Evaluation of AGA and Fukuoka Guidelines for EUS and surgical resection of incidental pancreatic cysts.

Objectives Management of asymptomatic pancreatic cysts is challenging. Guidelines by the American Gastroenterological Association (AGA) and International Association of Pancreatology (Fukuoka) seek to identify high-risk patients. We assessed performance of these guidelines in selecting patients for endoscopic ultrasound (EUS) and/or surgery. Methods PART I - We retrospectively studied 143 asymptomatic cysts with magnetic resonance imaging (MRI) followed by EUS. Appropriate selection for EUS was defined as: malignant cytology or surgical pathology, or development of concerning features on MRI as defined by the guidelines. PART II - We retrospectively studied 152 resected cysts to assess the performance of guidelines in selecting cysts for surgery using malignant histology as the outcome. Results PART I - Of 143 EUS, 43 (30.1 %) were male with median age 65.0 years (interquartile range [IQR] 58.0 - 73.0). AGA guideline demonstrated lower sensitivity (17.6 % versus 35.3 %, P = 0.03), higher specificity (94.5 % versus 66.1 %, p < 0.001), and higher accuracy (76.2 % versus 58.7 %, P = 0.002) than Fukuoka. There was no difference in positive predictive value (50.0 % versus 24.5 %, P = 0.15) and negative predictive value (78.6 % versus 76.6 %, p=0.75). PART II - Of 152 resected cysts, 45 (29.8 %) were male with median age 59.0 years (IQR 47.3 - 66.7). There was no difference in performance characteristics of the guidelines in selecting cysts for surgery. AGA and Fukuoka guidelines missed 25.0 % and 18.8 % of malignant cysts, respectively (P = 1.00). Conclusions For referral to EUS, the AGA guideline was highly specific compared to Fukuoka; both suffered from poor sensitivity, although the Fukuoka guideline was relatively more sensitive than AGA. For referral to surgery, both guidelines have modest sensitivity and specificity and miss a similar percentage of malignant lesions.

Diagnosis of Chronic Pancreatitis Incorporating Endosonographic Features, Demographics, and Behavioral Risk.

OBJECTIVES: Diagnosing chronic pancreatitis remains challenging. Endoscopic ultrasound (EUS) is utilized to evaluate pancreatic disease. Abnormal pancreas function test is considered the "nonhistologic" criterion standard for chronic pancreatitis. We derived a prediction model for abnormal endoscopic pancreatic function test (ePFT) by enriching EUS findings with patient demographic and pancreatitis behavioral risk characteristics. METHODS: Demographics, behavioral risk characteristics, EUS findings, and peak bicarbonate results were collected from patients evaluated for pancreatic disease. Abnormal ePFT was defined as peak bicarbonate of less than 75 mEq/L. We fit a logistic regression model and converted it to a risk score system. The risk score was validated using 1000 bootstrap simulations. RESULTS: A total of 176 patients were included; 61% were female with median age of 48 years (interquartile range, 38-57 years). Abnormal ePFT rate was 39.2% (69/176). Four variables formulated the risk score: alcohol or smoking status, number of parenchymal abnormalities, number of ductal abnormalities, and calcifications. Abnormal ePFT occurred in 10.7% with scores 4 or less versus 92.0% scoring 20 or greater. The model C-statistic was 0.78 (95% confidence interval, 0.71-0.85). CONCLUSIONS: Number of EUS pancreatic duct and parenchymal abnormalities, presence of calcification, and smoking/alcohol status were predictive of abnormal ePFT. This simple model has good discrimination for ePFT results.

The Utility of the Systemic Inflammatory Respsonse Syndrome Score on Admission in Children With Acute Pancreatitis.

OBJECTIVES: Pediatric patients with acute pancreatitis (AP) may meet criteria at admission for the systemic inflammatory response syndrome (SIRS). Early SIRS in adults with AP is associated with severe disease. Our aim was to evaluate the importance of SIRS in children presenting with AP on various outcomes. METHODS: This is a retrospective cohort study of children hospitalized with AP at Boston Children's Hospital in 2010. Increased length of stay (LOS) and/or admission to the intensive care unit (ICU) served as the primary outcomes. Statistical analyses of measures studied included the presence of SIRS, demographic, and clinical information present on admission. RESULTS: Fifty encounters, in which AP was the primary admitting diagnosis, were documented. Patients had a median LOS of 4.5 (interquartile range, 2-9) days. Systemic inflammatory response syndrome was present in 22 (44%) of 50 patients at admission. Systemic inflammatory response syndrome at admission was an independent predictor of increased LOS (odds ratio, 7.99; P = 0.045) as well as admission to the ICU (odds ratio, 12.06; P = 0.027). CONCLUSIONS: The presence of SIRS criteria on admission serves as a useful and easy-to-calculate predictor of increased LOS and admission to ICU in children with AP.

