Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts

Published:August 05, 2015DOI:

      Background and Aims

      The value of next-generation sequencing (NGS) of pancreatic cyst fluid relative to the clinical and imaging impression has not been well-studied. The aim of this study was to assess the impact of NGS on the clinical diagnosis from imaging and carcinoembryonic antigen (CEA) and thus the management of pancreatic cysts.


      Ninety-two pancreatic cyst fluids from 86 patients were analyzed by cytology, CEA, and targeted NGS. Cysts were classified by imaging as nonmucinous, mucinous, or not specified. NGS results were compared with the imaging impression stratified by CEA and cytology.


      NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels. The VHL gene in 2 intraductal papillary mucinous neoplasms (IPMNs) supported a serous cystadenoma. Twenty percent of cysts that were nonmucinous by imaging were mucinous by NGS. Of the 14 not-specific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%). A KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%. A KRAS mutation reclassified 19% of nonneoplastic cysts with nonmucinous CEA as mucinous. Seven cyst fluids (8%) had either a TP53 mutation or loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5 of 7 (71%) were clinically malignant, and high-grade cytology was detected in all 5. Overall, CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86% vs 57%).


      NGS of pancreatic cyst fluid impacts clinical diagnosis and patient management by defining, supporting, or changing the clinical diagnosis based on imaging and CEA. NGS was most valuable in identifying mucinous cysts with nonmucinous CEA. An added benefit is the potential to detect mutations late in the progression to malignancy that may increase the risk classification of the cyst based on imaging and cytology.


      CEA (carcinoembryonic antigen), EUS-FNA (EUS-guided FNA), HGA (high-grade atypia), IPMN (intraductal papillary mucinous neoplasm), NGS (next-generation sequencing), PCR (polymerase chain reaction), PDAC (pancreatic duct adenocarcinoma)
      To read this article in full you will need to make a payment


      Subscribe to Gastrointestinal Endoscopy
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect


