Proteomic Analysis of Human Bile from Malignant Biliary Stenosis

Dec 5, 2008 - We performed a proteomic analysis of bile samples from patients having a bilary stenosis caused by pancreatic adenocarcinoma. A total of...
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Proteomic Analysis of Human Bile from Malignant Biliary Stenosis Induced by Pancreatic Cancer Annarita Farina,*,† Jean-Marc Dumonceau,‡ Jean-Louis Frossard,‡ Antoine Hadengue,‡ Denis F. Hochstrasser,†,§ and Pierre Lescuyer†,§ Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Faculty of Medicine, Geneva University, Geneva CH-1211, Switzerland, Service of Gastroenterology and Hepatology, Geneva University Hospitals, Geneva CH-1211, Switzerland, and Laboratory Medicine Service, Department of Genetics and Laboratory Medicine, Geneva University Hospitals, Geneva CH-1211, Switzerland Received July 2, 2008

Stenosis of the common bile duct may be either due to benign (chronic pancreatitis) or malignant (cholangiocarcinoma, pancreatic adenocarcinoma) conditions. The benign nature of the stricture should be first confirmed in order to ensure appropriate therapy. Therefore, the identification of markers allowing discrimination between malignant and benign biliary stenosis would be very valuable in clinical practice. To this intent, we performed a proteomic analysis of bile samples from patients having a biliary stenosis caused by pancreatic adenocarcinoma. Bile samples were collected during endoscopic retrograde cholangiopancreatography and purified using different methods. The extracted proteins were then analyzed by SDS-PAGE and LC-MS/MS. A total of 127 proteins were identified, 34 of which have not been previously detected in proteomic studies of bile. Among them, several proteins have been described as potential biomarkers of pancreatic cancer. We extended our investigation by studying the expression of some of these pancreatic cancer markers in bile samples collected from patients with various etiologies of biliary stenosis including pancreatic cancer, cholangiocarcinoma, chronic pancreatitis, as well as gallstone-induced stenosis. Our data showed a conspicuous overexpression of CEACAM6 and MUC1 (CA19-9) in pancreatic cancer and cholangiocarcinoma samples, according to the hypothesis that bile fluid collects cancer-associated protein leaking from the tumor microenvironment. These results underline the interest of using bile as a source of biomarkers for the diagnosis of malignant biliary stenosis. Keywords: bile • pancreatic cancer • biliary stenosis • proteomics • mass spectrometry • biomarkers • CEACAM6 • MUC1 • CA 19-9.

Introduction Stenosis may obstruct the common bile duct, impede the outflow of bile from the liver, and finally cause jaundice. Common bile duct stenosis is most often caused by pancreatic adenocarcinoma, the fourth leading cause of cancer death in the United States.1 Other etiologies include primary bile duct carcinoma (i.e., cholangiocarcinoma) and benign diseases such as chronic pancreatitis and primary sclerosing cholangitis.2,3 Tools that are currently available to differentiate between benign and malignant biliary stenosis, including imaging techniques and pathological examination of endoscopic biliary samples, are plagued by a poor sensitivity and/or specificity.4,5 Resulting diagnostic uncertainties regularly lead to inadequate * To whom correspondence should be addressed: Dr. Annarita Farina, Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Faculty of Medicine, Geneva University, rue Michel Servet 1, Geneva CH-1211. Telephone, +41.22.3795140; fax, +41.22.3795926; e-mail, [email protected]. † Geneva University. ‡ Service of Gastroenterology and Hepatology, Geneva University Hospitals. § Department of Genetics and Laboratory Medicine, Geneva University Hospitals. 10.1021/pr8004925 CCC: $40.75

 2009 American Chemical Society

and potentially harmful disease management. Indeed, 5-10% of duodenopancreatectomies performed for a suspected malignancy reveal benign disease, while this surgical procedure may be associated with a high rate of morbidity and mortality that can reach in referral centers almost 40% and 1-5%, respectively.6,7 Many investigators, over the years, have sought to find accurate diagnostic markers of pancreatic cancer and other malignant causes of bile duct stenosis. Nevertheless, current standard serum markers, including sialylated Lewis antigen 19-9 (CA 19-9), have not proved of any benefit because of insufficient positive predictive value.8-10 More reliable markers, to be of useful diagnostic aid, are urgently needed. One potential source of malignant stenosis biomarkers is represented by bile which is the fluid bathing diseased cells. Proteins secreted/shed by tumoral cells of malignant stenoses should be in much higher concentration in bile than in the serum, and therefore, detection of cancer markers in bile might have a greater sensitivity. Recently, bile investigation using proteomic-based approaches has received substantial interest in cancer biomarker discovery.11 However, up to now, only a few proteomic studies have been performed on bile, probably Journal of Proteome Research 2009, 8, 159–169 159 Published on Web 12/05/2008

research articles because of its high concentration of various substances that can interfere with proteomic techniques, including bile acids, cholesterol, phospholipids, and bile pigments. Overall, three large-scale identifications of biliary proteins were published.12-14 These studies investigated bile samples from gallstones disease or cholangiocarcinoma. In particular, Kristiansen et al.12 identified several known cancer-associated proteins in bile from a patient having a cholangiocarcinoma and showed that some of them could be potential diagnostic markers of biliary tract carcinoma. Since to date no study specifically refers to bile protein composition in the occurrence of biliary stenosis related to pancreatic cancer, we performed a proteomic analysis of human bile from patients with biliary stenosis caused by pancreatic adenocarcinoma. The bile centrifugation pellet, which might contain floating cancer cells, was included in the analysis. This work provided a strong contribution to improve knowledge of the bile protein content with a large proportion of the identified proteins not previously described in bile. Interestingly, several of them are known to be associated with pancreatic cancer. In addition, we provided evidence of cancer biomarkers overexpression in bile by immunoblot analysis of samples from different etiologies of biliary stenosis.