Incidence and clinical sequelae of portal and hepatic venous thrombosis following percutaneous cryoablation of liver tumors.

PURPOSE: To assess the incidence and sequelae of portal and hepatic venous thrombosis after percutaneous cryoablation of hepatic tumors. METHODS: From November 1998 through December 2010, 223 hepatic tumors were cryoablated during 170 ablation procedures in 135 patients. 24-h post-procedure MR images were reviewed retrospectively by two abdominal radiologists in consensus to identify tumor ablations that developed one or more new portal or hepatic venous thromboses in or outside the ablation zone. On follow-up MRI and CT examinations the outcomes of thromboses were classified as resolved, partially recanalized, persistent, or propagated. RESULTS: Venous thrombosis developed in association with 54 (24%) of 223 tumor ablations treated during 53 (31%) ablation procedures in 39 (28.8%) patients (15 women, 24 men; age range 40-82 years, mean 59 years). Of these 54 thromboses, 49 (91%) were located in portal vein branches, four (7%) in both portal and hepatic vein branches, and one (2%) in a hepatic vein branch. Thrombosed veins were outside but abutted the ablation zone in 36 (66.7%), and within it in 18 (33.3%). On follow-up imaging (n = 49), thrombi resolved in 29 (59%), partially recanalized in two (4%), persisted in 18 (37%) and propagated from sub-segmental or segmental branches to the left or right portal branches in five (10%). No thrombus propagated to the main portal vein or inferior vena cava. CONCLUSION: Portal and hepatic vein branch thromboses are common in small branches following percutaneous cryoablation of hepatic tumors and most resolve spontaneously without sequelae.

The Atlanta Classification, Revised Atlanta Classification, and Determinant-Based Classification of Acute Pancreatitis: Which Is Best at Stratifying Outcomes?

OBJECTIVES: To determine which classification is more accurate in stratifying severity. METHODS: The study used a retrospective analysis of a prospective acute pancreatitis database (June 2005-December 2007). Acute pancreatitis severity was stratified according to the Atlanta classification (AC) 1992, the revised Atlanta classification (RAC) 2012, and the determinant-based classification (DBC) 2012. Receiver operating characteristic analysis (area under the curve) compared the accuracy of each classification. Logistic regression identified predictors of mortality. RESULTS: 338 patients were analyzed: 13% had persistent organ failure (POF) (>48 hours), of whom 37% had multisystem POF, and 11% had pancreatic necrosis, of whom 19% had infected necrosis. Mortality was 4.1%. For predicting mortality (area under the curve), the RAC (0.91) and DBC (0.92) were comparable (P = 0.404); both outperformed the AC (0.81) (P < 0.001). For intensive care unit admission, the RAC (0.85) and DBC (0.85) were comparable (P = 0.949); both outperformed the AC (0.79) (P < 0.05). There were 2 patients in the critical category of the DBC. Multisystem POF was an independent predictor of mortality (odds ratio, 75.0; 95% confidence interval, 13.7-410.6; P < 0.001), whereas single-system POF, sterile necrosis, and infected necrosis were not. CONCLUSION: The RAC and DBC were generally comparable in stratifying severity. The paucity of patients in the critical category in the DBC limits its utility. Neither classification accounts for the impact of multisystem POF, which was the strongest predictor of mortality.

Randomized Noninferiority Trial Comparing Diagnostic Yield of Cytopathologist-guided versus 7 passes for EUS-FNA of Pancreatic Masses.

