SELDI-TOF MS Analysis of Hepatocellular Carcinoma in an Australian Cohort.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common cause of cancer death worldwide. Resection offers the best chance of long-term survival, but a consistent adverse prognostic factor is the presence of microvascular invasion (MVI). In this study, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), a high throughput method of analyzing complex samples, was used to explore differentially expressed proteins between HCC and adjacent nontumour liver tissue (ANLT). These findings were correlated with clinical outcomes. MATERIALS AND METHODS: From 2002 to 2011, tumor and ANLT were collected from patients who underwent liver resection and these samples were later prepared for SELDI-TOF MS. Output data were then used to identify proteins capable of discriminating HCC from ANLT. Proteins from the multivariate analysis were then analyzed to determine prognostic factors and the m/z ratios of these proteins were entered into the ExPASy database to infer potential candidates. RESULTS: During the study period, 30 patients had SELDI-TOF MS performed on their HCC and ANLT samples. On multivariate analysis, a panel of four proteins-m/z 5840, m/z 8921, m/z 9961, and m/z 25,872-discriminated HCC from ANLT with an area under the ROC curve of 0.954 (P < 0.001). On prognostic factor assessment, decreased m/z 9961 was significantly associated with the presence of MVI (P = 0.025) and shorter disease-free survival (P = 0.045) in our patients. A potential candidate for this protein was coxsackievirus and adenovirus receptor, isoform 3 (CAR 3/7), which helps maintain tight junction integrity. CONCLUSIONS: Using SELDI TOF-MS, we identified a panel of four proteins with excellent discriminative capacity between HCC and ANLT. Of these, m/z 9961 was the only protein significantly associated with a known poor prognostic factor (presence of MVI) and survival (shorter disease-free survival). While loss of CAR 3/7 could lead to MVI, further research is warranted to validate the identity of protein m/z 9961.

Using patient-derived xenograft models of colorectal liver metastases to predict chemosensitivity.

BACKGROUND: Few in vivo models for colorectal cancer have been demonstrated to show external validity by accurately predicting clinical patient outcomes. Patient-derived xenograft (PDX) models of cancer have characteristics that might provide a form of translational research leading to personalized cancer care. The aim of this pilot study was to assess the feasibility of using PDXs as a platform for predicting patient colorectal liver metastases responses, in this case by correlating PDX and patient tumor responses to either folinic acid, fluorouracil plus oxaliplatin or folinic acid, fluorouracil plus irinotecan-based regimens. METHODS: Sixteen patients underwent potentially curative resection of colorectal liver metastases, and tumors were grafted into NOD.CB17-Prkdcscid/Arc mice. Mice were divided into groups to determine relative tumor growth in response to treatment. Tumors were analyzed by immunohistochemistry for Ki67 and Excision repair cross-complementation group 1. RESULTS: An engraftment rate of 81% was achieved. Overall, there was a 67% positive match rate between eligible patient and PDX chemosensitivity profiles. There was a significant difference in relative decrease in Ki67 expression between sensitive/stable versus resistant PDXs for both treatment regimens. There was no statistically significant correlation between baseline ERCC1 expression and response to Oxaliplatin + 5-Fluorouracil in the PDXs. CONCLUSIONS: This pilot study supports the feasibility of using PDX models of advanced colorectal cancer in larger studies to potentially predict patient chemosensitivity profiles.

A patient-derived subrenal capsule xenograft model can predict response to adjuvant therapy for cancers in the head of the pancreas.

