Purpose: Pancreatic cysts are frequently detected via imaging, and may have varying potential for malignancy, though most are benign. First line testing (imaging, cytology, and CEA) does not adequately detect malignancy, often resulting in excessive use surgery to manage these cysts. Mutational profiling of pancreatic cysts has emerged as a second-line testing alternative to aid in the determination of the risk of malignancy. We sought to determine the outcomes of patients who had previously been tested with integrated mutational profiling (PathFinderTG, RedPath Integrated Pathology, Pittsburgh, PA) in order to evaluate its performance characteristics. Methods: 362 patients with pancreatic cysts were tested using mutational profiling of pancreatic cyst fluid. Outcomes on 80 patients were determined from review of patient charts. These 80 patients ranged in age from 38 to 94 years (mean 69.6 years, 69% over 65 years). There were 60 females (75%) and 20 males (25%). Malignant diagnoses were confirmed by surgical pathology, positive cytology, or clinical cancer treatment. Benign outcomes were defined as surgical pathology with no malignancy or 2 years with no disease progression. In the present analysis, patients with less than 2 years followup were excluded. For patients who were tested serially over the followup period, the diagnosis from the first case was used as the reference. DNA for mutational profiling was extracted from pancreatic cyst fluid and tested for mutations in a panel of 16 microsatellites targeting 1p, 3p, 5q, 9p, 10q, 17p, 17q, 18q, 21q, 22q plus point mutation in KRAS. Diagnosis incorporated DNA quantity and quality, and integrated relevant imaging or cytologic findings. Overall diagnosis of biological behavior was categorized as Benign, statistically indolent low (SI-Low), statistically indolent high (SI-High), or Aggressive. Diagnoses of Benign and SI-Low were treated as negative for malignancy, and SI-High and Aggressive as positive. Results:Table 1 shows integrated mutational profiling results versus outcome for 80 cyst fluids. Sensitivity and specificity in cyst fluids were 90% and 97%, with accuracy of 96%. The relative risk (RR) of malignancy was 56.5 times greater among cysts diagnosed as positive than as negative.Table 1: No Caption available.Conclusion: Integrated molecular testing of pancreatic cyst fluid shows high overall accuracy and can be a useful addition to first-line testing to assess malignant potential of pancreatic cysts. Disclosure: Damien Mallat - No financial relationship with RedPath Eric Ellsworth - Employee of RedPath Integrated Pathology. Ann-Marie Terry - Employee of RedPath Integrated Pathology. Brendan Corcoran - Employee of RedPath Integrated Pathology Sydney. Finkelstein - Employee, Stockholder, Patent Holder of RedPath Integrated Pathology. This research was supported by an industry grant from RedPath Integrated Pathology paid costs for IRB submission, and paid staff time for retrieval of records. Dr. Mallat received no grants or payment for this study.
6Novel expression of vascular endothelial growth factor isoforms in the pancreas and pancreatic cystic lesions
Novel expression of vascular endothelial growth factor isoforms in the pancreas and pancreatic cystic lesions
Abstract B8: Proteomic analysis of cyst fluids from intraductal papillary mucinous neoplasms
Introduction: As a result of the increasing use and improvements in imaging technologies such as computed tomography and magnetic resonance imaging, the frequency at which pancreatic cysts are detected has been growing. Diagnosing the biological nature of such pancreatic cystic lesions remains a difficult challenge to clinicians, as diagnosis by cytology is insensitive and there are currently no reliable methods to measure the degree malignant potential of cysts. Therefore, new markers capable of differentiating benign from malignant IPMNs would assist in establishing an optimal therapeutic strategy for treatment by identifying patients who would benefit from surgery or surveillance. We applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics and multiple reaction monitoring (LC-MRM-MS) analyses on a collection of IPMN cyst fluid samples to identify and verify potential biomarker which may serve as diagnostic indicators of malignant potential and assist in risk stratification. Methods: A total of 15 IPMN pancreatic cyst fluid samples (5 adenomas, 5 carcinomas in situ and 5 invasive carcinomas) were obtained from freshly resected cysts and were analyzed by shotgun proteomics. Aliquots of each sample were reduced, alkylated and digested with trypsin and the resulting peptides were fractionated by isoelectric focusing (pI 3.5-4.7) into 15 fractions. Reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed using data-dependent scanning on a Thermo Orbitrap Velos instrument. Following LC-MS/MS analysis, tandem mass spectra were searched against the human IPI database (v.3.56) using the Myrimatch