remarkable different types of pancreatic lesions can evolve towards pancreatic cancer. These precursor lesions include: Pancreatic intraepithelial neoplasia (PanIN) that are microscopic lesions of the pancreas, Intraductal Papillary Mucinous Neoplasms (IPMN) and Mucinous Cystic Neoplasms (MCN) that are both macroscopic lesions. However, the cellular origin of these lesions is still a matter of debate. Classically, neoplasm initiation or progression is driven by several genetic and epigenetic alterations. The aim of this review is to assemble the current information on genetic mutations and epigenetic disorders that affect genes during pancreatic carcinogenesis. We will further discuss the interest of the genetic and epigenetic alterations for the diagnosis and prognosis of PDAC. Large genetic alterations (chromosomal deletion/amplification) and single point mutations are well described for carcinogenesis inducers. Mutations classically occur within key regions of the genome. Consequences are various and include activation of mitogenic pathways or silencing of apoptotic processes. Alterations of K-RAS, P16 and DPC4 genes are frequently observed in PDAC samples and have been described to arise gradually during carcinogenesis. DNA methylation is an epigenetic process involved in imprinting and X chromosome inactivation. Alteration of DNA methylation patterns leads to deregulation of gene expression, in the absence of mutation. Both genetic and epigenetic events influence genes and non-coding RNA expression, with dramatic effects on proliferation, survival and invasion. Besides improvement in our fundamental understanding of PDAC development, highlighting the molecular alterations that occur in pancreatic carcinogenesis could provide new clinical tools for early diagnosis of PDAC and the molecular basis for the development of new effective therapies.     

MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer

The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.     

Low Incidence of High-Grade Pancreatic Intraepithelial Neoplasia Lesions in a Crmp4 Gene–Deficient Mouse Model of Pancreatic Cancer

Pancreatic intraepithelial neoplasia (PanIN), the most common premalignant lesion of the pancreas, is a histologically well-defined precursor to invasive pancreatic ductal adenocarcinoma (PDAC). However, the molecular mechanisms underlying the progression of PanINs have not been fully elucidated. Previously, we demonstrated that the expression of collapsin response mediator protein 4 (CRMP4) in PDAC was associated with poor prognosis. The expression of CRMP4 was also augmented in a pancreatitis mouse model. However, the role of CRMP4 in the progression of PanIN lesions remains uncertain. In the present study, we examined the relationship between CRMP4 expression and progression of PanIN lesions using genetically engineered mouse models. PanIN lesions were induced by peritoneal injection of the cholecystokinin analog caerulein in LSL-KRAS^G12D; Pdx1-Cre (KC-Crmp4 wild-type, WT) mice and LSL-KRAS^G12D; Pdx1-Cre; Crmp4^−/− (KC-Crmp4 knockout, KO) mice. We analyzed pancreatic tissue sections from these mice and evaluated PanIN grade by hematoxylin and eosin staining. CRMP4 expression was examined and the cellular components assessed by immunohistochemistry using antibodies against CRMP4, CD3, and α-smooth muscle actin (SMA). The incidence of high-grade PanIN in KC-Crmp4 WT mice was higher than that in KC-Crmp4 KO animals. CRMP4 was expressed not only in epithelial cells but also in αSMA-positive cells in stromal areas of PanIN lesions. The CRMP4 expression in stromal areas correlated with PanIN grade in WT mice. These results suggested that the expression of CRMP4 in stromal cells may underlie the incidence or progression of PanIN.     

Adipocytes promote pancreatic cancer cell proliferation via glutamine transfer

Adipocytes promote progression of multiple cancers, but their role in pancreatic intraepithelial neoplasia (PanIN) and ductal adenocarcinoma (PDAC) is poorly defined. Nutrient transfer is a mechanism underlying stromal cell-cancer crosstalk. We studied the role of adipocytes in regulating in vitro PanIN and PDAC cell proliferation with a focus on glutamine metabolism. Murine 3T3L1 adipocytes were used to model adipocytes. Cell lines derived from PKCY mice were used to model PanIN and PDAC. Co-culture was used to study the effect of adipocytes on PanIN and PDAC cell proliferation in response to manipulation of glutamine metabolism. Glutamine secretion was measured with a bioanalyzer. Western blotting was used to study the effect of PanIN and PDAC cells on expression of glutamine-related enzymes in adipocytes. Adipocytes promote proliferation of PanIN and PDAC cells, an effect that was amplified in nutrient-poor conditions. Adipocytes secrete glutamine and rescue PanIN and PDAC cell proliferation in the absence of glutamine, an effect that was glutamine synthetase-dependent and involved PDAC cell-induced down-regulation of glutaminase expression in adipocytes. These findings suggest glutamine transfer as a potential mechanism underlying adipocyte-induced PanIN and PDAC cell proliferation.     