BACKGROUND: Although gemcitabine is commonly used as adjuvant therapy for pancreatic adenocarcinoma and pancreaticobiliary-type periampullary cancers, not all patients appear to benefit. This translational study evaluates the potential of a patient-derived subrenal capsule pancreatic cancer xenograft (SRCPCX) model to identify within eight weeks after surgery those tumours which will respond to gemcitabine. METHODS: SRCPCXs from 32 pancreatectomy patients were established in six to ten NOD/SCID mice per patient. After four weeks the mice were randomly assigned to receive gemcitabine or saline for four more weeks. After eight weeks, gemcitabine response in the grafts was evaluated by the percentage of tumour growth inhibition (%TGI), histological morphology and immunohistochemical markers (Ki-67, CK7 and cleaved caspase-3). These were collated into an Overall Response. Survival was assessed by Kaplan-Meier and Cox multivariate analyses. RESULTS: 375 of 450 pieces of tissue from 27 of 31 patients were evaluable. In 90% of patients, histopathological and immunostaining features of saline-treated control grafts were concordant with their original tumours. At follow up, six of 15 patients whose tumours had an Overall Response to gemcitabine died, compared with ten of 12 whose tumours did not respond (P = 0.025, Fisher's exact test). This was associated with improved survival on Kaplan-Meier analysis (P = 0.013). Cox multivariate analysis indicated that Overall Response, stage and grade were independent predictors of survival. CONCLUSION: This SRCPCX model retains major histopathological and immunohistochemical characteristics of the original tumour and when a combination of measures is used, enables early assessment of tumour sensitivity to gemcitabine in pancreatic cancers.

Discovery of diagnostic biomarkers for pancreatic cancer in immunodepleted serum by SELDI-TOF MS.

MOTIVATION: Reports of serum pancreatic cancer (PC) biomarkers using SELDI-TOF MS have been inconsistent because different chip surfaces and interference with high-abundant proteins. This study examines the influence of these factors on the detection of discriminating diagnostic biomarkers. METHODS: Serum from fourteen from patients with PC, disease controls (DC, n = 14) and healthy volunteers (HV, n = 14) were evaluated by SELDI using H50, IMAC, Q10 and CM10 chips. A further evaluation was undertaken after depletion of seven high-abundant proteins using spin cartridges. RESULTS: More protein peaks were detected in whole serum than in depleted serum for IMAC, H50 and Q10 chips: 60 vs 39, 56 vs 48 and 69 vs 65, respectively, while the CM10 found less peaks in serum (27 vs 47 peaks). However, there were more differentially expressed peaks in the depleted serum samples for PC vs DC and PC vs HV samples using the H50, Q10 and CM10 ProteinChip arrays, whereas for IMAC arrays, more discriminating peaks were seen in non-depleted serum. The highly significant peaks observed on Q10, CM10 and H50 are consistent with the previous finding of ApoA-I (m/z 27,910-28000) and ApoA-II (m/z 8758 and 17,240). In addition, a number of new discriminating protein peaks were found on different ProteinChip arrays, notably peaks at m/z 4280 and 7763 on IMAC arrays. CONCLUSION: This study confirms the diagnostic value of ApoA-I&II and identifies further potential diagnostic biomarkers for pancreatic cancer when multiple chip surfaces are used with depletion of the most highly-abundant proteins.

Cancer-associated stroma reveals prognostic biomarkers and novel insights into the tumour microenvironment of colorectal cancer and colorectal liver metastases.

BACKGROUND AND AIMS: Cancer-associated stroma (CAS) is emerging as a key determinant of metastasis in colorectal cancer (CRC); however, little is known about CAS in colorectal liver metastases (CRLM). This study aimed to validate the prognostic significance of stromal protein biomarkers in primary CRC and CRLM. Secondly, this study aimed to describe the transcriptome of the CAS of CRLM and identify novel targetable pathways of metastasis. METHODS: A case-control study design from a prospectively maintained database was adopted. The prognostic value of epithelial and stromal CALD1, IGFBP7, POSTN, FAP, TGF-ß and pSMAD2 expression was assessed by immunohistochemistry (IHC) in multivariate models. Pathway enrichment and sparse partial least square-discriminant analysis (sPLS-DA) were performed on a nested cohort after isolating epithelial tumour and CAS by laser capture microdissection. RESULTS: 110 CRCs with 124 paired CRLMs, and 110 matched non-metastatic control CRCs were included. Median follow-up was 62 and 45 months for primary and CRLM groups, respectively. Stromal FAP and POSTN were independent predictors for the development of CRLM. After CRLM resection, stromal IGFBP7 and POSTN were predictors of poorer survival. sPLS-DA on the nested cohort identified a number of novel targetable stromal genes and pathways that defined poor prognosis CRC and the CAS of CRLM. CONCLUSIONS: This study is the first to describe key differences in stromal gene expression between paired primary CRC and CRLM as well as identifying several targetable biomarkers and transcriptomic pathways whose relevance specifically in the CAS of CRC and CRLM have not been previously described.