Experimental Section Materials. Unless stated otherwise, all reagents and chemicals were of the highest purity currently available. Water was purified using a MilliQ system (Millipore, Bedford, MA). Glycine, sodium dodecyl sulfate (SDS), ethanol (EtOH), acetic acid (AcH), 2-propanol, 2-butanol, coomassie brilliant blue R-250, bromophenol blue sodium salt (BFB), disodium hydrogen phosphate dehydrate, trifluoroacetic acid (TFA), and Triton X-100 were from Fluka (Buchs, Switzerland). Sodium chloride, 1,4-dithioerythritol (DTE), methanol (MeOH), hydrochloric acid, chloroform (CHCl3), potassium chloride and glycerol were from Merck (Darmstadt, Germany). Acrylamide/piperazine diacrylamide (37.5:1) solution was from Biosolve (Valkenswaard, Netherlands). Ammonium bicarbonate, Trizma base (TRIS), iodoacetamide (IAM), peptide N-glycosidase F (PNGase F), potassium phosphate monobasic, formaldehyde, bovine serum albumin (BSA), Naphthol Blue Black (Amido Black) and porcine trypsin were from Sigma-Aldrich (St. Louis, MO). Ammonium persulfate (APS), 2-mercaptoethanol, N,N,N′,N′tetramethylethylenediamine(TEMED)andunstainedorprestained molecular weight standards (broad range) for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were from Bio-Rad (Hercules, CA). Tween 20 and nonfat dried milk powder were from AppliChem (Darmstadt, Germany). Acetone (AcO) was from Acros organics (Geel, Belgium). Dulbecco’s Phosphate-Buffered Saline (D-PBS) was from Gibco (Grand Island, NY). Sample Collection. Human bile samples were collected during endoscopic retrograde cholangiopancreatography (ERCP) from patients presented with biliary stenosis of different malignant etiologies: pancreatic adenocarcinoma (n ) 6), cholangiocarcinoma (n ) 3) (Table 1). Bile samples from patients having chronic pancreatitis (n ) 2) and gallstones (n ) 2) were also included in the study to be used as nonmalignant controls. A volume of 10-30 mL of bile was collected upstream to the bile duct stenosis before contrast medium injection. Immediately after collection, bile samples were transported on ice, aliquoted and stored at -80 °C until analysis. Two samples from malignant stenosis (pancreatic 160

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Farina et al. a

Table 1. Patients’ Characteristics

b

patient no.b

disease

sex

age

1 2 3 4 5 6 7 8 9 10 11 12 13

Chronic pancreatitis Chronic pancreatitis Pancreatic adenocarcinoma Pancreatic adenocarcinoma Pancreatic adenocarcinoma Pancreatic adenocarcinoma Pancreatic adenocarcinoma Pancreatic adenocarcinoma Gallstones Gallstones Cholangiocarcinoma Cholangiocarcinoma Cholangiocarcinoma

M M M M M F M F F F M M M

50 70 77 61 72 73 52 90 66 54 73 69 77

a Bold lines represent samples subjected to proteomic analysis. Number codes correspond to those of Figures 5 and 6.

adenocarcinoma) were used for proteomic investigation. All samples were subjected to immunoblot validation. Patient informed consent was obtained and the protocol was approved by the Ethical Committee of the Geneva University Hospital. Sample Preparation for Proteomic Analysis. Two samples from patients with pancreatic adenocarcinoma were subjected in parallel to proteomic analysis. Two milliliters of each sample was divided in four aliquots of 500 µL. All aliquots were centrifuged at 16 000g for 10 min at 4 °C. Centrifugation pellets, containing cellular debris, were washed twice in D-PBS, dissolved in SDS 10%, and then stored at -20 °C for further analysis. Each supernatant was treated with 125 µL of the solidphase, nonionic adsorbent, Cleanascite (Biotech Support Group, North Brunswick, NJ) and then ultrafiltrated onto a prerinsed YM-3 centricon filter unit (Millipore, Bedford, MA) as previously described.12 For both bile samples, one of the delipided/ desalted (d/d-bile) aliquots was stored at -20 °C until proteomic analysis. The three other ones were subjected to one of the following purification steps: (i) AcO/EtOH precipitation; (ii) MeOH/CHCl3 extraction; (iii) albumin/IgG immunodepletion (Figure 1). Protein precipitation was performed by adding 9 vol of ice-cold AcO/EtOH (1:1) solution to one of the d/dbile aliquots. After overnight incubation at -20 °C, the sample was centrifuged at 15 000g for 15 min at 4 °C, the supernatant was discarded and the pellet was dissolved in SDS 10% and stored at -20 °C. Protein extraction was performed by adding 1 vol of ice-cold MeOH/CHCl3 (1:2) solution to a d/d-bile aliquot. The mixture was gently shaken and maintained at 0 °C on melting ice for 30 min. Centrifugation at 7000g for 5 min at 4 °C separated the mixture into three phases. The lower organic phase was discarded, while the upper aqueous phase and the insoluble interphase were mixed and evaporated to dryness by vacuum centrifugation. The pellet was dissolved in SDS 10% and stored at -20 °C. Albumin and IgG immunodepletion of a d/d-bile aliquot was performed using ProteoPrep Immunoaffinity Albumin & IgG Depletion Kit (SigmaAldrich, St. Louis, MO), according to manufacturer’s instructions. Proteins were quantified using Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Gel-Electrophoresis and In-gel Enzymatic Digestion. Protein samples (50 µg) were fractionated by SDS-PAGE15 on 12.5%T, 2.6%C polyacrylamide gels (8 × 8 cm). Gel lanes were sliced into 30-50 pieces depending on the complexity of the sample and destained by incubation in 100 µL of 50 mM ammonium bicarbonate/30% AcN for 15 min at room temper-

Proteomic Analysis of Bile from Malignant Biliary Stenosis

Figure 1. Purification workflow. Bile from two patients with pancreatic adenocarcinoma was centrifuged and the resulting supernatant was subjected to lipid removal followed by a 3 kDa ultrafiltration. A delipided/desalted bile (d/d-bile) fraction was kept for further analysis. Other fractions were purified using one of three techniques: AcO/EtOH precipitation, MeOH/CHCl3 extraction or albumin/IgG immunodepletion. Purified fractions were then separated by SDS-PAGE, together with d/d-bile and pellets obtained from the first centrifugation. After migration, entire lanes were excised into slices and then subjected to in-gel digestion with PNGase F and trypsin. Deglycosylated tryptic peptides were finally analyzed by reverse-phase LC-MS/MS.