Antidiabetic Drug Metformin Prevents Progression of Pancreatic Cancer by Targeting in Part Cancer Stem Cells and mTOR Signaling

Epidemiologic studies have shown that diabetes mellitus is associated positively with increased risk of pancreatic ductal adenocarcinoma (PDAC), and recent meta-analysis studies showed that metformin, reduces the risk of pancreatic cancer (PC). We tested the effects of metformin on pancreatic intraepithelial neoplasia (PanIN) and their progression to PDAC in p48Cre/+.LSL-KrasG12D/+ transgenic mice. Mice fed control diet showed 80% and 62% incidence of PDAC in males and females, respectively. Male mice showed 20% and 26%, and female mice showed 7% and 0% PDAC incidence with 1000- and 2000-ppm metformin treatments, respectively. Both doses of metformin decreased pancreatic tumor weights by 34% to 49% (P < 0.03–0.001). The drug treatment caused suppression of PanIN 3 (carcinoma in situ) lesions by 28% to 39% (P < .002) and significant inhibition of carcinoma spread in the pancreas. The pancreatic tissue and/or serum of mice fed metformin showed a significant inhibition of mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), phosphorylated extracellular signal-regulated kinases (pErk), and insulin-like growth factor 1 (IGF-1) with an increase in phosphorylated 5′ adenosine monophosphate kinase (pAMPK), tuberous sclerosis complex 1 (TSC1, TSC2), C-protein and an autophagy related protein 2 (ATG2). The cancer stem cell (CSC) markers were significantly decreased (P < 0.04–0.0002) in the pancreatic tissue. These results suggest that biologic effects of metformin are mediated through decreased CSC markers cluster of differentiation 44 (CD44 and CD133), aldehyde dehydrogenase isoform 1 (ALDH1), and epithelial cell adhesion molecule (EPCAM) and modulation of the mTOR signaling pathway. Our preclinical data indicate that metformin has significant potential for use in clinical trials for PC chemoprevention.     

Increased B Cell-Activating Factor Promotes Tumor Invasion and Metastasis in Human Pancreatic Cancer

B cell-activating factor (BAFF) is a cytokine belonging to the tumor necrosis factor (TNF) superfamily. It has been reported that BAFF is elevated in patients with autoimmune pancreatitis and contributes to the malignant potential of blood cancers and solid tumors. In this study, clinical evidence of increased BAFF levels in patients with pancreatic ductal adenocarcinoma (PDAC) was obtained, and the roles and mechanisms of BAFF in PDAC were clarified in human tissues of PDAC and from in vitro data of PDAC cell lines. Serum levels of BAFF in patients with PDAC were significantly higher than in healthy subjects (p = 0.0121). Patients with UICC stage IV PDAC (T1-4, N0-1, M1) had significantly higher levels of serum BAFF compared to patients with PDAC (p = 0.0182). BAFF was remarkably expressed in infiltrating B lymphocytes surrounding pancreatic cancer in human pancreatic tissues, suggesting that BAFF may play a role in progression of pancreatic cancer. PDAC cell lines were cultured with human recombinant BAFF, and morphology and gene expression were analyzed; pancreatic cancer cells changed to a fibroblast-like morphology, and showed altered gene expression of E-cadherin, vimentin and Snail. These BAFF-induced changes reflect enhanced cell motility and invasion. BAFF-R-overexpressing cell clones confirmed the association between these BAFF-induced changes and epithelial-mesenchymal transition (EMT)-related genes. BAFF was elevated in patients with metastatic advanced PDAC and induced alterations in PDAC cells via regulation of EMT-related genes. Elucidation of the precise role and mechanism of control of BAFF may lead to new therapeutic approaches with the aim of improving pancreatic cancer survival.     