        • Laffan T.A.
        • Horton K.M.
        • Klein A.P.
        • et al.
        Prevalence of unsuspected pancreatic cysts on MDCT.
        AJR Am J Roentgenol. 2008; 191: 802-807
        • Spinelli K.S.
        • Fromwiller T.E.
        • Daniel R.A.
        • et al.
        Cystic pancreatic neoplasms: observe or operate.
        Ann Surg. 2004; 239 (discussion 7-9): 651-657
        • Fernández-del Castillo C.
        • Targarona J.
        • Thayer S.P.
        • et al.
        Incidental pancreatic cysts: clinicopathologic characteristics and comparison with symptomatic patients.
        Arch Surg. 2003; 138 (discussion 33-34): 427-433
        • Zhang X.M.
        • Mitchell D.G.
        • Dohke M.
        • et al.
        Pancreatic cysts: depiction on single-shot fast spin-echo MR images.
        Radiology. 2002; 223: 547-553
        • Pitman M.B.
        • Lewandrowski K.
        • Shen J.
        • et al.
        Pancreatic cysts: preoperative diagnosis and clinical management.
        Cancer Cytopathol. 2010; 118: 1-13
        • Kneuertz P.J.
        • Pitt H.A.
        • Bilimoria K.Y.
        • et al.
        Risk of morbidity and mortality following hepato-pancreato-biliary surgery.
        J Gastrointest Surg. 2012; 16: 1727-1735
        • Greenblatt D.Y.
        • Kelly K.J.
        • Rajamanickam V.
        • et al.
        Preoperative factors predict perioperative morbidity and mortality after pancreaticoduodenectomy.
        Ann Surg Oncol. 2011; 18: 2126-2135
        • Tanaka M.
        • Fernández-del Castillo C.
        • Adsay V.
        • et al.
        International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas.
        Pancreatology. 2012; 12: 183-197
        • Chebib I.
        • Yaeger K.
        • Mino-Kenudson M.
        • et al.
        The role of cytopathology and cyst fluid analysis in the preoperative diagnosis and management of pancreatic cysts >3 cm.
        Cancer Cytopathol. 2014; 122: 804-809
        • Sahani D.V.
        • Miller J.C.
        • del Castillo C.F.
        • et al.
        Cystic pancreatic lesions: classification and management.
        J Am Coll Radiol. 2009; 6: 376-380
        • Salvia R.
        • Malleo G.
        • Marchegiani G.
        • et al.
        Pancreatic resections for cystic neoplasms: from the surgeon's presumption to the pathologist's reality.
        Surgery. 2012; 152: S135-S142
        • Correa-Gallego C.
        • Ferrone C.R.
        • Thayer S.P.
        • et al.
        Incidental pancreatic cysts: Do we really know what we are watching?.
        Pancreatology. 2010; 10: 144-150
        • Khashab M.A.
        • Kim K.
        • Lennon A.M.
        • et al.
        Should we do EUS/FNA on patients with pancreatic cysts? The incremental diagnostic yield of EUS over CT/MRI for prediction of cystic neoplasms.
        Pancreas. 2013; 42: 717-721
        • Freeny P.C.
        • Saunders M.D.
        Moving beyond morphology: new insights into the characterization and management of cystic pancreatic lesions.
        Radiology. 2014; 272: 345-363
        • Pitman M.B.
        • Michaels P.J.
        • Deshpande V.
        • et al.
        Cytological and cyst fluid analysis of small (< or =3 cm) branch duct intraductal papillary mucinous neoplasms adds value to patient management decisions.
        Pancreatology. 2008; 8: 277-284
        • Genevay M.
        • Mino-Kenudson M.
        • Yaeger K.
        • et al.
        Cytology adds value to imaging studies for risk assessment of malignancy in pancreatic mucinous cysts.
        Ann Surg. 2011; 254: 977-983
        • Cizginer S.
        • Turner B.G.
        • Turner B.
        • et al.
        Cyst fluid carcinoembryonic antigen is an accurate diagnostic marker of pancreatic mucinous cysts.
        Pancreas. 2011; 40: 1024-1028
        • Pitman M.B.
        • Centeno B.A.
        • Genevay M.
        • et al.
        Grading epithelial atypia in endoscopic ultrasound-guided fine-needle aspiration of intraductal papillary mucinous neoplasms: an international interobserver concordance study.
        Cancer Cytopathol. 2013; 121: 729-736
        • Pitman M.B.
        • Centeno B.A.
        • Daglilar E.S.
        • et al.
        Cytological criteria of high-grade epithelial atypia in the cyst fluid of pancreatic intraductal papillary mucinous neoplasms.
        Cancer Cytopathol. 2014; 122: 40-47
        • Cho C.S.
        • Russ A.J.
        • Loeffler A.G.
        • et al.
        Preoperative classification of pancreatic cystic neoplasms: the clinical significance of diagnostic inaccuracy.
        Ann Surg Oncol. 2013; 20: 3112-3119
        • Miglio U.
        • Oldani A.
        • Mezzapelle R.
        • et al.
        KRAS mutational analysis in ductal adenocarcinoma of the pancreas and its clinical significance.
        Pathol Res Pract. 2014; 210: 307-311
        • Reid M.D.
        • Saka B.
        • Balci S.
        • et al.
        Molecular genetics of pancreatic neoplasms and their morphologic correlates: an update on recent advances and potential diagnostic applications.
        Am J Clin Pathol. 2014; 141: 168-180
        • Khalid A.
        • Zahid M.
        • Finkelstein S.D.
        • et al.
        Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study.
        Gastrointest Endosc. 2009; 69: 1095-1102
        • Shen J.
        • Brugge W.R.
        • Dimaio C.J.
        • et al.
        Molecular analysis of pancreatic cyst fluid: a comparative analysis with current practice of diagnosis.
        Cancer. 2009; 117: 217-227
        • Singhi A.D.
        • Nikiforova M.N.
        • Fasanella K.E.
        • et al.
        Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts.
        Clin Cancer Res. 2014; 20: 4381-4389
        • Kung J.S.
        • Lopez O.A.
        • McCoy E.E.