ature. Destaining solution was removed and fragments were then incubated for 35 min at 56 °C in 100 µL of 10 mM DTE in 50 mM ammonium bicarbonate. The DTE solution was then replaced by 100 µL of 55 mM IAM in 50 mM ammonium bicarbonate and gel fragments were incubated for 45 min at room temperature in the dark. Gel pieces were then washed twice for 10 min with 100 µL of 50 mM ammonium bicarbonate and for 10 min with 100 µL of 50 mM ammonium bicarbonate/ 30% AcN. Gel pieces were then dried for 30 min in a Concentrator 5301 vacuum centrifuge (Eppendorf AG, Hamburg, Germany). Dried pieces of gel were rehydrated for 30 min at 37 °C in 20 µL of a 0.025 U/µL of PNGase F solution. After further addition of 25 µL of milliQ water, gel pieces were incubated overnight at 37 °C. Tubes were briefly centrifuged and digested glycans were extracted four times by 30 min sonication in 200 µL of milliQ water (maintained below 30 °C). Gel pieces were then dried for 45 min in a vacuum centrifuge. Dried pieces of gel were then rehydrated for 45 min at 0 °C (on melting ice) in 20 µL of a 50 mM ammonium bicarbonate

research articles solution containing trypsin at 6.25 ng/µL. After overnight incubation at 37 °C, tubes were briefly centrifuged. Extraction of tryptic peptides was performed by 15 min sonication in 25 µL of 1% TFA at room temperature. The acidic supernatant containing peptides was transferred to a polypropylene tube. A second extraction was performed by 15 min sonication in 25 µL of 0.1% TFA/50% AcN at room temperature. The second acidic supernatant was pooled with the first one. The volume of pooled extracts was reduced to 5-10 µL under vacuum, diluted twice with 35 µL of 0.1% TFA, and then completely evaporated. Positive and negative extraction controls consisted of pieces of gels corresponding to a molecular weight marker or devoid of proteins, respectively. Liquid Chromatography-Tandem Mass Spectrometry. Peptides extracted following in-gel digestion were dissolved in 7 µL of 5% AcN/0.1% formic acid; 5 µL of sample was loaded for liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis. A precolumn (100 µm inner diameter, 3.0-3.5 cm long) was connected directly to an analytical column (75 µm inner diameter, 9-10 cm long). Both columns were packed in-house with 5 µm, 200 Å Magic C-18 (Michrom, Auburn, CA). A gradient from 4 to 56% solvent B in solvent A (Solvent A, 5% AcN/0.1% formic acid; Solvent B, 80% AcN/0.1% formic acid) was developed over 15 min at a flow rate of approximately 300 nL/min. The concentration of solvent B was increased up to 95% before returning to start conditions for re-equilibration of the column. The eluate was sprayed directly into the electrospray ionization (ESI) source of a quadrupole-ion-trap (LCQ) DecaXP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) with a spray voltage of 1.8-2.2 kV. Datadependent acquisition was used to automatically select 2 precursors for MS/MS from each MS spectrum (m/z range 400-1600). MS/MS spectra were acquired with a normalized collision energy of 35%, an activation Q of 0.25 and an isolation width of 4 m/z. The activation time was 30 ms. Dynamic exclusion was applied with a repeat count of 2, an exclusion time of 30 s, and an exclusion peak width of (1.5 Da. Wideband activation was also applied. Maximum injection times of 50 and 200 ms were used for MS and MS/MS acquisitions, respectively, and the corresponding automatic gain control targets were set to 108. Data Extraction and Database Interrogation. Peak lists were generated using Bioworks 3.1 software (Thermo Finnigan, San Jose, CA). The resulting .dta files from each analysis were automatically combined into a single text file. The resulting peak lists were searched against the UniProt combined SwissProt and TrEMBL database restricted to human entries using Mascot operating on a local server (Version: 2.2.03, Matrix Sciences, U.K.) and Phenyx Virtual Desktop (Version: 2.5, Gene Bio, Switzerland). Mascot was used with average mass selected, a precursor mass error of 2.0 Da and a peptide mass error of 1.0 Da. Trypsin was selected as the enzyme, with a single potential missed cleavage. ESI ion trap was selected as the instrument type, carbamidomethylated cysteine as fixed modifications and oxidized methionine and deamidated asparagine as a variable modification. For Phenyx, ESI ion trap was selected for the instrument type and LCQ for the algorithm. One search round was used, with trypsin selected as the enzyme, carbamidomethylated cysteine as fixed modification and oxidized methionine and deamidated asparagine as variable modifications. Database searching included deamidated asparagine because of the conversion of asparagine to aspartic acid where PNGase F has removed a glycan. One missed cleavage was Journal of Proteome Research • Vol. 8, No. 1, 2009 161