Mucin (Muc) expression during pancreatic cancer progression in spontaneous mouse model: potential implications for diagnosis and therapy

Pancreatic cancer (PC) is a lethal malignancy primarily driven by activated Kras mutations and characterized by the deregulation of several genes including mucins. Previous studies on mucins have identified their significant role in both benign and malignant human diseases including PC progression and metastasis. However, the initiation of MUC expression during PC remains unknown because of lack of early stage tumor tissues from PC patients. In the present study, we have evaluated stage specific expression patterns of mucins during mouse PC progression in (KrasG12D;Pdx1-Cre (KC)) murine PC model from pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma (PDAC) by immunohistochemistry and quantitative real-time PCR. In agreement with previous studies on human PC, we observed a progressive increase in the expression of mucins particularly Muc1, Muc4 and Muc5AC in the pancreas of KC (as early as PanIN I) mice with advancement of PanIN lesions and PDAC both at mRNA and protein levels. Additionally, mucin expression correlated with the increased expression of inflammatory cytokines IFN-γ (p?相似文献   

Expression analysis of PMP22/Gas3 in premalignant and malignant pancreatic lesions.

PMP22 is a structural protein of Schwann cells, but it also influences cell proliferation. In the present study, quantitative RT-PCR (QRT-PCR) and immunohistochemistry were used to determine PMP22 mRNA levels and to localize PMP22 in the normal pancreas (n=20), chronic pancreatitis (CP) (n=22), pancreatic ductal adenocarcinoma (PDAC) (n=31), intraductal papillary mucinous neoplasms (IPMN) (n=9), mucinous cystic tumors (MCN) (n=4), and in a panel of PanIN lesions (n=29). PMP22 mRNA levels were significantly higher in CP (3-fold) and PDAC (2.5-fold), compared to normal pancreatic tissues. PMP22 expression was restricted to nerves in the normal pancreas, while in CP and PDAC PMP22 was also expressed in PanIN lesions and in a small percentage of pancreatic cancer cells. PMP22 was weak to absent in the tumor cells of IPMNs and MCNs. PMP22 mRNA was present at different levels in cultured pancreatic cancer cells and up-regulated by transforming growth factor (TGF)-beta1 in 2 of 8 of these cell lines. In conclusion, PMP22 expression is present in both CP and PDAC tissues. Its expression in PanIN lesions and some pancreatic cancer cells in vitro and in vivo suggests a role of PMP22 in the neoplastic transformation process from the normal pancreas to pre-malignant lesions to pancreatic cancer.     

Acute pancreatitis accelerates initiation and progression to pancreatic cancer in mice expressing oncogenic Kras in the nestin cell lineage

Targeting of oncogenic Kras to the pancreatic Nestin-expressing embryonic progenitor cells and subsequently to the adult acinar compartment and Nestin-expressing cells is sufficient for the development of low grade pancreatic intraepithelial neoplasia (PanIN) between 2 and 4 months. The mice die around 6 month-old of unrelated causes, and it is therefore not possible to assess whether the lesions will progress to carcinoma. We now report that two brief episodes of caerulein-induced acute pancreatitis in 2 month-old mice causes rapid PanIN progression and pancreatic ductal adenocarcinoma (PDAC) development by 4 months of age. These events occur with similar frequency as observed in animals where the oncogene is targeted during embryogenesis to all pancreatic cell types. Thus, these data show that oncogenic Kras-driven PanIN originating in a non-ductal compartment can rapidly progress to PDAC when subjected to a brief inflammatory insult.     

Acute pancreatitis markedly accelerates pancreatic cancer progression in mice expressing oncogenic Kras

Chronic pancreatitis increases by 16-fold the risk of developing pancreatic ductal adenocarcinoma (PDAC), one of the deadliest human cancers. It also appears to accelerate cancer progression in genetically engineered mouse models. We now report that in a mouse model where oncogenic Kras is activated in all pancreatic cell types, two brief episodes of acute pancreatitis caused rapid PanIN progression and accelerated pancreatic cancer development. Thus, a brief inflammatory insult to the pancreas, when occurring in the context of oncogenic Kras^G12D, can initiate a cascade of events that dramatically enhances the risk for pancreatic malignant transformation.     