BACKGROUND AND AIM: To improve diagnostic yield of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in solid pancreatic lesions, on-site cytology review has been recommended. Because this is not widely available throughout the world, the aim of this study was to compare the diagnostic yield of EUS-FNA performed with rapid on-site evaluation (ROSE) versus 7 FNA passes without ROSE in pancreatic masses. METHODS: In this multicenter randomized noninferiority trial, patients were randomized to ROSE versus 7 passes into a solid pancreatic mass. On the basis of the absolute difference in diagnostic yield with 7 passes versus cytopathologist-guidance, the noninferiority margin for the difference in diagnostic yield was defined as -15%. Definite diagnosis was defined to include positive for malignancy, neoplastic cells present, and negative for malignancy. RESULTS: A total of 142 patients were randomized with 73 in the cytopathologist arm and 69 in the 7 passes arm. Diagnostic yield for definite diagnosis was 78.3% with 7 passes and 78.1% with cytopathology guidance. With an absolute difference 0.2%, 95% CI -14.4 to 14.6, performing 7 passes was noninferior to cytopathologist-guided EUS-FNA. There was no significant difference in complications or time to perform FNA. A median of 5 passes were performed with ROSE. The median charge with onsite cytopathology was significantly greater than performing 7 passes [$1058 (958, 1445) versus $375 (275, 460), p<0.001]. CONCLUSIONS: The diagnostic yield for performing 7 passes during EUS-FNA into solid pancreatic masses is noninferior with lower charge compared to cytopathologist-guidance. This article is protected by copyright. All rights reserved.

Evaluation of Qualitative Magnetic Resonance Imaging Features for Diagnosis of Chronic Pancreatitis.

OBJECTIVES: The purpose of this study was to determine qualitative pancreatic magnetic resonance imaging (MRI) features that must be present to predict abnormal pancreatic secretory function in patients evaluated for chronic pancreatitis (CP). METHODS: The MRIs of study subjects were reviewed by 2 abdominal radiologists; qualitative parenchyma and ductal features were recorded. Endoscopic pancreatic function test (ePFT) results (reference standard) were classified as normal (peak pancreatic fluid bicarbonate [HCO3-] ≥75 meq/L) or abnormal (<75). Abnormal ePFT was further classified as mild/moderate (74-65) and marked deficiency (<65). Statistical analysis was performed to assess the association between MRI features and abnormal ePFT. RESULTS: The study cohort was composed of 93 subjects, mean age 49 years (range, 18-78 years), 65% females. Univariate analysis identified 9 qualitative MRI features significantly (P < 0.05) associated with abnormal pancreatic secretory function. Number of MRI features increases as peak pancreatic fluid [HCO3-] decreases (Pearson r = -0.629; P = 0.001). Receiver operating characteristic curve analysis determined that a threshold of 6 or more associated MRI features 64% sensitive and 94% specific for marked bicarbonate deficiency. CONCLUSIONS: Qualitative MRI parenchymal and ductal features are associated with CP. Presence of 6 or more features results in a higher specificity for the diagnosis of CP in advanced disease.

Endosonography in the diagnosis and management of pancreatic cysts.

Rapid advances in radiologic technology and increased cross-sectional imaging have led to a sharp rise in incidental discoveries of pancreatic cystic lesions. These cystic lesions include non-neoplastic cysts with no risk of malignancy, neoplastic non-mucinous serous cystadenomas with little or no risk of malignancy, as well as neoplastic mucinous cysts and solid pseudopapillary neoplasms both with varying risk of malignancy. Accurate diagnosis is imperative as management is guided by symptoms and risk of malignancy. Endoscopic ultrasound (EUS) allows high resolution evaluation of cyst morphology and precise guidance for fine needle aspiration (FNA) of cyst fluid for cytological, chemical and molecular analysis. Initially, clinical evaluation and radiologic imaging, preferably with magnetic resonance imaging of the pancreas and magnetic resonance cholangiopancreatography, are performed. In asymptomatic patients where diagnosis is unclear and malignant risk is indeterminate, EUS-FNA should be used to confirm the presence or absence of high-risk features, differentiate mucinous from non-mucinous lesions, and diagnose malignancy. After analyzing the cyst fluid for viscosity, cyst fluid carcinoembryonic antigen, amylase, and cyst wall cytology should be obtained. DNA analysis may add useful information in diagnosing mucinous cysts when the previous studies are indeterminate. New molecular biomarkers are being investigated to improve diagnostic capabilities and management decisions in these challenging cystic lesions. Current guidelines recommend surgical pancreatic resection as the standard of care for symptomatic cysts and those with high-risk features associated with malignancy. EUS-guided cyst ablation is a promising minimally invasive, relatively low-risk alternative to both surgery and surveillance.

Differential expression of GNAS and KRAS mutations in pancreatic cysts.