research articles allowed and the normal cleavage mode was used. The round was selected in “turbo” search mode. The minimum peptide length allowed was 6 amino acids and the parent ion tolerance was 2.0 Da. The acceptance criteria were AC score 7.0, peptide Z-score 7.0, peptide p value 1.0 × 10-6. Proteins that were identified as human proteins with 2 or more high-scoring peptides from Mascot and Phenyx were accepted to be true matches. “High-scoring peptides” corresponded to peptides that were above the threshold in Mascot searches (5% probability of false match for each peptide above this score) and above a peptide score of 7.0 for Phenyx searches using the LCQ scoring algorithm. Data obtained from all analyzed samples were merged to maximize the bile protein content coverage (Supplemental Table 1 in Supporting Information). Contribution of Bile Pretreatments to Protein Identification. To evaluate the benefit of the different purification workflows, we assigned identified proteins to a fractionation method on the basis of the following criteria (Table 2, column 6). Proteins containing at least two unique peptides that were identified in more than one sample were reported as detected in the sample with highest number of peptides. If the same number of peptides matched two or more fractions, the highest score one was selected. In rare cases, when all parameters corresponded, or scores were from two different softwares, the protein was attributed to the sample subjected to fewer purification steps. Gene Ontology Analysis. The classification of newly identified proteins was performed according to the Gene Ontology (GO) hierarchy using the EBI’s Sequence Retrieval System (SRS) (http://srs.ebi.ac.uk/ accessed on October 9, 2008) for GOID retrieving and the GO Terms Classifications Counter (http:// www.animalgenome.org/bioinfo/tools/catego/ accessed on October 9, 2008) for clusterization using the GO_Slim classification method. The gene ontology file was downloaded from the gene ontology database (http://www.animalgenome.org/bioinfo/ tools/catego/.goslim/GO_slim accessed on May 19, 2008) and was manually edited to obtain two separate classifications for cellular localization. Western Blot. All samples, from both (malignant and nonmalignant) etiologies, were subjected to immunoblot analysis. A volume of 0.75 µL of crude bile from each sample was used for SDS-PAGE. Proteins were transferred onto 0.2 µm pore size nitrocellulose membrane Protran BA83 (PerkinElmer Life And Analytical Sciences, Waltham, MA) for 2 h in Towbin buffer (12.4 mM TRIS/100 mM glycine/10% EtOH) at 100 V. Protein transfer was confirmed by Amido Black staining. The membrane was blocked for 60 min with 5% nonfat dried milk powder in phosphate-buffered saline supplemented with 0.05% Tween-20 (PBS-T). The membrane was incubated overnight at room temperature with primary antibody diluted in 1% milkPBS-T. After washing with 1% milk/PBS-T, the blot was incubated 60 min at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody diluted in 1% milk/PBS-T. Finally, the blot was washed with PBS-T and immunogenic proteins were visualized using the BM Chemiluminescence Blotting Substrate (POD) (Roche, Mannheim, Germany). Primary antibodies were used at the following dilutions: mouse monoclonal anti- carcinoembrionic antigen cell adhesion molecule 6 (CEACAM6) (Abcam, Cambridge, U.K.) 1:1000; mouse monoclonal anti-mucin 1 (MUC1) carbohydrate antigen 19-9 (CA 19-9) (AnaSpec, San Jose, CA) 1:800; rabbit polyclonal anti-annexin IV (ANXIV) (Santa Cruz Biotechnology) 162

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Farina et al. 1:200; mouse monoclonal anti-osteopontin (OPN) (Abcam) 1:1000; mouse monoclonal anti-matrylisin (MMP7) (Abcam) 1:200. HRP-conjugated goat anti-mouse and goat anti-rabbit immunoglobulins (Dako, Glostrup, Denmark) were used at a dilution of 1:2000. Chemiluminescent signal areas were compared by using ImageQuant TL software v2005 (1.1.0.1) (Nonlinear Dynamics, Durham, NC).

Results Sample Preparation and Fractionation for Proteomic Analysis. In addition to desalting and delipidation steps, we tested a series of additional purification protocols (Figure 1). Improved bands resolution was obtained by adding MeOH/ CHCl3 extraction or AcO/EtOH precipitation to the basic workflow (Figure 2, Lanes 2 and 3, respectively). Other fractionation methods, such as albumin/IgG immunodepletion (Figure 2, Lane 4), still suffered the effect of migration/staining interferences, mostly at the bottom of the gel, in correspondence of low molecular weight (6-20 kDa) proteins. Identification of Bile Proteins by One-Dimensional Gel Electrophoresis and LC-MS/MS. Merged data from LC-MS/ MS analysis of deglycosylated tryptic peptides obtained from in situ digestion of sliced gel lanes from the two analyzed samples are detailed in the Supplemental Table 1 in Supporting Information. A total of 127 proteins were identified on the basis of more than one unique tryptic peptide (Table 2). It is noteworthy that immunoglobulins and keratins were not included in the list presented in Table 2. In addition, 54 singlehit proteins were identified but they were not considered for further data analyses (Supplemental Table 2 in Supporting Information). Comparison of our data with previous results from Kristiansen et al.,12 Zhou et al.,13 and Guerrier et al.14 indicated that, among the 127 proteins identified in the present work, 34 (27%) represented new identifications in bile. However, comparison of our results with previously published data was biased by the fact that reported lists from previous proteomic studies on bile include proteins identified with a single peptide.12-14 Contribution of Bile Pretreatments to Protein Identification. The majority (43.3%) of identified proteins, assigned on the basis of criteria described in the Experimental Section, originated from the analysis of centrifugation pellet fractions. Albumin/IgG depleted samples and untreated (d/d-bile) contributed to the list with 31.5% and 24.4% of total proteins, respectively. Finally, only a minimal part of the assigned proteins (0.8%) were from the MeOH/CHCl3 purified specimens, and no identification was attributed to the AcO/EtOH precipitation fractions (Figure 3A). The contribution of the different workflows to the identification of new proteins was also evaluated. These data showed that centrifugation pellet and d/d-bile were the main contributors, providing 19 (55.9%) and 11 (32.4%) newly identified proteins, respectively. Immunodepleted samples contributed only for 3 (8.8%) new identifications, while 1 newly identified protein (2.9%) was from MeOH/CHCl3 extraction (Figure 3B). Gene Ontology Classification. The subcellular distribution of newly identified proteins was annotated according to the GO hierarchy. Intracellular proteins represented the most abundant population (82.9%) (Figure 4A). Among them, 33.9% of protein annotations were recognized as belonging to cytoplasm, 18.9% to nucleus and 15.0% to plasma membrane. Other minor fractions were identified as chromosomes (11.8%),

research articles

Proteomic Analysis of Bile from Malignant Biliary Stenosis a

Table 2. List of Multiple-Hit Proteins Identified from Bile Samples no.

AC

ID

name

no. peptides

fractionb

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

A0M8Q4 A0M8Q9 A2BDF6 P15144 P07355 P09525 P08758 P04114 P02749 P04003 O15484 P20160 P00751 P08603 P10909 P01034 P27487 P12724 EF1A2 P02671 P02675 P02679 P02751 P19440 Q93077 Q8IUE6 P62807 P69905 P68871 P02042 P04196 P02545 P61626 P09237 P05164 P01833 P00747 O43490 Q0VAS5 Q6FHB6 Q8IX02 O00391 P09651 P22626 P04843