algorithm and the search results filtered using IDPicker at a 5% FDR. A spectral counting approach was applied to detect statistically significant differences in protein abundances with the Quasitel algorithm. MRM assays were configured for differential proteins and further verified in a new collection of unfractionated IPMN cyst fluids with varying degrees of dysplasia (mild, moderate, severe and invasive carcinoma). LC-MRM-MS was performed on peptide samples and were analyzed in triplicate (4 peptides per protein and 5 transitions per peptides) on a triple quadrupole mass spectrometer (TSQ Vantage, Thermo Fisher Scientific, Waltham MA) equipped with an Eksigent nanoLC Ultra 1D Plus pump and AS-2 autosampler (Eksigent Technologies, Dublin CA). A single isotopically labeled peptide was spiked into the samples which served as the normalization reference for all of the peptides in the analysis. All data analysis was performed using Skyline. Preliminary Results: A total of 373,673 spectra were confidently identified, which corresponded to 5,259 protein groups across all biological and technical replicates. An average of 3,534, 3,393, and 4,597 protein groups were identified in the adenomas, carcinomas in situ, and invasive carcinomas, respectively. Spectral count data indicated approximately 50 proteins differentially expressed between benign adenomas and carcinoma in situ and 176 proteins different between adenomas and invasive cancers. From these data, a list of 30 potential candidates was generated, which included proteins upregulated in carcinoma in situ and invasive cancer, as well as several down-regulated proteins, which included common digestive enzymes, such as amylase 2B and elastases. In addition, several MUC isoforms and CEACAM were also targeted, as these have been suggested to distinguish nonmucinous from mucinous cystic lesions. The preliminary MRM data are consistent with the trends observed in the spectral count data from shotgun analyses. The analyses of pancreatic cyst fluids using proteomics platforms may lead to biomarkers that can assist clinicians in differentiating benign from malignant cysts, thus leading to improved diagnosis and treatment. Citation Format: Lisa J. Zimmerman, Cristina R. Ferrone, Nipun Merchant, Robbert J. C. Slebos, Daniel C. Liebler. Proteomic analysis of cyst fluids from intraductal papillary mucinous neoplasms [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B8.
Read more144Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts
Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts
57Glycomic and Proteomic Profiling of Pancreatic Cyst Fluids Identifies Hyperfucosylated Lactosamines on the N-linked Glycans of Overexpressed Glycoproteins
Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on β-lactosamine extensions. Following the observation of these "hyperfucosylated" glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic α-amylase, which were 20- and 22-fold more abundant, respectively, following A. aurantia lectin enrichment.
Read more77Pancreatic cyst fluid harbors a unique microbiome
BackgroundIt is clear that specific intestinal bacteria are involved in the development of different premalignant conditions along the gastrointestinal tract. An analysis of the microbial constituents in the context of pancreatic cystic lesions has, however, as yet not been performed. This consideration prompted us to explore whether endoscopically obtained pancreatic cyst fluids (PCF) contain bacterial DNA and to determine the genera of bacteria present in such material.MethodsTotal DNA was isolated from 69 PCF samples. Bacterial 16S rRNA gene-specific PCR was performed followed by Sanger sequencing and de novo deep sequencing for the V3-V4 variable region of 16S rRNA gene.ResultsWe observed that 98.2% of the samples were positive in conventional PCR, and that 100% of selected PCF samples (n = 33) were positive for bacterial microbiota as determined by next generation sequencing (NGS). Comprehensive NGS data analysis of PCF showed the presence of 408 genera of bacteria, of which 17 bacterial genera were uniquely abundant to PCF, when compared to the Human Microbiome Project (HMP) database and 15 bacterial microbiota were uniquely abundant in HMP only. Bacteroides spp., Escherichia/Shigella spp., and Acidaminococcus spp. which were predominant in PCF, while also a substantial Staphylococcus spp. and Fusobacterium spp. component was detected.ConclusionThese results reveal and characterize an apparently specific bacterial ecosystem in pancreatic cyst fluid samples and may reflect the local microbiota in the pancreas. Some taxa with potential deleterious functions are present in the bacterial abundance profiles, suggesting that the unique microbiome in this specific niche may contribute to neoplastic processes in the pancreas. Further studies are needed to explore the intricate relationship between pathophysiological status in the host pancreas and its microbiota.
