Proteomic profiling of cholangiocarcinoma: diagnostic potential of SELDI-TOF MS in malignant bile duct stricture.

Proteomic techniques promise to improve the diagnosis of cholangiocarcinoma (CC) in both tissue and serum as histological diagnosis and existing serum markers exhibit poor sensitivities. We explored the use of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to identify potential protein biomarkers of CC. Twenty-two resected CC samples were compared with adjacent noninvolved bile duct tissue. Serum from patients with CC (n=20) was compared with patients with benign disease (n=20), and healthy volunteers (n=25). Samples were analyzed on hydrophobic protein chips via SELDI-TOF MS, and classification models were developed using logistic regression and cross-validation analysis. Univariate analysis revealed 14 individual peaks differentially expressed between CC and bile duct tissue, 4 peaks between CC and benign disease, and 12 peaks between CC and sera of healthy volunteers. The 4,462 mass-to-charge serum peak had superior discriminatory ability to carbohydrate antigen 19.9 (CA19.9) and carcinoembryonic antigen (CEA) (P=.004; receiver operating characteristic [ROC] area under the curve [AUC]=0.76, 0.73, and 0.70, respectively). The training models developed panels of peaks that distinguished CC from bile duct tissue (92.5% sensitivity, 92.3% specificity; ROC AUC=0.96), CC from benign serum (65.0% sensitivity, 70.0% specificity; ROC AUC=0.83), and CC from sera of healthy volunteers (75.0% sensitivity, 100% specificity; ROC AUC=0.92). Serum results were further improved with the inclusion of CA19.9 and CEA (ROC AUC=0.86 and 0.99 for CC vs benign and healthy volunteer serum, respectively). In conclusion, biomarker panels are capable of distinguishing CC from nonmalignant tissue; serum markers have important diagnostic implications for unknown bile duct stricture.

Proteomic classification of pancreatic adenocarcinoma tissue using protein chip technology.

BACKGROUND & AIMS: Pancreatic adenocarcinoma is a most devastating cancer that presents late and is rapidly progressive. This study aimed to identify unique, tissue-specific protein biomarkers capable of differentiating pancreatic adenocarcinoma (PC) from adjacent uninvolved pancreatic tissue (AP), benign pancreatic disease (B), and nonmalignant tumor tissue (NM). METHODS: Tissue samples representing PC (n = 31), AP (n = 44), and B (n = 19) tissue were analyzed on hydrophobic protein chip arrays by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Training models were developed using logistic regression and validated using the 10-fold cross-validation approach. RESULTS: The hydrophobic protein chip array revealed 13 protein peaks differentially expressed between PC and AP (receiver operating characteristic [ROC] area under the curve [AUC], 0.64-0.85), 8 between PC and B (ROC AUC, 0.67-0.78), and 12 between PC and NM tissue (ROC AUC, 0.63-0.81). Logistic regression and cross-validation identified overlapping panels of peaks to develop a training model that distinguished PC from AP (77.4% sensitivity, 84.1% specificity), B (83.9% sensitivity, 78.9% specificity), and NM tissue (58.1% sensitivity, 90.5% specificity). The final panels selected correctly classified 80.6% of PC and 88.6% of AP samples (ROC AUC, 0.92), 93.5% of PC and 89.5% of B samples (ROC AUC, 0.99), and 71.0% of PC and 92.1% of NM samples (ROC AUC, 0.91). CONCLUSIONS: This study used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry to discover a number of protein panels that can distinguish effectively between pancreatic adenocarcinoma, benign, and adjacent pancreatic tissue. Identification of these proteins will add to our understanding of the biology of pancreatic cancer. Furthermore, these protein panels may have important diagnostic implications.

Assessment of HER-2 status in pancreatic adenocarcinoma: correlation of immunohistochemistry, quantitative real-time RT-PCR, and FISH with aneuploidy and survival.