A0M8Q4_HUMAN A0M8Q9_HUMAN A2BDF6_HUMAN AMPN_HUMAN ANXA2_HUMAN ANXA4_HUMAN ANXA5_HUMAN APOB_HUMAN APOH_HUMAN C4BP_HUMAN CAN5_HUMAN CAP7_HUMAN CFAB_HUMAN CFAH_HUMAN CLUS_HUMAN CYTC_HUMAN DPP4_HUMAN ECP_HUMAN EF1A2_HUMAN FIBA_HUMAN FIBB_HUMAN FIBG_HUMAN FINC_HUMAN GGT1_HUMAN H2A1B_HUMAN H2A2B_HUMAN H2B1C_HUMAN HBA_HUMAN HBB_HUMAN HBD_HUMAN HRG_HUMAN LMNA_HUMAN LYSC_HUMAN MMP7_HUMAN PERM_HUMAN PIGR_HUMAN PLMN_HUMAN PROM1_HUMAN Q0VAS5_HUMAN Q6FHB6_HUMAN Q8IX02_HUMAN QSOX1_HUMAN ROA1_HUMAN ROA2_HUMAN RPN1_HUMAN

4 4 2 34 20 11 3 4 9 5 2 2 3 10 3 2 10 2 2 5 8 6 13 3 3 2 4 9 12 11 7 11 5 5 2 20 3 2 4 11 2 2 2 2 2

cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp cp

46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83

P06702 P60903 P02743 P27105 P24821 P01033 P16035 P62988 P21796 P04004 P63104 P60709 P63261 P02768 P02647 P02649 P61769 P00915 P00918 P00450 P02741 P02792 P04406 P06396 P00738 Q9Y624 P01042 P07195 P10451 Q9BYE9 P30086 P00558 P62937 P32119 Q49AS2 Q4VJB6 Q5T8M8 Q6PYX1

S10A9_HUMAN S10AA_HUMAN SAMP_HUMAN STOM_HUMAN TENA_HUMAN TIMP1_HUMAN TIMP2_HUMAN UBIQ_HUMAN VDAC1_HUMAN VTNC_HUMAN 1433Z_HUMAN ACTB_HUMAN ACTG_HUMAN ALBU_HUMAN APOA1_HUMAN APOE_HUMAN B2MG_HUMAN CAH1_HUMAN CAH2_HUMAN CERU_HUMAN CRP_HUMAN FRIL_HUMAN G3P_HUMAN GELS_HUMAN HPT_HUMAN JAM1_HUMAN KNG1_HUMAN LDHB_HUMAN OSTP_HUMAN PCDLK_HUMAN PEBP1_HUMAN PGK1_HUMAN PPIA_HUMAN PRDX2_HUMAN Q49AS2_HUMAN Q4VJB6_HUMAN Q5T8M8_HUMAN Q6PYX1_HUMAN

C3 protein (C2 segment protein) C1 segment protein (Fragment) Histone cluster 2, H3a Aminopeptidase N Annexin A2 (Annexin-2) Annexin A4 Annexin A5 Apolipoprotein B-100 Beta-2-glycoprotein 1 C4b-binding protein alpha chain Calpain-5. Azurocidin Complement factor B Complement factor H Clusterin Cystatin-C Dipeptidyl peptidase 4 Eosinophil cationic protein Elongation factor 1-alpha 2 Fibrinogen alpha chain Fibrinogen beta chain Fibrinogen gamma chain Fibronectin Gamma-glutamyltranspeptidase 1 Histone H2A type 1-C Histone H2A type 2-B Histone H2B type 1-C/E/F/G/I Hemoglobin subunit alpha Hemoglobin subunit beta Hemoglobin subunit delta Histidine-rich glycoprotein Lamin-A/C Lysozyme C Matrilysin Myeloperoxidase Polymeric-immunoglobulin receptor Plasminogen Prominin-1 Histone H4 ANXA13 protein Lactoferrin Sulfhydryl oxidase 1 Heterogeneous nuclear ribonucleoprotein A1 Heterogeneous nuclear ribonucleoproteins A2/B1 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase 67 kDa subunit Protein S100-A9 Protein S100-A10 Serum amyloid P-component Erythrocyte band 7 integral membrane protein Tenascin Metalloproteinase inhibitor 1 Metalloproteinase inhibitor 2 Ubiquitin Voltage-dependent anion-selective channel protein 1 Vitronectin 14-3-3 protein zeta/delta Actin, cytoplasmic 1 Actin, cytoplasmic 2 Serum albumin Apolipoprotein A-I Apolipoprotein E Beta-2-microglobulin Carbonic anhydrase 1 Carbonic anhydrase 2 Ceruloplasmin C-reactive protein Ferritin light chain Glyceraldehyde-3-phosphate dehydrogenase Gelsolin Haptoglobin Junctional adhesion molecule A Kininogen-1 L-lactate dehydrogenase B chain Osteopontin Protocadherin LKC Phosphatidylethanolamine-binding protein Phosphoglycerate kinase 1 Peptidyl-prolyl cis-trans isomerase A Peroxiredoxin-2 ELK2, member of ETS oncogene family, pseudogene 1 14-3-3 protein epsilon isoform transcript variant 1 Actin, alpha 1, skeletal muscle Hepatitis B virus receptor binding protein

2 2 8 4 5 3 2 2 4 2 2 14 14 39 6 10 2 9 7 5 2 2 7 2 5 3 3 2 2 9 2 2 3 3 3 4 8 6

cp cp cp cp cp cp cp cp cp cp d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d d/d

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Table 2. Continued no.

AC

ID

name

no. peptides

fractionb

84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127

Q8IZI0 P02753 P02766 P02763 P19652 P01009 P04217 A2BHY4 P01023 P01011 P17174 Q9BYF1 O60218 P02760 P01019 P01008 P05090 O75882 P07339 P13688 P40199 P01024 094760 P23141 Q9Y6R7 Q9HC38 P09211 P02790 P05155 P05154 Q08380 P40925 P15941 Q9HC84 P80188 Q16651 Q13228 P04179 P60174 P02787 O95497 P02774 P25311 Q9NP55