Laminin,gamma 2 (LAMC2): A Promising New Putative Pancreatic Cancer Biomarker Identified by Proteomic Analysis of Pancreatic Adenocarcinoma Tissues

In pancreatic cancer, the incidence and mortality curves coincide. One major reason for this high mortality rate in pancreatic ductal adenocarcinoma (PDAC) patients is the dearth of effective diagnostic, prognostic, and disease-monitoring biomarkers. Unfortunately, existing tumor markers, as well as current imaging modalities, are not sufficiently sensitive and/or specific for early-stage diagnosis. There is, therefore, an urgent need for improved serum markers of the disease. Herein, we performed Orbitrap® mass spectrometry proteomic analysis of four PDAC tissues and their adjacent benign tissues and identified a total of 2190 nonredundant proteins. Sixteen promising candidates were selected for further scrutiny using a systematic scoring algorithm. Our preliminary serum verification of the top four candidates (DSP, LAMC2, GP73, and DSG2) in 20 patients diagnosed with pancreatic cancer and 20 with benign pancreatic cysts, showed a significant (p < 0.05) elevation of LAMC2 in pancreatic cancer serum. Extensive validation of LAMC2 in healthy, benign, and PDAC sera from geographically diverse cohorts (n = 425) (Japan, Europe, and USA) demonstrated a significant increase in levels in early-stage PDAC compared with benign diseases. The sensitivity of LAMC2 was comparable to CA19.9 in all data sets, with an AUC value greater than 0.85 in discriminating healthy patients from early-stage PDAC patients. LAMC2 exhibited diagnostic complementarity with CA19.9 by showing significant (p < 0.001 in two out of three cohorts) elevation in PDAC patients with clinically low CA19.9 levels.Pancreatic ductal adenocarcinoma (PDAC)¹ is one of the most devastating cancers and the fourth leading cause of cancer-related deaths in North America (1). Ninety-five percent of patients will not survive beyond five years; this high mortality rate is primarily attributed to the lack of effective diagnostic techniques and treatment regimens. The hallmark features of pancreatic cancer (PC) are late presentation and aggressive metastatic progression (2, 3). The National Cancer Institute statistics estimate that approximately $1.9 billion is being spent in the United States alone each year on PC diagnosis and treatment. PDAC is classified into resectable (∼10–20%), locally advanced unresectable (∼30–40%), and metastatic (∼50%) (3). PDAC diagnosed at resectable stage can possibly be cured with complete surgical removal. This could improve the survival rates and considerably lower treatment costs. It is projected that 20–40% of patients with resectable PDAC survive more than five years after complete surgical removal, highlighting the importance of early-stage diagnosis. Unfortunately, carbohydrate antigen 19–9 (CA19.9), the current standard serum tumor marker for PDAC, has certain limitations as an early detection biomarker (its sensitivity for small tumors {<3 cm} is ∼50% and it is significantly elevated in many benign conditions (e.g. biliary obstruction, hepatic cirrhosis, chronic pancreatitis)) (4, 5). In light of the scarcity of other, more reliable markers, CA19.9 is currently used in the clinic as a prognostic and surveillance marker. Undoubtedly, the need for a more reliable consistent biomarker (or biomarker panel) for early PDAC diagnosis remains unmet. In pursuit of novel PDAC biomarker candidates, we have previously delineated the proteomes of malignant pancreatic ascitic fluids, pools of pancreatic juice, and pancreatic cancer cell lines (BxPC3, CAPAN, CFPAC1, MIA-Paca2, PANC1, and SU.86.86). We identified a panel of five potential candidate biomarkers, which, in combination, slightly outperformed CA19.9 in a pilot verification study (40 individuals; 20 healthy, and 20 PDAC) (6).From a different perspective, in the current study, we deployed a comparative quantitative tissue proteomic methodology to compare the proteome of malignant pancreatic tissues with that of their adjacent normal counterparts. A total of 2190 nonredundant proteins were identified, which were further scrutinized using a systematic scoring algorithm based on their quantified cancer-versus-normal ratios, on their identification in malignant pancreatic ascites fluid, on their cancer-specific nature, and on their tissue-expression profiles. Our analysis resulted in sixteen promising candidate biomarkers, which fulfilled our criteria and selected for further validation studies. In a multistep validation approach, the selected candidates were first verified in serum samples obtained from 20 patients with benign pancreatic diseases and 20 patients with pancreatic cancer, using commercially available ELISA kits. The best candidate (LAMC2) was further tested in three geographically diverse cohorts from Germany, Japan, and the US composed of 435 serum samples from healthy, benign, and early and late stage cancer patients. Our approach brought to light a previously unknown, promising PDAC candidate biomarker, LAMC2.     