HER-2 is a transmembrane growth factor receptor recognized in overexpression as an independent adverse prognostic factor in several cancers. This study measured HER-2 overexpression in pancreatic adenocarcinoma at the genetic, transcriptional, and translational level. Expression was gauged with regard to stage, grade, and survival. Pancreatic adenocarcinoma samples (n = 30) were analyzed with immunohistochemical labeling for HER-2 protein, Quantitative real-time reverse transcriptase polymerase chain reaction (Q-RT-PCR) measurement of HER-2 mRNA and fluorescence in situ hybridization (FISH) analysis of HER-2 gene expression. HER-2 expression in benign pancreatic lesions (n = 10) provided a control. Five (17%) of the pancreatic adenocarcinomas scored maximal 3+ immunohistochemistry (IHC) labeling, seven (23%) had significantly increased expression of HER-2 mRNA, while only one (3%) exhibited low level HER-2 gene amplification. Ten (33%) tumors demonstrated aneuploidy. In general, concordance between methodologies was poor, but the best agreement was seen between FISH aneuploidy status and Q-RT-PCR mRNA overexpression (80% agreement), followed by IHC and Q-RT-PCR (73% agreement). The least agreement was seen between IHC and FISH aneuploidy status (67% agreement). Tumor stage was positively associated with HER-2 mRNA and protein expression, but tumor grade and other patient characteristics did not reach statistical significance. A poor survival outcome was demonstrated with positive HER-2 status in all three measures of overexpression (Kaplan-Meier log-rank score; P < 0.01 [IHC], P = 0.05 [Q-RT-PCR], P = 0.02 [FISH]). Discordance in expression at the nuclear, cytoplasmic, and cell surface levels highlights the limitations of immunohistochemical evaluation alone and stresses the need for further evaluation of response to anti-HER-2 targeted therapies in tumors displaying overexpression in gene copy, mRNA, and receptor protein.

Significant overexpression of urokinase-type plasminogen activator in pancreatic adenocarcinoma using real-time quantitative reverse transcription polymerase chain reaction.

BACKGROUND AND AIMS: Overexpression of urokinase-type plasminogen activator (uPA) has been shown to be strongly associated with an increased metastatic potential and poor prognosis in a variety of human malignancies. It was hypothesized that uPA would be overexpressed in highly metastatic pancreatic cancer. The aims of this study were to analyze uPA mRNA expression in pancreatic cancer and to correlate this to the expression of uPA protein and to the stage of the disease. METHODS: Twenty-one pancreatic adenocarcinoma, six ampullary carcinoma and 10 benign mucinous cystadenoma samples, all with adjacent normal tissue, were collected. uPA mRNA was measured using real-time quantitative reverse transcription polymerase chain reaction. Localization of uPA within normal and pancreatic tumor sections was subsequently confirmed using immunohistochemistry. RESULTS: The median and range of the ratios of uPA mRNA measures between tumor tissue and non-involved pancreatic tissue was 17.1 (1.4-653.6) for pancreatic adenocarcinoma (P < 0.001), 3.9 (0.7-7.7) for ampullary carcinoma (P = 0.055) and 1.9 (0.6-5.9) for mucinous cystadenoma tissue (P = 0.052). uPA low tumors were associated with an exuberant stromal reaction, whereas uPA high tumors showed little stromal response. Immunohistochemistry confirmed that uPA protein was more prevalent in pancreatic adenocarcinoma tissue than in normal tissue and that it was membrane-bound. uPA mRNA expression was significantly associated with poorly differentiated pancreatic cancers (P < 0.05) and positively associated with tumor stage. CONCLUSIONS: These observations suggest that significant overexpression of uPA correlates closely to the rapid progression and invasiveness of pancreatic cancer and that uPA may provide a future therapeutic target for pancreatic cancer treatment.

A study of parenteral versus enteral nutrition following caecal ligation and puncture in the rat: Influence on survival and tissue protein turnover.

BACKGROUND & AIMS: Methods of nutritional management in abdominal sepsis remain controversial. METHODS: Sprague Dawley rats were either fed via a central line in the right internal jugular vein or duodenally via a gastrostomy tube, and were randomised to undergo either caecal ligation and puncture (CLP) or laparotomy only. Post-operatively, animals received either parenteral nutrition, enteral nutrition or saline only (parenteral and enteral nutrition protocols were isocaloric and isonitrogenous). After 72 h, fractional rate of protein synthesis (Ks, %/day) was measured in gastrocnemius muscle and liver, and protein breakdown was measured in incubated epitrochlearis muscles. Serum insulin-like growth factor-I (IGF-I), acid-labile subunit (ALS) and IGF binding protein-1 (IGFBP-1) levels were determined by specific radioimmunoassay methods. RESULTS: After CLP, when compared with starved animals, only enteral nutrition resulted in a significant decrease in survival to 72 h (P < 0.001). Parenteral nutrition, but not enteral nutrition, increased muscle (P = 0.02) and liver (P < 0.001) Ks, IGF-I (P < 0.001) and ALS levels (P < 0.001), whereas both parenteral and enteral nutrition reduced IGFBP-1 levels (P < 0.001). Neither enteral nor parenteral nutrition reduced protein breakdown in septic animals. CONCLUSIONS: In this model of severe abdominal sepsis where gut function cannot be assessed, enteral nutrition was associated with increased mortality and was less effective than parenteral nutrition in augmenting muscle and liver protein synthesis.