Q8IZI0_HUMAN RETBP_HUMAN TTHY_HUMAN A1AG1_HUMAN A1AG2_HUMAN A1AT_HUMAN A1BG_HUMAN A2BHY4_HUMAN A2MG_HUMAN AACT_HUMAN AATC_HUMAN ACE2_HUMAN AK1BA_HUMAN AMBP_HUMAN ANGT_HUMAN ANT3_HUMAN APOD_HUMAN ATRN_HUMAN CATD_HUMAN CEAM1_HUMAN CEAM6_HUMAN CO3_HUMAN DDAH1_HUMAN EST1_HUMAN FCGBP_HUMAN GLOD4_HUMAN GSTP1_HUMAN HEMO_HUMAN IC1_HUMAN IPSP_HUMAN LG3BP_HUMAN MDHC_HUMAN MUC1_HUMAN MUC5B_HUMAN NGAL_HUMAN PRSS8_HUMAN SBP1_HUMAN SODM_HUMAN TPIS_HUMAN TRFE_HUMAN VNN1_HUMAN VTDB_HUMAN ZA2G_HUMAN PLUNC_HUMAN

Hemoglobin beta chain variant Hb-I_Toulouse Plasma retinol-binding protein Transthyretin Alpha-1-acid glycoprotein 1 Alpha-1-acid glycoprotein 2 Alpha-1-antitrypsin Alpha-1B-glycoprotein Complement component C4B Alpha-2-macroglobulin Alpha-1-antichymotrypsin Aspartate aminotransferase, cytoplasmic Angiotensin-converting enzyme 2 Aldo-keto reductase family 1 member B10 AMBP protein (Contains: Alpha-1-microglobulin) Angiotensinogen Antithrombin-III Apolipoprotein D Attractin Cathepsin D Carcinoembryonic antigen-related cell adhesion molecule 1 Carcinoembryonic antigen-related cell adhesion molecule 6 Complement C3 N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 Liver carboxylesterase 1 IgGFc-binding protein Glyoxalase domain-containing protein 4 Glutathione S-transferase P Hemopexin Plasma protease C1 inhibitor Plasma serine protease inhibitor Galectin-3-binding protein Malate dehydrogenase, cytoplasmic Mucin-1 Mucin-5B Neutrophil gelatinase- associated lipocalin Prostasin Selenium-binding protein 1 Superoxide dismutase [Mn], mitochondrial Triosephosphate isomerase Serotransferrin Pantetheinase Vitamin D-binding protein Zinc-alpha-2-glycoprotein Protein Plunc

7 4 7 3 3 13 4 15 13 6 4 2 10 5 3 4 2 4 2 2 3 16 2 3 6 2 4 6 12 5 7 3 3 31 8 2 5 3 2 8 4 4 5 5

d/d d/d d/d d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+id d/d+m/c

a Bold lines represent proteins not described in previous proteomics studies of bile. b Cp, pellet obtained by centrifugation of crude bile; d/d, delipided/ desalted bile; d/d+id, delipided/desalted bile treated for albumin/IgG immunodepletion; d/d+m/c, delipided/desalted bile subjected to MeOH/CHCl3 extraction.

mitochondria (7.9%), endoplasmic reticulum (5.5%), cytoskeleton (4.7%) and Golgi apparatus (2.4%) (Figure 4B). Immunoblotting Analysis. Several proteins previously described as being associated with pancreatic cancer (Table 3) were further investigated using immunoblots. Crude bile samples from malignant and nonmalignant conditions were probed using specific antibodies against CEACAM6 and MUC1 (CA 19-9) (Figure 5A). The concentration of CEACAM6 was higher in malignant compared to nonmalignant samples, as evidenced by the strong signal apparent in 8 of the 9 samples from patients having cancer (pancreatic adenocarcinoma and cholangiocarcinoma). The last pancreatic cancer sample was also positive but with a fainter signal. Among nonmalignant controls, a single one from a patient with chronic pancreatitis showed a feeble signal similar to the lowest signal from the cancer series (Figure 6). Antibodies against MUC1 (CA 19-9) showed a lower sensitivity for malignancy detection (7/9 positive samples) but a higher specificity since no signal was observed in the nonmalignant samples. For both proteins, comparison between untreated bile and the two centrifugation fractions (supernatant and pellet) showed that the positive signal was observed in the supernatant only (Figure 5B). Other cancer-associated proteins identified in bile samples, ANX IV, OPN and MMP7, disclosed no differential expression between 164

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Figure 2. Coomassie-stained gel of bile fractions. Fifty micrograms of each bile fraction, obtained as described in Figure 1, was loaded onto a homemade 12.5%T polyacrylamide gel (8 × 8 cm) for separation. Proteins were stained with Coomassie Brilliant Blue. Lane M, molecular weight markers; lane 1, delipided/desalted-bile (d/d-bile); lane 2, d/d-bile after AcO/EtOH precipitation; lane 3, d/d-bile after MeOH/CHCl3 extraction; lane 4, d/d-bile after albumin/IgG immunodepletion; lane 5, pellet from centrifugation of crude bile.

Proteomic Analysis of Bile from Malignant Biliary Stenosis

Figure 3. Contribution of the different bile pretreatments to protein identification. (A) Pie chart showing the relative contribution of each analyzed bile fraction to the identification of all multiple-hit proteins identified in this study (n ) 127). (B) Pie chart showing the relative contribution of each analyzed bile fraction to the identification of proteins that had not been described in previous large-scale proteomic studies of human bile (n ) 34). d/d, delipided/desalted bile; d/d+a/e, delipided/desalted bile after AcO/EtOH precipitation; d/d+m/c, delipided/desalted bile after MeOH/CHCl3 extraction; d/d+id, delipided/desalted bile after albumin/IgG immunodepletion; cp, centrifugation pellet obtained from crude bile.

cancer and noncancer samples (Supplemental Figure 1 in Supporting Information).