Read moreSupplementary Table S3 from Discovery and Validation of Biomarkers That Distinguish Mucinous and Nonmucinous Pancreatic Cysts
<p>The biomarker candidates previously reported in pancreatic cyst fluids.</p>
IDENTIFICATION OF UNIQUE DIFFERENTIAL PROTEOMIC BIOMARKERS IN PANCREATIC CYSTIC NEOPLASM FLUID
Background: Current preoperative differentiation of premalignant and malignant pancreatic cysts from benign pancreatic cysts based on imaging and cyst fluid analysis is inadequate. This inability to accurately differentiate has resulted in unnecessary surgical resections. This is the first study applying proteomic technology to identify novel proteins or protein patterns in pancreatic cyst fluid which may more accurately predict pancreatic cyst pathology preoperatively and help triage patient care. Methods: Pancreatic cyst fluid was collected by endoscopic ultrasound guided FNA from patients with pathologically confirmed pancreatic mucinous and non-mucinous cysts, centrifuged and stored immediately at −80°C. Fluid from representative pancreatic cysts was prepared for proteomic analysis using centrifugation, acetone, ethanol and TCA precipitation, followed by solubilization into highly chaotropic nonionic detergents and buffer for 2-D electrophoresis and 2 dimensional liquid chromatography (PF2D: 1-D chromatofocussing, 2D-reverse phase). Spots from 2D gels were robotically excised, digested and mass spectrometry was used to identify unique differential proteins using liquid chromatography tandem mass spectrometry (LC-MS/MS) and MALTI-TOF. Proteins were identified using Mascot search engine and Sequest search algorithms. Results: 122 pancreatic cyst fluid samples were collected, stored and processed according to the protocol. Initial protein quantification for ranged from 1 to 5 mg/ml. Pancreatic cyst fluid from 5 samples (2 mucinous and 3 serous cystadenomas) was used for initial proteomic analysis (2D gel electrophoresis (2DE:(3)), PF2D(2)). Initial analysis shows a striking differential protein profile between serous and mucinous samples (by PF2D) in both dimensions (1-D chromatofocussing and 2-D reverse phase). Comparison of two-dimensional maps of the pancreatic serous and mucinous cystadenomas show few overlaps thus suggesting many proteins unique to each cystic neoplasm. Interpretable 2-D electrophoresis gels were obtained from pancreatic cyst fluid. Using 2-D Electrophoresis we have already identified over 20 unique cellular and membrane proteins using LC-MS/MS. Through 2-D electrophoresis gel we have identified cellular and membrane proteins using LC-MS/MS. Chromatofocussed fractions were processed over a second dimension HPRP column to produce over 700 fractions in each experiment. The second dimension reverse phase fractions for serous cyst show a very different protein profile than mucinous cysts. Conclusions: To our knowledge, this is the first report detailing the proteome of pancreatic cystic neoplasm fluid. Differential protein expression was found between pancreatic mucinous and serous cystadenoma fluid. Identification of specific protein patterns or proteins may be used as highly accurate serum based or cyst fluid based biomarkers.
Read moreAmplified Sciences PanCyst Pro Panel to accurately identify non-mucinous pancreatic cyst fluid.