CONTEXT: KRAS mutations play an important role in pancreatic cancer. GNAS mutations were discovered in intraductal papillary mucinous neoplasms (IPMN). OBJECTIVES: Our aim was to identify the frequency of KRAS and GNAS mutations in pancreatic cystic neoplasms and pancreatic ductal adenocarcinoma (PDAC). METHODS: Sixty-eight surgically resected formalin fixed, paraffin embedded pancreatic specimens were analyzed, including: 1) benign (20 serous cystadenoma (SCA)), 2) pre-malignant (10 mucinous cystic neoplasm (MCN), 10 branch duct intraductal papillary mucinous neoplasm (BD-IPMN), 9 main duct IPMN (MD-IPMN)), 3) malignant (19 PDAC). Total nucleic acid extraction was performed. KRAS codon 12/13 and GNAS codon 201 mutations were interrogated via targeted sequencing using the Ion Torrent's Personal Genome Machine (PGM). RESULTS: Mean age of 68 patients was 61.9±8.4 with 72% female. KRAS and GNAS mutations were more common in PDAC and IPMN. KRAS mutations predominated in PDAC compared to pancreatic cysts (16/19, 84% versus 10/49, 20%; P<0.001). GNAS mutations were more common in IPMN compared to non-IPMN lesions (8/19, 42% versus 2/49, 4%; P=0.0003). No GNAS mutations were detected in PDAC and MCN while 2 SCA carried GNAS mutations. Double mutations with KRAS and GNAS were only present in IPMN (5/19 versus 0/30 SCA and MCN, P=0.006). CONCLUSIONS: KRAS and GNAS mutations were more common in PDAC and IPMN with KRAS mutations primarily in PDAC and GNAS mutations more frequent in IPMN. No GNAS mutations occurred in MCN and double mutations were only present in IPMN.

Investigating MicroRNA Expression Profiles in Pancreatic Cystic Neoplasms.

OBJECTIVES: Current diagnostic tools for pancreatic cysts fail to reliably differentiate mucinous from nonmucinous cysts. Reliable biomarkers are needed. MicroRNAs (miRNA) may offer insights into pancreatic cysts. Our aims were to (1) identify miRNAs that distinguish benign from both premalignant cysts and malignant pancreatic lesions using formalin-fixed, paraffin-embedded (FFPE) pathology specimens; (2) identify miRNAs that distinguish mucinous cystic neoplasm (MCN) from branch duct-intraductal papillary mucinous neoplasm (BD-IPMN). METHODS: A total of 69 FFPE pancreatic specimens were identified: (1) benign (20 serous cystadenoma (SCA)), (2) premalignant (10 MCN, 10 BD-IPMN, 10 main duct IPMN (MD-IPMN)), and (3) malignant (19 pancreatic ductal adenocarcinoma (PDAC)). Total nucleic acid extraction was performed followed by miRNA expression profiling of 378 miRNAs interrogated using TaqMan MicroRNA Arrays Pool A and verification of candidate miRNAs. Bioinformatics was used to generate classifiers. RESULTS: MiRNA profiling of 69 FFPE specimens yielded 35 differentially expressed miRNA candidates. Four different 4-miRNA panels differentiated among the lesions: one panel separated SCA from MCN, BD-IPMN, MD-IPMN, and PDAC with sensitivity 85% (62, 97), specificity 100% (93, 100), a second panel distinguished MCN from SCA, BD-IPMN, MD-IPMN, and PDAC with sensitivity and specificity 100% (100, 100), a third panel differentiated PDAC from IPMN with sensitivity 95% (76, 100) and specificity 85% (72, 96), and the final panel diagnosed MCN from BD-IPMN with sensitivity and specificity approaching 100%. CONCLUSIONS: MiRNA profiling of surgical pathology specimens differentiates serous cystadenoma from both premalignant pancreatic cystic neoplasms and PDAC and MCN from BD-IPMN.

A proteomic comparison of formalin-fixed paraffin-embedded pancreatic tissue from autoimmune pancreatitis, chronic pancreatitis, and pancreatic cancer.