Pancreatic adenocarcinoma preferentially takes up and is suppressed by synthetic nanoparticles carrying apolipoprotein A-II and a lipid gemcitabine prodrug in mice.

We hypothesised that synthetic HDL nanoparticles carrying a gemcitabine prodrug and apolipoprotein A-II (sHDLGemA2) would target scavenger receptor-B1 (SR-B1) to preferentially and safely deliver gemcitabine into pancreatic ductal adenocarcinoma (PDAC). We designed, manufactured and characterised sHDLGemA2 nanoparticles sized ~130 nm, incorporating 20 mol% of a gemcitabine prodrug within the lipid bilayer, which strengthens on adding ApoA-II. We measured their ability to inhibit growth in cell lines and cell-derived and patient-derived murine PDAC xenografts. Fluorescent-labelled sHDLGemA2 delivered gemcitabine inside xenografts. Xenograft levels of active gemcitabine after sHDLGemA2 were similar to levels after high-dose free gemcitabine. Growth inhibition in mice receiving 4.5 mg gemcitabine/kg/d, carried in sHDLGemA2, was equivalent to inhibition after high-dose (75 mg/kg/d) free gemcitabine, and greater than inhibition after low-dose (4.5 mg/kg/d) free gemcitabine. sHDLGemA2 slowed growth in semi-resistant cells and a resistant human xenograft. sHDLGemA2 targeted xenografts more effectively than sHDLGemA1. SR-B1 was over-expressed in PDAC cells and xenografts. Targeting by ApoA-II was suppressed by anti-SR-B1. Because sHDLGemA2 provided only ~6% of the free gemcitabine dose for an equivalent response, patient side effects can be greatly reduced, and the sHDLGemA2 concept should be developed through clinical trials.

Biomimetic Gemcitabine-Lipid Prodrug Nanoparticles for Pancreatic Cancer.

Gemcitabine (Gem) is a key drug for pancreatic cancer, yet limited by high systemic toxicity, low bioavailability and poor pharmacokinetic profiles. To overcome these limitations, Gem prodrug amphiphiles were synthesised with oleyl, linoleyl and phytanyl chains. Self-assembly and lyotropic mesophase behaviour of these amphiphiles were examined using polarised optical microscopy and Synchrotron SAXS (SSAXS). Gem-phytanyl was found to form liquid crystalline inverse cubic mesophase. This prodrug was combined with phospholipids and cholesterol to create biomimetic Gem-lipid prodrug nanoparticles (Gem-LPNP), verified by SSAXS and cryo-TEM to form liposomes. In vitro testing of the Gem-LPNP in several pancreatic cancer cell lines showed lower toxicity than Gem. However, in a cell line-derived pancreatic cancer mouse model Gem-LPNP displayed greater tumour growth inhibition than Gem using a fraction (<6 %) of the clinical dose and without any systemic toxicity. The easy production, improved efficacy and low toxicity of Gem-LPNP represents a promising new nanomedicine for pancreatic cancer.

Parenteral versus enteral nutrition: effect on serum cytokines and the hepatic expression of mRNA of suppressor of cytokine signaling proteins, insulin-like growth factor-1 and the growth hormone receptor in rodent sepsis.