617 Background: Pancreatic cysts are rarely symptomatic, and, consequently, are detected incidentally during routine imaging analyses for unrelated disorders. Guidelines for diagnosis of pancreatic cysts rely on imaging and supportive analyses of fluid extracted during endoscopic ultrasound with fine-needle aspiration (EUS-FNA). Unfortunately, currently available protein markers and assays are not sufficiently accurate to rule-out patients in whom a non-mucinous pancreatic cyst has formed. Since non-mucinous pancreatic cysts seldom progress to cancer, this patient population is at risk of being subjected to the severe physical, emotional, and financial burdens associated with unwarranted surveillance, testing, and possible surgical intervention. Furthermore, identification of mucinous pancreatic cysts does not establish the risk of progression to cancer (rule-in) or the need for surgical intervention. Methods: To address the unmet needs in pancreatic cyst diagnosis, Amplified Sciences has combined a panel of markers with a clinically translated surface enhanced Raman spectroscopy (SERS)-based protease activity assay as a rule-out diagnostic tool. The tri-analyte panel was tested on a cohort of 185 retrospective samples gathered from three separate institutions (UPMC, IU Health, and UCSF) and consumed only 12 µL of cyst fluid per sample. The goals of this study were to establish the negative predictive value (NPV) of this panel prior to translation to a CLIA-regulated laboratory and create a preliminary algorithm for analysis. Results: The results of this statistically powered study show that protease activity outperforms carcinoembryonic antigen (CEA), a current standard diagnostic marker for pancreatic cyst fluid, in sensitivity, specificity, and NPV. Furthermore, in combination, the tri-analyte panel identifies non-mucinous pancreatic cysts with 99% sensitivity and a negative predictive value >95%. Conclusions: These results support the use of this panel in clinical settings as a diagnostic tool to rule-out patients from surgical intervention, as well as the use of SERS as an optical detection mode for clinical diagnostics. Future work will continue to address the unmet need for improved diagnostic information through multiplexed analyses of markers that consume comparatively minimal volume of pancreatic cyst fluid samples. Studies will also seek to build on the SERS platform by introducing more dyes, substrates, assays, and analytical methods. Amplified Sciences would like to acknowledge significant efforts to build the clinical samples cohort from Gina Zhu, Kelli Ifuku, and Dr. Kimberly Kirkwood from USCF, Christine Decapite and Dr. Randall Brand from UPMC, and Michele Yip-Schneider and Dr. C. Max Schmidt from IU Health.
Read moreA-097 Body Fluid Validation for 11 Analytes on a Chemistry Analyzer
Background Most clinical chemistry assays are intended for use in serum, plasma, urine, and/or CSF with no performance characteristics on body fluids. However, testing of body fluids can be medically necessary for certain diagnoses. Such as determining the cause of fluid buildup in peritoneal spaces, pleural effusions, whether effusions are transudates or exudates, and even diagnosing some joint disorders. To provide these medically necessary tests, the burden is on the laboratory to perform regulatory-required validations. Herein is the validation of 11 clinical chemistry analytes in several common body fluids. Methods Mixing and stability studies were performed for the following analytes on the Roche cobas c702 (Roche Diagnostics), using the methods intended for use with serum/plasma samples: Albumin, Amylase, Bilirubin, Urea Nitrogen, Cholesterol, Creatinine, Glucose, Lactate Dehydrogenase, Lipase, Protein, and Triglycerides. Six individual fluid samples were mixed with the intended use matrix in 3 ratios: 1:3, 1:1, and 3:1. The following matrices were evaluated: pleural, peritoneal, JP drainage, synovial, and pancreatic cyst fluids. Stabilities were tested for 7 days at room temperature (RT) and 2-8°C. A maximum dilution study was also performed for Amylase. Results Mixing Study: The results are shown in the table below: Stability: For most body fluids, analytes were stable for 7 days at RT and 2-8 °C. The following analytes displayed shorter stability at RT: Bilirubin (peritoneal: 2 days, pleural: 1 day, JP drainage: 1 day), Urea Nitrogen (JP drain fluid: 2 days), Lipase (peritoneal fluid 5 days), and Triglycerides (peritoneal fluid: 5 days). Lactate dehydrogenase in pleural fluid showed a stability of 5 days at RT and 2 days at 2-8 °C. Maximum Dilution A 1:505 compounded manual and automated dilution displayed a &lt;7% bias. Conclusions No sample matrix interferences were observed for any of the body fluids-analyte pairs tested allowing for this medically necessary testing.