        • et al.
        Fluid genetic analyses predict the biological behavior of pancreatic cysts: three-year experience.
        JOP. 2014; 15: 427-432
        • Wu J.
        • Matthaei H.
        • Maitra A.
        • et al.
        Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development.
        Sci Transl Med. 2011; 3: 92ra66
        • Reid M.D.
        • Choi H.
        • Balci S.
        • et al.
        Serous cystic neoplasms of the pancreas: clinicopathologic and molecular characteristics.
        Semin Diagn Pathol. 2014; 31: 475-483
        • Charlesworth M.
        • Verbeke C.S.
        • Falk G.A.
        • et al.
        Pancreatic lesions in von Hippel-Lindau disease? A systematic review and meta-synthesis of the literature.
        J Gastrointest Surg. 2012; 16: 1422-1428
        • Takano S.
        • Fukasawa M.
        • Maekawa S.
        • et al.
        Deep sequencing of cancer-related genes revealed GNAS mutations to be associated with intraductal papillary mucinous neoplasms and its main pancreatic duct dilation.
        PLoS One. 2014; 9: e98718
        • Zheng Z.
        • Liebers M.
        • Zhelyazkova B.
        • et al.
        Anchored multiplex PCR for targeted next-generation sequencing.
        Nat Med. 2014; 20: 1479-1484
        • Brugge W.R.
        • Lewandrowski K.
        • Lee-Lewandrowski E.
        • et al.
        Diagnosis of pancreatic cystic neoplasms: a report of the cooperative pancreatic cyst study.
        Gastroenterology. 2004; 126: 1330-1336
        • Li H.
        • Durbin R.
        Fast and accurate short read alignment with Burrows-Wheeler transform.
        Bioinformatics. 2009; 25: 1754-1760
        • Cibulskis K.
        • Lawrence M.S.
        • Carter S.L.
        • et al.
        Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples.
        Nat Biotechnol. 2013; 31: 213-219
        • Jiao Y.
        • Shi C.
        • Edil B.H.
        • et al.
        DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors.
        Science. 2011; 331: 1199-1203
        • Al-Haddad M.
        • DeWitt J.
        • Sherman S.
        • et al.
        Performance characteristics of molecular (DNA) analysis for the diagnosis of mucinous pancreatic cysts.
        Gastrointest Endosc. 2014; 79: 79-87
        • Amato E.
        • Molin M.D.
        • Mafficini A.
        • et al.
        Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas.
        J Pathol. 2014; 233: 217-227
        • Wu J.
        • Jiao Y.
        • Dal Molin M.
        • et al.
        Whole-exome sequencing of neoplastic cysts of the pancreas reveals recurrent mutations in components of ubiquitin-dependent pathways.
        Proc Natl Acad Sci USA. 2011; 108: 21188-21193
        • Nikiforova M.N.
        • Khalid A.
        • Fasanella K.E.
        • et al.
        Integration of KRAS testing in the diagnosis of pancreatic cystic lesions: a clinical experience of 618 pancreatic cysts.
        Mod Pathol. 2013; 26: 1478-1487
        • Toll A.D.
        • Kowalski T.
        • Loren D.
        • et al.
        The added value of molecular testing in small pancreatic cysts.
        JOP. 2010; 11: 582-586
        • Layfield L.J.
        • Ehya H.
        • Filie A.C.
        • et al.
        Utilization of ancillary studies in the cytologic diagnosis of biliary and pancreatic lesions: The Papanicolaou Society of Cytopathology Guidelines.
        CytoJournal. 2014; 11: 4
        • Winner M.
        • Sethi A.
        • Poneros J.M.
        • et al.
        The role of molecular analysis in the diagnosis and surveillance of pancreatic cystic neoplasms.
        JOP. 2015; 16: 143-149
        • Ahmad N.A.
        • Kochman M.L.
        • Brensinger C.
        • et al.
        Interobserver agreement among endosonographers for the diagnosis of neoplastic versus non-neoplastic pancreatic cystic lesions.
        Gastrointest Endosc. 2003; 58: 59-64
        • Sigel C.S.
        • Edelweiss M.
        • Tong L.C.
        • et al.
        Low interobserver agreement in cytology grading of mucinous pancreatic neoplasms.
        Cancer Cytopathol. 2014; 123: 40-50
        • Do R.K.
        • Katz S.S.
        • Gollub M.J.
        • et al.
        Interobserver agreement for detection of malignant features of intraductal papillary mucinous neoplasms of the pancreas on MDCT.
        AJR Am J Roentgenol. 2014; 203: 973-979
        • de Jong K.
        • van Hooft J.E.
        • Nio C.Y.
        • et al.
        Accuracy of preoperative workup in a prospective series of surgically resected cystic pancreatic lesions.
        Scand J Gastroenterol. 2012; 47: 1056-1063
        • Lennon A.M.
        • Wolfgang C.L.
        • Canto M.I.
        • et al.
        The early detection of pancreatic cancer: What will it take to diagnose and treat curable pancreatic neoplasia?.
        Cancer Res. 2014; 74: 3381-3389
        • Hruban R.H.
        • Takaori K.
        • Klimstra D.S.
        • et al.
        An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms.
        Am J Surg Pathol. 2004; 28: 977-987
        • Murphy S.J.
        • Hart S.N.
        • Lima J.F.
        • et al.
        Genetic alterations associated with progression from pancreatic intraepithelial neoplasia to invasive pancreatic tumor.
        Gastroenterology. 2013; 145: 1098-1109.e1
        • Jones S.
        • Zhang X.
        • Parsons D.W.
        • et al.
        Core signaling pathways in human pancreatic cancers revealed by global genomic analyses.
        Science. 2008; 321: 1801-1806
        • Gaujoux S.
        • Brennan M.F.
        • Gonen M.
        • et al.
        Cystic lesions of the pancreas: changes in the presentation and management of 1,424 patients at a single institution over a 15-year time period.
        J Am Coll Surg. 2011; 212 (discussion, 3): 590-600