INTRODUCTION: Early nutrition is recommended for patients with sepsis, but data are conflicting regarding the optimum route of delivery. Enteral nutrition (EN), compared with parenteral nutrition (PN), results in poorer achievement of nutritional goals but may be associated with fewer infections. Mechanisms underlying differential effects of the feeding route on patient outcomes are not understood, but probably involve the immune system and the anabolic response to nutrients. We studied the effect of nutrition and the route of delivery of nutrition on cytokine profiles, the growth hormone-insulin-like growth factor-1 (IGF-I) axis and a potential mechanism for immune and anabolic system interaction, the suppressors of cytokine signaling (SOCS), in rodents with and without sepsis. METHODS: Male Sprague-Dawley rats were randomized to laparotomy (Sham) or to cecal ligation and puncture (CLP), with postoperative saline infusion (Starve), with EN or with PN for 72 hours. Serum levels of IL-6 and IL-10 were measured by immunoassay, and hepatic expressions of cytokine-inducible SH2-containing protein, SOCS-2, SOCS-3, IGF-I and the growth hormone receptor (GHR) were measured by real-time quantitative PCR. RESULTS: IL-6 was detectable in all groups, but was only present in all animals receiving CLP-PN. IL-10 was detectable in all but one CLP-PN rat, one CLP-EN rat, approximately 50% of the CLP-Starve rats and no sham-operated rats. Cytokine-inducible SH2-containing protein mRNA was increased in the CLP-EN group compared with the Sham-EN group and the other CLP groups (P < 0.05). SOCS-2 mRNA was decreased in CLP-PN rats compared with Sham-PN rats (P = 0.07). SOCS-3 mRNA was increased with CLP compared with sham operation (P < 0.03). IGF-I mRNA (P < 0.05) and GHR mRNA (P < 0.03) were greater in the fed CLP animals and in the Sham-PN group compared with the starved rats. CONCLUSION: In established sepsis, nutrition and the route of administration of nutrition influences the circulating cytokine patterns and expression of mRNA of SOCS proteins, GHR and IGF-I. The choice of the administration route of nutrition may influence cellular mechanisms that govern the response to hormones and mediators, which further influence the response to nutrients. These findings may be important in the design and analysis of clinical trials of nutritional interventions in sepsis in man.

Classification of pancreatic cystic lesions using SELDI-TOF mass spectrometry.

BACKGROUND: The diagnosis of pancreatic cystic lesions is problematical with difficulties arising in the differentiation between malignant, premalignant or benign lesions. This preliminary study aimed to analyse pancreatic cyst fluid, using a proteomic approach, to generate reproducible protein profiles to assist in the classification of malignant and non-carcinoma samples. METHODS: Pancreatic cyst fluid samples from patients with pancreatic adenocarcinoma and non-carcinoma cystic lesions were analysed on hydrophobic protein chip arrays by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). RESULTS: Differential protein expression profiles were observed between pancreatic adenocarcinoma and non-carcinoma cyst fluid samples using SELDI-TOF MS, with 12 protein peaks differentially expressed between pancreatic adenocarcinoma and non-carcinoma. Additionally, unique patterns were observed between the different subtypes of non-carcinoma samples as well as malignant adenocarcinoma. CONCLUSIONS: In this preliminary study we used SELDI-TOF MS to identify protein expression profiles of pancreatic cyst fluid, showing a potential to aid in the differential diagnosis of pancreatic cystic lesions.

Patient-derived xenograft models of colorectal cancer in pre-clinical research: a systematic review.

AIMS: We sought to objectively assess the internal and external validity of patient-derived xenograft (PDX) models as a platform in pre-clinical research into colorectal cancer (CRC). Metastatic disease is the most common cause of death from CRC, and despite significant research, the results of current combination chemotherapy and targeted therapies have been underwhelming for most of this patient group. One of the key factors limiting the success of translational CRC research is the biologically inaccurate models in which new therapies are developed. METHODS: We used the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) checklist and SYRCLE (Systematic Review Centre for Laboratory animal Experimentation) guidelines to search Ovid MEDLINE and Embase databases up to July 2015 to identify studies involving PDX models of CRC where the model had been validated across multiple parameters. Data was extracted including host mouse strain, engraftment rate, site of engraftment, donor tumour source and development of metastases in the model. RESULTS: Thirteen articles satisfied the inclusion criteria. There was significant heterogeneity amongst the included studies, but overall the median engraftment rate was high (70%) and PDX models faithfully recapitulated the characteristics of their patient tumours on the microscopic, genetic and functional levels. CONCLUSIONS: PDX models of CRC have a reasonable internal validity and a high external validity. Developments in xenografting technology are broadening the applications of the PDX platform. However, the included studies could be improved by standardising reporting standards and closed following the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines.