Read more72Predicting the Grade of Dysplasia of Pancreatic Cystic Neoplasms Using Cyst Fluid DNA Methylation Markers
Purpose: Pancreatic cysts are common and pose diagnostic and management challenges. Pancreatic cyst fluid markers have the potential to aid in the management of cysts with concerning imaging findings. Our aim was to evaluate cyst fluid methylated DNA markers for their accuracy for predicting the histologic grade of neoplastic pancreatic cysts.Experimental Design: Pancreatic cyst fluid samples from 183 patients (29 discovery and 154 validation) aspirated after surgical resection were analyzed for methylated DNA at selected genes (SOX17, BNIP3, FOXE1, PTCHD2, SLIT2, EYA4, and SFRP1) using methylation-specific droplet-digital PCR (dd-QMSP). Methylated DNA levels were evaluated for their accuracy at predicting the grade of dysplasia of the pancreatic cyst.Results: All six markers evaluated in the validation set could accurately distinguish high-risk cystic neoplasms (with high-grade dysplasia and/or associated invasive cancer) from low-risk cysts (lower grades of dysplasia) with accuracies from 79.8% to 83.6%. Methylated SOX17 had the highest overall accuracy as a single marker (sensitivity, 78.4%; specificity, 85.6%; accuracy 83.6%, cutoff; 25 methylated DNA molecules/μL cyst fluid). The best four-gene combination had 84.3% sensitivity, 89.4% specificity, and 88.0% accuracy at distinguishing cysts with high-grade dysplasia and/or invasive cancer from those without. All six markers were independent predictors of having invasive cancer/high-grade dysplasia after adjusting for clinical/imaging factors known to be associated with grade of dysplasia. The combination of methylated SOX17 with cytology better predicted neoplastic grade than cytology alone.Conclusions: A panel of methylated gene markers quantified by dd-QMSP can be used to predict the grade of dysplasia of pancreatic cysts. Clin Cancer Res; 23(14); 3935-44. ©2017 AACR.
Read more112Elevated microRNA miR-21 Levels in Pancreatic Cyst Fluid Are Predictive of Mucinous Precursor Lesions of Ductal Adenocarcinoma
Elevated microRNA miR-21 Levels in Pancreatic Cyst Fluid Are Predictive of Mucinous Precursor Lesions of Ductal Adenocarcinoma
Read moreQuistes pancreáticos: valor agregado de EUS-FNA sobre EUS y RMI. Experiencia de un hospital universitario de alta complejidad
Background. Endoscopic ultrasound (EUS) and magnetic resonance imaging (MRI) are valuable tools in the assessment of cystic pancreatic lesions (CPLs). However, preoperative diagnosis of CPLs still remains a challenge. EUS-guided fine needle aspiration (FNA) provides a method to obtain cyst fluid for analysis to gain additional information. Aim. Compare the concordance between the diagnosis of the cyst by FNA, with that obtained by two diagnostic imaging methods (EUS morphology and MRI). Evaluate if EUS-FNA offered any benefit in cases of inconclusive MRI. Material and methods. We performed a retrospective analysis of a prospectively collected database at a tertiary-care academic medical center between January 2015 and December 2018. All patients who were referred for EUS were reviewed, and patients with diagnosis of pancreatic cysts were included. Imaging, pancreatic cyst fluid (PCF) and follow-up were analyzed. Results. A total of 2238 EUS were performed during the study period and 319 of them had a final diagnosis of pancreatic cyst. FNA was performed on 139 cysts: 62 were diagnosed as mucinous by PCF. The agreement between the diagnosis of the cyst by FNA, with that obtained by EUS morphology was 89.2% (Kappa 0.78, p < 0.001). The concordance between the diagnosis of the cyst by FNA and that obtained by MRI was 72.66 (Kappa 0.41, p < 0.001). From the 319 patients with pancreatic cysts, 60 (18.8%) had inconclusive results on MRI and EUS morphology was able to make a diagnosis in 31 of them. When we analyzed the 139 punctured cysts, MRI was indeterminate in 40 patients and FNA diagnosed 36 of the 40 patients (90%). Discussion. In our study, EUS +/- FNA was superior to MRI specially in cases of inconclusive MRI.