CONTEXT: Formalin-fixed paraffin-embedded (FFPE) tissue is a standard for specimen preservation, and as such FFPE tissue banks are an untapped resource of histologically-characterized specimens for retrospective biomarker investigation for pancreatic disease. OBJECTIVES: We use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to compare FFPE specimens from three different diseases of the exocrine pancreas. DESIGN: We investigated the proteomic profile of FFPE pancreatic tissue from 9 archived specimens that were histologically classified as: autoimmune pancreatitis (n=3), chronic pancreatitis (n=3), and pancreatic cancer (n=3), using LC-MS/MS. SETTING: This is a proteomic analysis experiment of FFPE pancreatic tissue in an academic center. PATIENTS: FFPE tissue specimens were provided by Dana-Farber/Harvard Cancer Center (Boston, MA, USA). INTERVENTIONS: FFPE tissue specimens were collected via routine surgical resection procedures. MAIN OUTCOME MEASURES: We compared proteins identified from chronic pancreatitis, autoimmune pancreatitis, and pancreatic cancer FFPE pancreatic tissue. RESULTS: We identified 386 non-redundant proteins from 9 specimens. Following our filtering criteria, 73, 29, and 53 proteins were identified exclusively in autoimmune pancreatitis, chronic pancreatitis, and pancreatic cancer specimens, respectively. CONCLUSIONS: We report that differentially-expressed proteins can be identified among FFPE tissues specimens originating from individuals with different histological diagnoses. These proteins merit further confirmation with a greater number of specimens and orthogonal validation, such as immunohistochemistry. The mass spectrometry-based methodology used herein has the potential to enhance diagnostic biomarker and therapeutic target discovery, further advancing pancreatic research.

Urinary 1H-NMR metabolomics can distinguish pancreatitis patients from healthy controls.

CONTEXT: The characterization of the urinary metabolome may yield biomarkers indicative of pancreatitis. OBJECTIVES: We establish a non-invasive technique to compare urinary metabolic profiles in patients with acute and chronic pancreatitis to healthy controls. METHODS: Urine was obtained from healthy controls (HC, n=5), inpatients with mild acute pancreatitis (AP, n=5), and outpatients with chronic pancreatitis (CP, n=5). Proton nuclear magnetic resonance spectra were obtained for each sample. Metabolites were identified and quantified in each spectrum; resulting concentrations were normalized to account for differences in dilution among samples. Kruskal-Wallis test, post-hoc Mann-Whitney U tests, and principal component analysis were performed to identify metabolites that discriminate healthy controls, acute pancreatitis, and chronic pancreatitis. RESULTS: Sixty metabolites were identified and quantified; five were found to differ significantly (P<0.05) among the three groups. Of these, citrate and adenosine remained significant after validation by random permutation. Principal component analysis demonstrated that healthy control urine samples can be differentiated from patients with chronic pancreatitis or acute pancreatitis; chronic pancreatitis patients could not be distinguished from acute pancreatitis patients. CONCLUSIONS: This metabolomic investigation demonstrates that this non-invasive technique offers insight into the metabolic states of pancreatitis. Although the identified metabolites cannot conclusively be defined as biomarkers of disease, future studies will validate our findings in larger patient cohorts.

Analysis of endoscopic pancreatic function test (ePFT)-collected pancreatic fluid proteins precipitated via ultracentrifugation.

CONTEXT: We have shown previously that trichloroacetic acid precipitation is an effective method of protein extraction from pancreatic fluid for downstream biomarker discovery, compared to other common extraction methods tested. OBJECTIVE: We aim to assess the utility of ultracentrifugation as an alternative method of protein extraction from pancreatic fluid. DESIGN: Proteins extracted from trichloroacetic acid- and ultracentrifugation-precipitated pancreatic fluid were identified using mass spectrometry techniques (in-gel tryptic digestion followed by liquid chromatography-tandem mass spectrometry; GeLC-MS/MS). Data were analyzed using Proteome Discoverer and Scaffold 3. SETTING: This is a proteomic analysis experiment of endoscopically collected fluid in an academic center. PATIENTS: The study population included adult patients referred to the Center for Pancreatic Disease at Brigham and Women's Hospital, Boston, MA, USA for the evaluation of abdominal pain and gastrointestinal symptoms. INTERVENTIONS: Secretin-stimulated pancreatic fluid was collected as standard of care for the evaluation of abdominal pain and gastrointestinal symptoms. MAIN OUTCOME MEASURES: We compared proteins identified via standard trichloroacetic acid precipitation and this alternative ultracentrifugation strategy. RESULTS: A subset of pancreatic fluid proteins was identified via the ultracentrifugation method. Of these proteins, similar numbers were obtained from fully tryptic or semi-tryptic database searching. Proteins identified in the ultracentrifugation-precipitated samples included previously identified biomarker candidates of chronic pancreatitis. CONCLUSIONS: This alternative ultracentrifugation strategy requires less time and fewer handling procedures than standard trichloroacetic acid precipitation, at the expense of higher sample volume. As such, this method is well suited for targeted assays (i.e., dot blotting or targeted mass spectrometry) if the protein of interest is among those readily identified by ultracentrifugation-promoted precipitation.