Upstream and Downstream Co-inhibition of Mitogen-Activated Protein Kinase and PI3K/Akt/mTOR Pathways in Pancreatic Ductal Adenocarcinoma.

BACKGROUND: Extensive cross talk exists between PI3K/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, and both are upregulated in pancreatic ductal adenocarcinoma (PDAC). Our previous study suggested that epidermal growth factor receptor inhibitor erlotinib which acts upstream of these pathways acts synergistically with PI3K inhibitors in PDAC. Horizontal combined blockade upstream and downstream of these two pathways is therefore explored. METHODS: Erlotinib paired with PI3K inhibitor (BYL719) was tested against erlotinib plus dual PI3K/mTOR inhibitor BEZ-235, and MEK inhibitor (PD98059) plus BEZ235, on five primary PDAC cell lines and on two pairs of parent and erlotinib-resistant (ER) cell lines. A range of in vitro assays including cell proliferation, Western blotting, migration, clonogenic, cell cycle, and apopotic assays was used to test for the efficacy of combined blockade. RESULTS: Dual downstream blockade of the MAPK and PAM pathways was more effective in attenuating downstream molecular signals. Synergy was demonstrated for erlotinib and BEZ235 and for PD-98059 and BEZ-235. This resulted in a trend of increased growth cell cycle arrest, apoptosis, cell proliferation, and colony and migration suppression. This combination showed more efficacy in cell lines with acquired resistance to erlotinib. CONCLUSIONS: The additional mTOR blockade provided by BEZ235 in combined blockade resulted in increased anticancer effect. The hypersensitivity of ER cell lines to additional mTOR blockade suggested PAM pathway oncogenic dependence via mTOR. Dual downstream combined blockade of MAPK and PAM pathways with MEK and PI3K/mTOR inhibitor appeared most effective and represents an attractive therapeutic strategy against pancreatic cancer and its associated drug resistance.

Apolipoprotein A-II Plus Lipid Emulsion Enhance Cell Growth via SR-B1 and Target Pancreatic Cancer In Vitro and In Vivo.

BACKGROUND: Apolipoprotein A-II (ApoA-II) is down regulated in the sera of pancreatic ductal adenocarcinoma (PDAC) patients, which may be due to increase utilization of high density lipoprotein (HDL) lipid by pancreatic cancer tissue. This study examined the influence of exogenous ApoA-II on lipid uptake and cell growth in pancreatic cancer (PC) both in vitro and in vivo. METHODS: Cryo transmission electron microscopy (TEM) examined ApoA-II's influence on morphology of SMOFLipid emulsion. The influence of ApoA-II on proliferation of cancer cell lines was determined by incubating them with lipid+/-ApoA-II and anti-SR-B1 antibody. Lipid was labeled with the fluorophore, DiD, to trace lipid uptake by cancer cells in vitro by confocal microscopy and in vivo in PDAC patient derived xenograft tumours (PDXT) by fluorescence imaging. Scavenger receptor class B type-1(SR-B1) expression in PDAC cell lines and in PDAC PDXT was measured by western blotting and immunohistochemistry, respectively. RESULTS: ApoA-II spontaneously converted lipid emulsion into very small unilamellar rHDL like vesicles (rHDL/A-II) and enhanced lipid uptake in PANC-1, CFPAC-1 and primary tumour cells as shown by confocal microscopy. SR-B1 expression was 13.2, 10.6, 3.1 and 2.3 fold higher in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cell lines than the normal pancreatic cell line (HPDE6) and 3.7 fold greater in PDAC tissue than in normal pancreas. ApoA-II plus lipid significantly increased the uptake of labeled lipid and promoted cell growth in PANC-1, MIAPaCa-2, CFPAC-1 and BxPC3 cells which was inhibited by anti SR-B1 antibody. Further, ApoA-II increased the uptake of lipid in xenografts by 3.4 fold. CONCLUSION: Our data suggest that ApoA-II enhance targeting potential of lipid in pancreatic cancer which may have imaging and drug delivery potentialities.