Read moreBenign Pancreatic Cystic Neoplasms: Does F/U EUS-FNA Confirm or Alter the Diagnosis
Benign Pancreatic Cystic Neoplasms: Does F/U EUS-FNA Confirm or Alter the Diagnosis
17Quantitative proteomic analysis of pancreatic cyst fluid proteins associated with malignancy in intraductal papillary mucinous neoplasms
BackgroundThe application of advanced imaging technologies for identifying pancreatic cysts has become widespread. However, accurately differentiating between low-grade dysplasia (LGD), high-grade dysplasia (HGD), and invasive intraductal papillary mucinous neoplasms (IPMNs) remains a diagnostic challenge with current biomarkers, necessitating the development of novel biomarkers that can distinguish IPMN malignancy.MethodsCyst fluid samples were collected from nine IPMN patients (3 LGD, 3 HGD, and 3 invasive IPMN) during their pancreatectomies. An integrated proteomics approach that combines filter-aided sample preparation, stage tip-based high-pH fractionation, and high-resolution MS was applied to acquire in-depth proteomic data of pancreatic cyst fluid and discover marker candidates for IPMN malignancy. Biological processes of differentially expressed proteins that are related to pancreatic cysts and aggressive malignancy were analyzed using bioinformatics tools such as gene ontology analysis and Ingenuity pathway analysis. In order to confirm the validity of the marker candidates, 19 cyst fluid samples were analyzed by western blot.ResultsA dataset of 2992 proteins was constructed from pancreatic cyst fluid samples. A subsequent analysis found 2963 identified proteins in individual samples, 2837 of which were quantifiable. Differentially expressed proteins between histological grades of IPMN were associated with pancreatic diseases and malignancy according to ingenuity pathway analysis. Eighteen biomarker candidates that were differentially expressed across IPMN histological grades were discovered—7 DEPs that were upregulated and 11 that were downregulated in more malignant grades. HOOK1 and PTPN6 were validated by western blot in an independent cohort, the results of which were consistent with our proteomic data.ConclusionsThis study demonstrates that novel biomarker candidates for IPMN malignancy can be discovered through proteomic analysis of pancreatic cyst fluid.
Read moreEndoscopic Ultrasound (EUS) Guided Pancreatic Fluid Aspiration for DNA Analysis of KRAS and GNAS Mutations for the Evaluation of Pancreatic Cystic Neoplasia
Purpose: Discriminating benign from premalignant cysts of the pancreas is limited by the performance of cyst fluid aspirate and cytology. The presence of KRAS mutations in cyst fluid has been reported to increase the yield of cyst fluid aspirate for the presence of neoplastic cysts. Recently, mutations in GNAS have been found to have an oncogenic role in intraductal papillary mucinous neoplasms (IPMN) through promotion of adenyl cyclase activity and cAMP levels. Our aim was to evaluate whether analysis of cyst fluid DNA obtained by EUS for GNAS mutations can enhance the preoperative diagnosis of neoplastic pancreatic cysts. Methods: Endoscopic ultrasound-guided pancreatic cyst aspirates were collected during a 24-month period and investigated for cytology, carcinoembryonic antigen (CEA) level, and molecular analysis. KRAS and GNAS point mutations were detected by dideoxy chain termination sequencing (Applied Biosystems) of extracted DNA (qiagen) from cyst fluid. The cyst fluid analysis was compared to the final surgical pathology or cytology. Results: We identified 23 patients (11 male, mean age = 68.2 ± 13.8 [SD] years) with pancreatic cysts who underwent EUS-guided cyst aspiration. The mean cyst diameter was 2.3 cm (range 1-8 cm). Cysts were located in the head (n=10), body (n=8), and tail (n=5) of the pancreas. The cohort consisted of four serous cystadenomas (SCA), three mucinous cyst neoplasms (MCN), one pseudocyst, five solid-cystic adenocarcinoma (PDA), two solid-cystic endocrine neoplasms, one lymphoepithelial cyst, and seven IPMNs. The mean cyst fluid CEA levels in IPMNs was 3,895 ng/mL (range 22-24,747); 3/7 patients with IPMN had CEA levels <192 ng/mL. Mutations in KRAS were present in 5/7 (71%) IPMNs; LOH was demonstrated in three of these. Three of seven (43%) IPMNs carried a mutation in GNAS, all of which harbored degrees of dysplasia. The mutations in GNAS occurred at codon 201. All IPMNs carried a mutation in at least one of the two oncogenes. No mutations were identified in the SCA, in contrast to the IPMNs (p<0.01). GNAS mutations were not identified in any of the MCNs or PDA (p<0.005 when compared to IPMNs), although KRAS mutations were found in 1/3 (33%) of MCNs and 3/5 (60%) of PDAs. Cyst fluid CEA >192 ng/mL had a sensitivity of 57% to predict the presence of an IPMN. The sensitivity of KRAS mutation in cyst fluid was 71% for the diagnosis of IPMN, but with the addition of GNAS mutations, all IPMNs were identified (sensitivity = 100%, p<0.05). Conclusion: The presence of GNAS in combination with KRAS in pancreatic cystic fluid improves the sensitivity for diagnosis of IPMN when compared to CEA or KRAS alone. This demonstrates the feasibility of GNAS analysis from pancreatic cyst fluid obtained by EUS guided aspiration.
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