Cross-species analysis of nicotine-induced proteomic alterations in pancreatic cells.

Toxic compounds in tobacco, such as nicotine, may adversely affect pancreatic function. We aim to determine nicotine-induced protein alterations in pancreatic cells, thereby revealing links between nicotine exposure and pancreatic disease. We compared the proteomic alterations induced by nicotine treatment in cultured pancreatic cells (mouse, rat, and human stellate cells and human duct cells) using MS-based techniques, specifically SDS-PAGE (gel) coupled with LC-MS/MS and spectral counting. We identified thousands of proteins in pancreatic cells, hundreds of which were identified exclusively or in higher abundance in either nicotine-treated or untreated cells. Interspecies comparisons of stellate cell proteins revealed several differentially abundant proteins (in nicotine treated versus untreated cells) common among the three species. Proteins appearing in all nicotine-treated stellate cells include amyloid beta (A4), procollagen type VI alpha 1, integral membrane protein 2B, and toll-interacting protein. Proteins that were differentially expressed upon nicotine treatment across cell lines were enriched in certain pathways, including nicotinic acetylcholine receptor, cytokine, and integrin signaling. At this analytical depth, we conclude that similar pathways are affected by nicotine, but alterations at the protein level among stellate cells of different species vary. Further interrogation of such pathways will lead to insights into the potential effect of nicotine on pancreatic cells at the biomolecular level and the extension of this concept to the effect of nicotine on pancreatic disease.

Mass spectrometry-based quantitative proteomic profiling of human pancreatic and hepatic stellate cell lines.

The functions of the liver and the pancreas differ; however, chronic inflammation in both organs is associated with fibrosis. Evidence suggests that fibrosis in both organs is partially regulated by organ-specific stellate cells. We explore the proteome of human hepatic stellate cells (hHSC) and human pancreatic stellate cells (hPaSC) using mass spectrometry (MS)-based quantitative proteomics to investigate pathophysiologic mechanisms. Proteins were isolated from whole cell lysates of immortalized hHSC and hPaSC. These proteins were tryptically digested, labeled with tandem mass tags (TMT), fractionated by OFFGEL, and subjected to MS. Proteins significantly different in abundance (P<0.05) were classified via gene ontology (GO) analysis. We identified 1223 proteins and among them, 1222 proteins were quantifiable. Statistical analysis determined that 177 proteins were of higher abundance in hHSC, while 157 were of higher abundance in hPaSC. GO classification revealed that proteins of relatively higher abundance in hHSC were associated with protein production, while those of relatively higher abundance in hPaSC were involved in cell structure. Future studies using the methodologies established herein, but with further upstream fractionation and/or use of enhanced MS instrumentation will allow greater proteome coverage, achieving a comprehensive proteomic analysis of hHSC and hPaSC.

Post-translational modifications of pancreatic fluid proteins collected via the endoscopic pancreatic function test (ePFT).

BACKGROUND: Early diagnosis of chronic pancreatitis by mass spectrometry-based proteomics may result in therapies to retard or modify disease progression. We aimed to identify differences in posttranslational modifications (PTMs) in pancreatic fluid proteins from individuals with chronic pancreatitis (n=9) and non-pancreatitis controls (n=9). METHODS: We collected proteomic data from pancreatic fluid using mass spectrometry techniques. We performed database searches with emphasis on PTMs using ProteinPilot. We compared the frequency of specific PTMs in pancreatic fluid between cohorts and also to those identified in bile, gastroduodenal fluid, urine, and pancreatic duct and stellate cell lysates. RESULTS: We identified 97 PTMs in endoscopically-collected pancreatic fluid, of which 11 were identified exclusively in one cohort and 9 others were significantly different in frequency between cohorts. Comparing pancreatic fluid with other specimens revealed differences in specific PTM frequencies, indicating that the identified PTMs were not merely artifacts of sample processing. CONCLUSIONS: We determined PTMs of proteins extracted from pancreatic fluid which differed in frequency in chronic pancreatitis patients verses controls. Such PTMs may serve as biomarker candidates of chronic pancreatitis upon validation with larger cohorts. The analysis of the PTM profile of pancreatic fluid proteins offers an alternative method to standard protein-based biomarker discovery. BIOLOGICAL SIGNIFICANCE: The early diagnosis of chronic pancreatitis is paramount in developing strategies to modify, retard, or halt disease progression. In the present study, we compared post-transitional modifications (PTMs) of proteins extracted from pancreatic fluid of chronic pancreatitis patients verses a control cohort. With many mass spectrometry-based proteomics workflows aimed to identify and quantify proteins, data for PTMs typically comes gratis, in that such data are collected during protein sequencing and, as such, require only downstream bioinformatics processing. We identified a total of 20 PTMs which were exclusive to or significantly different between cohorts. Upon validation with larger cohorts and enrichment of these PTMs may serve as biomarker candidates of chronic pancreatitis. PTM profiling of pancreatic fluid proteins is complementary to standard protein-based biomarker discovery, and may be readily applied to studies of pancreatic disease. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Cigarette smoking impairs pancreatic duct cell bicarbonate secretion.