Salivary MicroRNA in Pancreatic Cancer Patients

Background

Pancreatic cancer is the fourth leading cause of cancer death in Western countries, with the lowest 1-year survival rate among commonly diagnosed cancers. Reliable biomarkers for pancreatic cancer diagnosis are lacking and are urgently needed to allow for curative surgery. As microRNA (miRNA) recently emerged as candidate biomarkers for this disease, we explored in the present pilot study the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery, precancerous lesions, inflammatory disease or cancer-free patients as a potential early diagnostic tool.

Methods

Whole saliva samples from patients with pancreatic cancer (n = 7), pancreatitis (n = 4), IPMN (n = 2), or healthy controls (n = 4) were obtained during endoscopic examination. After total RNA isolation, expression of 94 candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm. Human-derived pancreatic cancer cells were xenografted in athymic mice as an experimental model of pancreatic cancer.

Results

We identified hsa-miR-21, hsa-miR-23a, hsa-miR-23b and miR-29c as being significantly upregulated in saliva of pancreatic cancer patients compared to control, showing sensitivities of 71.4%, 85.7%, 85,7% and 57%, respectively and excellent specificity (100%). Interestingly, hsa-miR-23a and hsa-miR23b are overexpressed in the saliva of patients with pancreatic cancer precursor lesions. We found that hsa-miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis as compared to the control group, with sensitivity of 100% and 75%, and specificity of 100% and 80%, respectively. Last hsa-miR-216 was upregulated in cancer patients as compared to patients diagnosed with pancreatitis, with sensitivity of 50% and specificity of 100%. In experimental models of PDAC, salivary microRNA detection precedes systemic detection of cancer cells markers.

Conclusions

Our novel findings indicate that salivary miRNA are discriminatory in pancreatic cancer patients that are not eligible for surgery. In addition, we demonstrate in experimental models that salivary miRNA detection precedes systemic detection of cancer cells markers. This study stems for the use of salivary miRNA as biomarker for the early diagnosis of patients with unresectable pancreatic cancer.     

The Proteome of Pancreatic Cancer‐Derived Exosomes Reveals Signatures Rich in Key Signaling Pathways

Exosomes are membrane‐bound vesicles that traffic small molecular cargos. These cargos participate in cell–cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. Performed in this study are comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. Analysis of composition of microbiomes (ANCOM) determined a cluster of 98 enriched pancreatic cancer exosome core proteins (ePC‐ECPs). Further, these proteins are predicted by ingenuity pathway analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell‐to‐cell signaling and interaction in particular (top three p‐value significant pathways). Significant enrichment of canonical pathways of acute phase response signaling (inflammatory response signaling pathways) and FXR and RXR activation in biosynthetic pathways are also predicted; 97 ePC‐ECPs are associated with cancer and among them, 34 are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer‐associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted.     

Elevating pancreatic cystic lesion stratification: Current and future pancreatic cancer biomarker(s)

Pancreatic ductal adenocarcinoma (PDAC) is an incredibly deadly disease with a 5-year survival rate of 9%. The presence of pancreatic cystic lesions (PCLs) confers an increased likelihood of future pancreatic cancer in patients placing them in a high-risk category. Discerning concurrent malignancy and risk of future PCL progression to cancer must be carefully and accurately determined to improve survival outcomes and avoid unnecessary morbidity of pancreatic resection. Unfortunately, current image-based guidelines are inadequate to distinguish benign from malignant lesions. There continues to be a need for accurate molecular and imaging biomarker(s) capable of identifying malignant PCLs and predicting the malignant potential of PCLs to enable risk stratification and effective intervention management. This review provides an update on the current status of biomarkers from pancreatic cystic fluid, pancreatic juice, and seromic molecular analyses and discusses the potential of radiomics for differentiating PCLs harboring cancer from those that do not.     