Discussion Contribution to the Repertoire of Human Bile Proteins. The proteomic analysis of bile from patients with malignant stenosis caused by pancreatic adenocarcinoma allowed the identification of a total of 127 proteins with at least two unique tryptic peptides. Of them, 34 were described in bile for the first time. This result is likely attributable to several novelties in our approach. First, we analyzed for the first time bile samples collected from patients suffering from pancreatic cancer. Second, in contrast to previous studies, we included in the analysis the pellet fraction from crude bile centrifugation. Indeed, we assumed that this fraction could contain floating cancer cells representing a potential source of biomarkers.4,5 In addition, several sample preparation strategies were used to optimize the bile proteins purification workflow. This fluid, in fact, is known to contain high concentrations of hydrophobic molecules and salts, which may interfere with protein identification. Previous studies established the effectiveness of lipid and salts removal in enhancing protein recovery from bile, but several contaminants still remain after such purifications. To circumvent interferences and enrich for presence of lowabundance and hydrophobic proteins, we used a combination of different purification methods: AcO/EtOH precipitation,

research articles MeOH/CHCl3 extraction and albumin/IgG immunodepletion. However, the contribution of AcO/EtOH precipitation and MeOH/CHCl3 extraction was limited both in term of total protein identifications and in term of newly identified proteins in bile. A significant part of protein identifications was attributed to immunodepleted samples, but in contrast, this protocol allowed the detection of only a small percentage of new proteins. Interestingly, most of the newly identified proteins were detected in pellet fractions, likely because centrifugation pellet was not included in previous proteomic analyses of bile. Finally, a significant series of new identifications came from bile that was only delipided and desalted. This could be attributable to intrinsic differences between patients and/or sampling techniques. In fact, bile samples subjected to proteomic analysis in the present study were collected close to the stricture using ERCP in patients with pancreatic adenocarcinoma. In comparison, patients with gallstones or cholangiocarcinoma were analyzed in previous studies, with bile collected from the gallbladder during cholecystectomy in two studies. The GO classification showed that intracellular proteins represent the uppermost population in newly identified proteins, including membrane and nuclear proteins identified from the pellet. The main explanation is the inclusion in the analytical workflow of the centrifugation pellet, which is likely to contain cells shed by the bile duct walls, including potential tumor cells. However, the presence of intracellular proteins in our bile samples could also be explained by ERCP itself or by pancreatic adenocarcinoma. It is indeed possible that the endoscope caused microlesions during its passage into the bile duct, thereby inducing the release in bile of blood and tissue proteins. In addition, inflammation and necrosis related to pancreatic cancer could lead to the release of tissue leakage markers in bile. Discussion of Several Cancer-Associated Proteins Newly Identified in Bile. Bibliographic searches of all identified proteins revealed that a number of them had previously been described as cancer-associated proteins (Table 3). Here, we discuss a restricted part of those directly involved in studies of pancreatic cancer; namely, CEACAM6, MMP 7, MUC1, and OPN. CEACAM6 is a membrane receptor linked to glycosylphosphatidylinositol (GPI) showing aberrant expression in various types of cancer,16-20 including pancreatic adenocarcinomas.21,22 Particularly, the correlation between CEACAM6 overexpression and the modulation of pancreatic adenocarcinoma cellular invasiveness was clearly demonstrated.21 MMP7 belongs to the class of matrix metalloproteinases that are zinccontaining enzymes showing the common ability to degrade various components of the extracellular matrix. Both in vitro and in vivo investigations have shown that increased MMPs levels are associated with invasive and metastatic potentials in several malignant tumors, such as renal cell carcinoma.23 Moreover, Kuhlmann et al.24 showed that plasma MMP7 levels discriminated between patients with pancreatic carcinoma and those with chronic pancreatitis or other benign diseases. MUC1 is a membrane protein, highly expressed in tumor cells of the digestive tract, which carries the CA 19-9 antigen (2,6 sialosylfucosyl-lactotetraose) in association with cancer development. In the last two decades, a number of reports addressed the role of CA 19-9 in the diagnosis and/or prognosis of pancreatic cancer.25-28 Nowadays tests based on monoclonal antibodies raised against cancer antigen 19-9 are widely used in clinical practice. OPN is a phosphoprotein secreted by T-lymphocytes, Journal of Proteome Research • Vol. 8, No. 1, 2009 165

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Figure 4. Gene ontology classification. Pie charts showing the classification of newly identified bile proteins (n ) 34) according to their localization (A) and intracellular distribution (B).

Figure 5. Immunoblot analysis of CEACAM6 and MUC1 (CA 19-9). A volume of 0.75 µL of crude bile from each specimen was loaded onto a homemade 12.5%T polyacrylamide gel for separation. Proteins were electroblotted onto a nitrocellulose membrane. Immunodetection was performed using either a mouse monoclonal anti-CEACAM6 or a mouse monoclonal anti-CA 19-9 antibody. (A) Lanes 1 and 2, bile from patients with chronic pancreatitis; lanes 3-8, bile from patients with pancreatic adenocarcinoma; lanes 9 and 10, bile from patients with gallstones; lanes 11-13, bile from patients with cholangiocarcinoma. (B) Lane 5b, 5p, 5s, respectively: crude bile, centrifugation pellet and supernatant from a patient with pancreatic adenocarcinoma (corresponding to lane 5 of section A). Patient numbers correspond to those of Table 1.

Figure 6. CEACAM6 immunoblot signal areas. Chart showing the CEACAM6 immunoblot signal areas comparison for bile samples from chronic pancreatitis, pancreatic adenocarcinoma, gallstones and cholangiocarcinoma. Patient numbers correspond to those of Table 1 and Figure 5.

epidermal cells, bone cells, macrophages, and endothelial cells. It has been implicated as an important mediator of tumor metastasis and investigated for use as a biomarker or potential therapeutic target. High levels of OPN expression correlate with tumor invasion, progression or metastasis in breast,29,30 gastric,31,32 lung,33,34 prostate,35,36 and colon cancer.37,38 Concerning pancreatic cancer, Kolb et al.39 have shown that recombinant human OPN significantly increased the invasive166

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ness of pancreatic cancer cells and that its downregulation by specific small interference RNA (siRNA) molecules decreased pancreatic cancer cell invasion. These examples strongly support the use of bile as a useful source for the identification candidate biomarkers for malignant biliary stenosis. Differential Expression in Bile of Cancer Biomarkers Detected by Proteomic Analysis. To assess the ability to measure differences in the concentration of cancer biomarkers

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Proteomic Analysis of Bile from Malignant Biliary Stenosis Table 3. List of Proteins Described in the Literature As Associated with Cancera ID

name

ANXA2_HUMAN ANXA4_HUMAN DPP4_HUMAN ECP_HUMAN

Annexin A2 (Annexin-2) Annexin A4 Dipeptidyl peptidase 4 Eosinophil cationic protein Elongation factor 1-alpha 2 Matrilysin Prominin-1 Heterogeneous nuclear ribonucleoproteins A2/B1 Protein S100-A9 Protein S100-A10 Tenascin 14-3-3 protein zeta/delta Junctional adhesion molecule A Osteopontin Protocadherin LKC Aldo-keto reductase family 1 member B10 Attractin Cathepsin D Carcinoembryonic antigen-related cell adhesion molecule 1 Carcinoembryonic antigen-related cell adhesion molecule 6 N(G),N(G)-dimethylarginine dimethylaminohydrolase 1 Glutathione S-transferase P Galectin-3-binding protein Mucin-1 Neutrophil gelatinaseassociated lipocalin Prostasin Protein Plunc