Cotargeting of epidermal growth factor receptor and PI3K overcomes PI3K-Akt oncogenic dependence in pancreatic ductal adenocarcinoma.

PURPOSE: PI3K-Akt is overexpressed in 50% to 70% of pancreatic ductal adenocarcinoma (PDAC). The hypothesis of this study is that PI3K and EGFR coinhibition may be effective in PDAC with upregulated PI3K-Akt signaling. EXPERIMENTAL DESIGN: Multiple inhibitors were tested on five PDAC cell lines. EGFR inhibitor (EGFRi)-resistant cell lines were found to have significantly overexpressed AKT2 gene, total Akt, and pAkt. In vitro erlotinib-resistant (ER) cell models (BxPC-ER and PANC-ER) with highly constitutively active PI3K-Akt were developed. These and their respective parent cell lines were tested for sensitivity to erlotinib, IGFIR inhibitor NVP-AEW541 (AEW), and PI3K-alpha inhibitor NVP-BYL719 (BYL), alone or in combination, by RTK-phosphoarray, Western blotting, immunofluorescence, qRT-PCR, cell proliferation, cell cycle, clonogenic, apoptosis, and migration assays. Erlotinib plus BYL was tested in vivo. RESULTS: Erlotinib acted synergistically with BYL in BxPC-ER (synergy index, SI = 1.71) and PANC-ER (SI = 1.44). Treatment of ER cell lines showing upregulated PI3K-Akt with erlotinib plus BYL caused significant G1 cell-cycle arrest (71%, P < 0.001; 58%, P = 0.003), inhibition of colony formation (69% and 72%, both P < 0.001), and necrosis and apoptosis (75% and 53%, both P < 0.001), more so compared with parent cell lines. In primary patient-derived tumor subrenal capsule (n = 90) and subcutaneous (n = 22) xenografts, erlotinib plus BYL significantly reduced tumor volume (P = 0.005). Strong pEGFR and pAkt immunostaining (2+/3+) was correlated with high and low responses, respectively, to both erlotinib and erlotinib plus BYL. CONCLUSION: PDAC with increased expression of the PI3K-Akt pathway was susceptible to PI3K-EGFR coinhibition, suggesting oncogenic dependence. Erlotinib plus BYL should be considered for a clinical study in PDAC; further evaluation of pEGFR and pAkt expression as potential positive and negative predictive biomarkers is warranted.

Suppression of urokinase plasminogen activator receptor inhibits proliferation and migration of pancreatic adenocarcinoma cells via regulation of ERK/p38 signaling.

Pancreatic ductal adenocarcinoma (PDAC) expresses high levels of urokinase-type plasminogen activator (uPA), its receptor (uPAR) and plasminogen activator inhibitor (PAI)-2, which may play an important role in PDAC progression. The overexpression of uPAR predicted short survival in PDAC patients. In this study, two different PDAC cell lines were used to examine the effect of small interfering (si) RNAs to uPAR, uPA and PAI-2 on proliferation, apoptosis, migration and MAP kinase activation. In both PDAC cell lines, siRNA to uPAR significantly inhibited cell proliferation and migration and stimulated apoptosis, to a greater extent than uPA siRNA. When either PDAC cell line was treated with uPAR siRNA, the level of phosphorylated ERK (p-ERK) decreased substantially, whereas phosphorylated p38 (p-p38) increased when compared to non-silencing control, uPA siRNA or PAI-2 siRNA treatment. This resulted in enhancement of the p-p38/p-ERK ratio which favors cancer cell arrest. Interestingly, uPAR protein expression was suppressed by p-ERK inhibition and stimulated with p-p38 inhibition, suggesting the presence of a positive feedback loop between uPAR and ERK. In summary, our data indicate that, of the uPA system, uPAR exerts the strongest effects on PDAC cells, by acting through the ERK signaling pathway via a positive feedback loop. Disruption of this loop with uPAR siRNA or inhibitor of p-ERK, inhibits PDAC proliferation and migration and promotes apoptosis. These findings suggest that uPAR strongly contributes to PDAC progression and may be considered as a potential anti-pancreatic cancer target.