LCN2 and TIMP1 as Potential Serum Markers for the Early Detection of Familial Pancreatic Cancer

High-risk individuals of familial pancreatic cancer (FPC) families are considered to be good candidates for screening programs to detect early PC or its high-grade precursor lesions, especially pancreatic intraepithelial neoplasia (PanIN) 2/3 lesions. There is a definite need for diagnostic markers as neither reliable imaging methods nor biomarkers are available to detect these lesions. On the basis of a literature search, the potential serum markers neutrophil gelatinase-associated lipocalin (LCN2), metallopeptidase inhibitor 1 (TIMP1), chemokine (C-X-C motif) ligand 16 (CXCL16), IGFBP4, and iC3a, which were first tested in transgenic KrasLSL.^G12D/+;p53^R172H/+;Pdx1-Cre mice, were identified. ELISA analyses of LCN2, TIMP1, and CXCL16 revealed significantly higher levels in mice with PanIN2/3 lesions or PC compared to mice with normal pancreata or PanIN1 lesions. Analysis of preoperative human serum samples from patients with sporadic PC (n = 61), hereditary PC (n = 24), chronic pancreatitis (n = 28), pancreatic neuroendocrine tumors (n = 11), and FPC patients with histologically proven multifocal PanIN2/3 lesions (n = 3), as well as healthy control subjects (n = 20), confirmed significantly higher serum levels of LCN2 and TIMP1 in patients with PC and multifocal PanIN2/3 lesions. The combination of LCN2 and TIMP1 as a diagnostic test for the detection of PC had a sensitivity, specificity, and positive predictive value of 100% each. Although this preliminary finding needs to be validated in a large series of individuals at high risk for FPC, serum measurement of LCN2 and TIMP1 might be a promising screening tool.     

Pyruvate Kinase M2 and Lactate Dehydrogenase A Are Overexpressed in Pancreatic Cancer and Correlate with Poor Outcome

Pancreatic cancer has a 5-year survival rate of less than 4%. Despite advances in diagnostic technology, pancreatic cancer continues to be diagnosed at a late and incurable stage. Accurate biomarkers for early diagnosis and to predict treatment response are urgently needed. Since alteration of glucose metabolism is one of the hallmarks of cancer cells, we proposed that pyruvate kinase type M2 (M2PK) and lactate dehydrogenase A (LDHA) enzymes could represent novel diagnostic markers and potential therapeutic targets in pancreatic cancer. In 266 tissue sections from normal pancreas, pancreatic cystic neoplasms, pancreatic intraepithelial neoplasia (PanIN) and cancer, we evaluated the expression of PKM2, LDHA, Ki-67 and CD8+ by immunohistochemistry and correlated these markers with clinicopathological characteristics and patient survival. PKM2 and LDHA expression was also assessed by Western blot in 10 human pancreatic cancer cell lines. PKM2 expression increased progressively from cyst through PanIN to cancer, whereas LDHA was overexpressed throughout the carcinogenic process. All but one cell line showed high expression of both proteins. Patients with strong PKM2 and LDHA expression had significantly worse survival than those with weak PKM2 and/or LDHA expression (7.0 months vs. 27.9 months, respectively, p = 0.003, log rank test). The expression of both PKM2 and LDHA correlated directly with Ki-67 expression, and inversely with intratumoral CD8+ cell count. PKM2 was significantly overexpressed in poorly differentiated tumours and both PKM2 and LDHA were overexpressed in larger tumours. Multivariable analysis showed that combined expression of PKM2 and LDHA was an independent poor prognostic marker for survival. In conclusion, our results demonstrate a high expression pattern of two major glycolytic enzymes during pancreatic carcinogenesis, with increased expression in aggressive tumours and a significant adverse effect on survival.     

Molecular Imaging of Pancreatic Duct Adenocarcinoma Using a Type 2 Cannabinoid Receptor-Targeted Near-Infrared Fluorescent Probe

Imaging probes targeting type 2 cannabinoid receptor (CB₂R) overexpressed in pancreatic duct adenocarcinoma (PDAC) tissue have the potential to improve early detection and surgical outcome of PDAC. The aim of our study was to evaluate the molecular imaging potential of a CB₂R-targeted near-infrared (NIR) fluorescent probe (NIR760-XLP6) for PDAC. CB₂R overexpression was observed in both PDAC patient tissues and various pancreatic cancer cell lines. In vitro fluorescence imaging indicated specific binding of NIR760-XLP6 to CB₂R in human PDAC PANC-1 cells. In a xenograft mouse tumor model, NIR760-XLP6 showed remarkable 50- (ex vivo) and 3.2-fold (in vivo) tumor to normal contrast enhancement with minimal liver and kidney uptake. In a PDAC lymph node metastasis model, significant signal contrast was observed in bilateral axillary lymph nodes with PDAC metastasis after injection of the probe. In conclusion, NIR760-XLP6 exhibits promising characteristics for imaging PDAC, and CB₂R appears to be an attractive target for PDAC imaging.