EF1A2_HUMAN MMP7_HUMAN PROM1_HUMAN ROA2_HUMAN

S10A9_HUMAN S10AA_HUMAN TENA_HUMAN 1433Z_HUMAN JAM1_HUMAN OSTP_HUMAN PCDLK_HUMAN AK1BA_HUMAN ATRN_HUMAN CATD_HUMAN CEAM1_HUMAN

CEAM6_HUMAN

DDAH1_HUMAN GSTP1_HUMAN LG3BP_HUMAN MUC1_HUMAN NGAL_HUMAN PRSS8_HUMAN PLUNC_HUMAN

ref

b

43 44 45 46 47 23 48 49

50 51 52 53 54 55 56 57 58 59 60

22

61 62 63 64 65 66 67

a

Bold lines represent proteins that were directly involved in studies on pancreatic cancer. b Only one reference has been cited for each protein.

detected in bile, we performed immunodetection on bile sample from patients having biliary stenosis of various etiologies. Among the potential biomarkers listed in Table 3, we selected CEACAM6, whose cancer-associated overexpression was well-described, and MUC1 (CA 19-9), a marker widely used inclinicalpracticeforthediagnosisofpancreaticadenocarcinoma. Immunoblot analysis showed an increased concentration of CEACAM6 in all malignant bile samples (pancreatic adenocarcinoma and cholangiocarcinoma). The positivity of one of the four benign controls, corresponding to a patient with a pancreatitis, accords to the data describing the presence of chronic pancreatitis false-positives.40 However, except for one pancreatic adenocarcinoma, expression levels in cancer samples were much higher than in the positive chronic pancreatitis. This result is, to our knowledge, the first evidence of differential expression of CEACAM6 as a function of cancer in bile fluid. Moreover, in these preliminary experiments, the sensitivity of CEACAM6 immunoblot on bile fluid, positive in 9 out of 9 cancer samples, was better than the one of CA 19-9, which was positive in only 7 out of 9 cancer samples. This result could be

explained by the fact that sialylated Lewis antigens are not expressed in 7-10% of the populations.41 However, all benign controls (gallstones and chronic pancreatitis) were found negative for CA 19-9. It is noteworthy that, for both antigens, the analysis of the centrifugation fractions points to the release of the marker in the bile fluid supernatant rather than its association with cellular debris in the pellet. These findings probably explain the failure of previous attempts to correlate MUC1 expression to the amount of exfoliated cancer cells by bile cytology.42

Conclusions Our approach, consisting in combination of data obtained from different purification methods, proved to be suitable for large-scale protein identification and detection of several cancer-associated proteins in bile. These cancer-associated proteins could be the leading candidates for further quantitative investigation of bile samples. Moreover, our study confirms the ability to detect differential expression of cancer markers in bile, underlining the interest of using this fluid as a source of new biomarkers. We believe that the present study will enhance efforts to generate and expand knowledge about bile fluid and so aid the discovery of more reliable diagnostics for malignant biliary stenosis.

Acknowledgment. We thank Dr. Yohann Coute´ and Prof. Jacques M. P. Deshusses for expert help with, respectively, mass spectrometric data analysis and protein purification methods. We are also very grateful to Alexandre Hainard for technical assistance. This project was supported by funds from the “Projets Recherche & De´veloppement” of Geneva University Hospitals, HUG. Supporting Information Available: Supplemental Table 1, 2 and Supplemental Figure 1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) American Cancer Society. Cancer Facts and Figures, 2008; American Cancer Society:Atlanta, GA, 2008. (2) Hall, J. G.; Pappas, T. N. Current management of biliary strictures. J. Gastrointest. Surg. 2004, 8 (8), 1098–110. (3) Vitale, G. C.; George, M.; McIntyre, K.; Larson, G. M.; Wieman, T. J. Endoscopic management of benign and malignant biliary strictures. Am. J. Surg. 1996, 171 (6), 553–7. (4) Iitsuka, Y.; Hiraoka, H.; Kimura, A.; Kodoh, H.; Koga, S. Diagnostic significance of bile cytology in obstructive jaundice. Jpn. J. Surg. 1984, 14 (3), 207–11. (5) Mohandas, K. M.; Swaroop, V. S.; Gullar, S. U.; Dave, U. R.; Jagannath, P.; DeSouza, L. J. Diagnosis of malignant obstructive jaundice by bile cytology: results improved by dilating the bile duct strictures. Gastrointest. Endosc. 1994, 40 (2 Pt 1), 150–4. (6) Simunovic, M.; To, T.; Theriault, M.; Langer, B. Relation between hospital surgical volume and outcome for pancreatic resection for neoplasm in a publicly funded health care system. Can. Med Assoc. J. 1999, 160, 643–8. (7) Wolfson, D.; Barkin, J. S.; Chari, S. T.; Clain, J. E.; Bell, R. H.; Alexakis, N.; Neoptolemos, J. P. Management of pancreatic masses. Pancreas 2005, 31, 203–17. (8) Goonetilleke, K. S.; Siriwardena, A. K. Systematic review of carbohydrate antigen (CA 19-9) as a biochemical marker in the diagnosis of pancreatic cancer. Eur. J. Surg. Oncol. 2007, 33 (3), 266–70. (9) Le´vy, P. Adenocarcinoma of the pancreas: are CA 19-9 assays useful. Presse Med. 2008, 37 (1 Pt 2), 88–94. (10) Daram, S. R. CA 19-9: not a magic marker for pancreatic cancer. South. Med. J. 2006, 99 (3), 205. (11) Bonney, G. K.; Craven, R. A.; Prasad, R.; Melcher, A. F.; Selby, P. J.; Banks, R. E. Circulating markers of biliary malignancy: opportunities in proteomics. Lancet Oncol. 2008, 9 (2), 149–58.

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