OBJECTIVE: To compare pancreatic duct cell function in smokers (current and past) and never smokers by measurement of secretin-stimulated peak bicarbonate concentration ([HCO3-]) in endoscopic collected pancreatic fluid (PF). METHODS: This retrospective study was cross-sectional in design, recording demographic information (age, gender, etc.), smoking status (former, current, never), alcohol intake, clinical data (imaging, endoscopy), and laboratory results (peak PF [HCO3-]) from subjects evaluated for pancreatic disease at a tertiary pancreas center. Univariate and multivariate statistical analysis (SAS Version 9.2, Cary, NC, USA) was performed to assess the relationship between cigarette smoking and secretin-stimulated pancreatic fluid bicarbonate concentration. RESULTS: A total of 131 subjects underwent pancreatic fluid collection (endoscopic pancreatic function test, ePFT) for bicarbonate analysis: 25.2% (33 out of 131) past smokers, 31.3% (41 out of 131) current smokers, and 43.5% (57 out of 131) were never smokers. Measures of Association: The mean peak PF [HCO3-] in never smokers (81.3 ± 18.5 mEq/L) was statistically higher (indicating better duct cell function) when compared to past smokers (66.8 ± 24.7 mEq/L, P=0.005) and current smokers (70.0 ± 20.2 mEq/L, P=0.005). However, the mean peak [HCO3-] in past smokers was not statistically different from that in current smokers (P=0.575), and therefore, the two smoking groups were combined to form a single "smokers cohort". When compared to the never smokers, the smokers cohort was older (P=0.037) and had a greater proportion of subjects with definite chronic pancreatitis imaging (P=0.010), alcohol consumption ≥20 g/day (P=0.012), and abnormal peak PF [HCO3-] (P<0.001). Risk-Based Estimates: Cigarette smoking (risk ratio, RR: 2.2, 95% CI: 1.3-3.5; P<0.001), diagnosis of definite chronic pancreatitis imaging (RR: 2.2, 95% CI: 1.6-3.2; P<0.001) and alcohol consumption ≥20 g/day (RR: 1.6, 95% CI: 1.1-2.4; P=0.033) were all associated with low mean peak PF [HCO3-] (indicating duct cell secretory dysfunction). Multivariate Analysis: Smoking (odds ratio, OR: 3.8, 95% CI: 1.6-9.1; P=0.003) and definite chronic pancreatitis imaging (OR: 5.7, 95% CI: 2.2-14.8; P<0.001) were determined to be independent predictors of low peak PF [HCO3-], controlling for age, gender, and alcohol intake. Furthermore there was no interaction between smoking status and alcohol intake in predicting duct cell dysfunction (P=0.571). CONCLUSION: Measurement of pancreatic fluid bicarbonate in smokers reveals that cigarette smoking (past and current) is an independent risk factor for pancreatic duct cell secretory dysfunction (low PF [HCO3-]). Furthermore, the risk of duct cell dysfunction in subjects who smoked was approximately twice the risk (RR: 2.2) in never smokers. Further in depth, translational research approaches to pancreatic fluid analysis may help unravel mechanisms of cigarette smoking induced pancreatic duct cell injury.

Subcellular fractionation enhances proteome coverage of pancreatic duct cells.