Prognostic significance of growth factors and the urokinase-type plasminogen activator system in pancreatic ductal adenocarcinoma.

OBJECTIVES: To determine the diagnostic and prognostic significance of growth factors and the urokinase-type plasminogen activator (uPA) system in pancreatic ductal adenocarcinoma (PDAC) using a multigene assay. METHODS: Messenger RNA (mRNA) expression of 15 genes from epidermal growth factor receptor, insulin-like growth factor (IGF), and uPA families were measured in 46 PDAC tissue samples using quantitative real-time reverse transcription-polymerase chain reaction. These results were compared with those of the uninvolved adjacent (AP) tissue and benign mucinous cystadenomas (BMC). The mRNA expression was evaluated using logistic regression and receiver operating characteristic area under the curve (ROC AUC) analyses. Their relationship with prognosis was tested by Cox regression multivariate analysis. RESULTS: All genes were overexpressed in most of the PDAC tissue. When compared with AP tissue, the median expression values for IGF-binding protein 3 (IGFBP-3) and uPA receptor (uPAR) was 9.8- and 9.6-fold, respectively. Expression levels of uPA, uPAR, IGF-I, and IGFBP-3 mRNA were significantly greater in PDAC than in BMC. The IGFBP-3 mRNA expression demonstrated greatest ROC AUC values for PDAC versus AP tissue (ROC AUC, 0.745; 95% confidence interval [CI], 0.65-0.86); whereas ROC AUC values were greatest for uPAR when PDAC was compared with BMC (ROC AUC, 0.846; 95% CI, 0.76-0.94). The combination of uPA, uPAR, and IGF-I significantly improved discriminatory power (ROC AUC, 0.965; 95% CI, 0.93-1.00). The IGFBP-3, uPA, plasminogen activator inhibitor-2, and International Union Against Cancer stage had a significant influence on survival, but the effect of IGFBP-3 was lost after multivariate stepwise analysis. CONCLUSIONS: These results indicate that there is an influence of IGF system in tumor progression from BMC to PDAC, whereas the uPA/uPAR system has the greater influence on survival in PDAC.

High expression of plasminogen activator inhibitor-2 (PAI-2) is a predictor of improved survival in patients with pancreatic adenocarcinoma.

OBJECTIVE: Recent findings suggest that the urokinase-type plasminogen activator (uPA), its receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1), and -2 (PAI-2) play key roles in cancer invasion. The prognostic value of components of this system is well established in breast cancer. However, little is known of its involvement in pancreatic cancer (PC). METHODS: Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used on tissue-banked specimens and immunohistochemistry (IHC) on paraffin specimens was used to measure expression of uPA, uPAR, PAI-1, and PAI-2 proteins in 46 PC and 12 cystadenoma specimens. Results were related to survival using Cox's proportional hazards testing. RESULTS: Increased expression of uPA, uPAR, and PAI-1 in PC tissue were independently associated with a higher Union Internationale Contre le Cancer [International Union Against Cancer (UICC)] tumor stage (P < 0.001) and were intercorrelated (P < 0.001). Overexpression of uPAR indicated reduced survival (P = 0.03). Conversely, PAI-2 messenger ribonucleic acid (mRNA) overexpression, which occurred in 21 of 46 tumors, negatively correlated with tumor size (P = 0.008) and survival (P < 0.007) but not with uPA, uPAR, or tumor stage. There was good agreement between PAI-2 mRNA value and IHC score (P < 0.001). Using Cox's stepwise analysis, PAI-2 mRNA value (HR = 0.24; P = 0.001) and UICC tumor stage (HR = 2.014; P = 0.001) independently predicted survival. An IHC score for PAI-2 of 3+ or 4+ also independently predicted improved survival (HR = 2.72; P = 0.025). CONCLUSIONS: The uPA/uPAR/PAI-1 system is activated in advanced pancreatic cancer and may account for the tumor's aggressive behavior, whereas PAI-2 expression appears to be independent of uPA/uPAR/PAI-1 and is associated with improved prognosis. Because of its intercorrelation with mRNA expression, PAI-2 IHC may be used as an indicator of survival.