OBJECTIVES: Subcellular fractionation of whole cell lysates offers a means of simplifying protein mixtures, potentially permitting greater depth of proteomic analysis. Here we compare proteins identified from pancreatic duct cells (PaDC) following organelle enrichment to those identified from PaDC whole cell lysates to determine if the additional procedures of subcellular fractionation increase proteome coverage. METHODS: We used differential centrifugation to enrich for nuclear, mitochondrial, membrane, and cytosolic proteins. We then compared - via mass spectrometry-based analysis - the number of proteins identified from these four fractions with four biological replicates of PaDC whole cell lysates. RESULTS: We identified similar numbers of proteins among all samples investigated. In total, 1658 non-redundant proteins were identified in the replicate samples, while 2196 were identified in the subcellular fractionation samples, corresponding to a 30% increase. Additionally, we noted that each organelle fraction was in fact enriched with proteins specific to the targeted organelle. CONCLUSIONS: Subcellular fractionation of PaDC resulted in greater proteome coverage compared to PaDC whole cell lysate analysis. Although more labor intensive and time consuming, subcellular fractionation provides greater proteome coverage, and enriches for compartmentalized sub-populations of proteins. Application of this subcellular fractionation strategy allows for a greater depth of proteomic analysis and thus a better understanding of the cellular mechanisms of pancreatic disease.

Short Gel, Long Gradient Liquid Chromatography Tandem Mass Spectrometry to Discover Urinary Biomarkers of Chronic Pancreatitis.

BACKGROUND: Chronic pancreatitis (CP) is currently diagnosed using invasive endoscopic as well as radiation and non-radiation-based imaging techniques. However, urine can be safely and non-invasively collected and as such may offer a superior alternative to current techniques of CP diagnosis. We use mass spectrometry-based methods to discover proteins which are exclusive to or differentially abundant in urine of chronic pancreatitis patients. METHODS: We have performed a comparative quantitative proteomic analysis of urine collected from 5 healthy controls and 5 severe CP patients. Proteins from urine were fractionated briefly on SDS-PAGE and subsequently digested in-gel with trypsin. The resulting peptides were fractionated for 3 hours by reversed-phase liquid chromatography in-line with a mass spectrometer. ProteinPilot software and the QSPEC algorithm identified proteins and determined statistically significant differences between cohorts. In addition, we used a third cohort of non-CP disease patients to filter out those proteins which may be indicative of an ailment other than CP. RESULTS: We identified over 600 proteins from urine, of which several hundred were either exclusive to or differ quantitatively between healthy controls and severe CP patients. Members of the cathepsin protein family were of significantly higher abundance in the severe CP cohort. In addition, we have identified a core set of 50 proteins in all 15 samples, 25 of which showed no significant difference among the cohorts. CONCLUSIONS: Proteomic analysis identified differentially abundant proteins in healthy controls and severe CP patients. Such proteins represent an initial set of targets for directed proteomics experiments for further validation studies. However, larger cohorts will be required to determine if these differences have statistically significant diagnostic potential.

Utility of commercial DNA analysis in detecting malignancy within pancreatic cysts.

CONTEXT: Pancreatic cysts raise concern because of their malignant potential. Our aims were to assess accuracy of DNA analysis in detecting malignant pancreatic cysts at EUS-FNA and to determine whether DNA analysis added to imaging and cyst fluid studies enhanced International Association of Pancreatology (IAP) guidelines for resection of pancreatic cysts. METHODS: This is a retrospective study including pancreatic cysts undergoing EUS-FNA and DNA analysis with k-ras and loss of heterozygosity testing. Diagnostic models of 2006 and 2012 IAP guidelines, DNA analysis alone, and DNA combined with 2012 IAP guidelines were developed, and area under receiver operator characteristic curves (AUC) compared to determine the added value of DNA for detecting malignant cysts at the time of EUS-FNA. RESULTS: Two-hundreds and fifty-seven patients were included with 8 (3.1%) malignant cysts. Solid component (P<0.001), main pancreatic duct dilation (P=0.012), suspicious or malignant cytology (P=0.001), and high DNA quantity (P<0.001) were associated with malignancy. Concurrent high amplitude k-ras with loss of heterozygosity mutations was highly specific (98.4%) though insensitive (12.5%) for malignancy. The 2012 IAP guideline (AUC=0.87; 95% CI: 0.82-0.91) was superior to 2006 IAP guideline (AUC=0.54; 95% CI: 0.47-0.60) and DNA analysis alone (AUC=0.60; 95% CI: 0.53-0.66) for detecting malignant cysts (P=0.004 and P=0.002, respectively). Addition of DNA did not improve performance of the 2012 IAP guideline (AUC=0.84; 95% CI: 0.79-0.88). CONCLUSIONS: Commercial DNA analysis does not add useful information beyond imaging and cytology for detection of malignant pancreatic cysts. The 2012 IAP guideline better predicted malignant cysts than the 2006 IAP guideline.