Molecular Cytology Applications on Pancreas and Biliary Tract

  • Rene GerhardEmail author
  • Roseann I. Wu
  • Norge Vergara


Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) and endoscopic retrograde cholangiopancreatography (ERCP) are today’s standard of care methods for obtaining a tissue diagnosis from the pancreatobiliary tract. The cytologic examination of material collected by either EUS-FNA or ERCP is usually sufficient for diagnosing the majority of pathologic conditions associated with the pancreatobiliary system. In addition to the essential role of cytomorphology for patient diagnosis and management, ancillary tests such as immunocytochemistry and molecular testing can be used to support the morphologic impression. Indeed, immunocytochemical stains for pancreatic neuroendocrine tumors, fluorescence in situ hybridization (FISH) for the evaluation of biliary tract strictures, and molecular analysis of KRAS gene mutations to support the diagnosis of pancreatobiliary carcinomas have been accepted as reliable ancillary tests applied to cytologic specimens. However, controversies exist regarding the routine implementation of such tests due to limitations such as the need for a substantial amount of diagnostic material, the labor-intensive and time-consuming nature of some of the techniques, the moderate specificity, difficulties in interpreting the results, and the relative costs of molecular methods. Currently, there is not sufficient clinical evidence to support widespread adoption of most molecular tests. Nevertheless, recent studies and new techniques, such as next-generation sequencing (NGS), could provide more comprehensive knowledge of the molecular landscape of pancreatobiliary tumors that may result in the discovery of biomarkers for future targeted therapies.


  1. 1.
    Vilmann P, Jacobsen GK, Henriksen FW, et al. Endoscopic ultrasonography with guided fine needle aspiration biopsy in pancreatic disease. Gastrointest Endosc. 1992;38:172–3.CrossRefPubMedGoogle Scholar
  2. 2.
    Shi C, Daniels JA, Hruban RH. Molecular characterization of pancreatic neoplasms. Adv Anat Pathol. 2008;15:185–95.CrossRefPubMedGoogle Scholar
  3. 3.
    Hong SM, Park JY, Hruban RH, et al. Molecular signatures of pancreatic cancer. Arch Pathol Lab Med. 2011;135:716–27.PubMedPubMedCentralGoogle Scholar
  4. 4.
    Reid MD, 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;141:168–80.CrossRefPubMedGoogle Scholar
  5. 5.
    Klimstra DS. Nonductal neoplasms of the pancreas. Mod Pathol. 2007;20(Suppl 1):S94–112.CrossRefPubMedGoogle Scholar
  6. 6.
    Hruban RH, Klimstra DS. Adenocarcinoma of the pancreas. Semin Diagn Pathol. 2014;31:443–51.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Cowan RW, Maitra A. Genetic progression of pancreatic cancer. Cancer J. 2014;20:80–4.CrossRefPubMedGoogle Scholar
  8. 8.
    Layfield LJ, Ehya H, Filie AC, et al. Utilization of ancillary studies in the cytologic diagnosis of biliary and pancreatic lesions: the Papanicolaou Society of Cytopathology guidelines for pancreatobiliary cytology. Diagn Cytopathol. 2014;42:351–62.CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tanaka Y, Kato K, Notohara K, et al. Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res. 2001;61:8401–4.PubMedGoogle Scholar
  10. 10.
    Abraham SC, Klimstra DS, Wilentz RE, et al. Solid-pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations. Am J Pathol. 2002;160:1361–9.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    La Rosa S, Sessa F, Capella C. Acinar cell carcinoma of the pancreas: overview of clinicopathologic features and insights into the molecular pathology. Front Med. 2015;2:41.Google Scholar
  12. 12.
    Chmielecki J, Hutchinson KE, Frampton GM, et al. Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF fusions and frequent inactivation of DNA repair genes. Cancer Discov. 2014;4:1398–405.CrossRefPubMedGoogle Scholar
  13. 13.
    Bournet B, Gayral M, Torrisani J, et al. Role of endoscopic ultrasound in the molecular diagnosis of pancreatic cancer. World J Gastroenterol. 2014;20:10758–68.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Bournet B, Souque A, Senesse P, et al. Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with KRAS mutation assay to distinguish pancreatic cancer from pseudotumoral chronic pancreatitis. Endoscopy. 2009;41:552–7.CrossRefPubMedGoogle Scholar
  15. 15.
    Khalid A, McGrath K, Pal R, et al. Microdissection based genotyping improves the accuracy of EUS guided FNA of pancreatic tumors. Gastrointest Endosc. 2004;59:94.CrossRefGoogle Scholar
  16. 16.
    de Biase D, de Luca C, Gragnano G, et al. Fully automated PCR detection of KRAS mutations on pancreatic endoscopic fine-needle aspirates. J Clin Pathol. 2016.
  17. 17.
    Bournet B, Pointreau A, Souque A, et al. Gene expression signature of advanced pancreatic ductal adenocarcinoma using low density array on endoscopic ultrasound-guided fine needle aspiration samples. Pancreatology. 2012;12:27–34.CrossRefPubMedGoogle Scholar
  18. 18.
    Brand RE, Adai AT, Centeno BA, et al. A microRNA-based test improves endoscopic ultrasound-guided cytologic diagnosis of pancreatic cancer. Clin Gastroenterol Hepatol. 2014;12:1717–23.CrossRefPubMedGoogle Scholar
  19. 19.
    Kubiliun N, Ribeiro A, Fan YS, et al. EUS-FNA with rescue fluorescence in situ hybridization for the diagnosis of pancreatic carcinoma in patients with inconclusive on-site cytopathology results. Gastrointest Endosc. 2011;74:541–7.CrossRefPubMedGoogle Scholar
  20. 20.
    Winter JM, Ting AH, Vilardell F, et al. Absence of E-cadherin expression distinguishes noncohesive from cohesive pancreatic cancer. Clin Cancer Res. 2008;14:412–8.CrossRefPubMedGoogle Scholar
  21. 21.
    Higashi M, Yokoyama S, Yamamoto T, et al. Mucin expression in endoscopic ultrasound-guided fine-needle aspiration specimens is a useful prognostic factor in pancreatic ductal adenocarcinoma. Pancreas. 2015;44:728–34.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Carpizo DR, Allen PJ, Brennan MF. Current management of cystic neoplasms of the pancreas. Surgeon. 2008;6:298–307.CrossRefPubMedGoogle Scholar
  23. 23.
    Laffan TA, Horton KM, Klein AP, et al. Prevalence of unsuspected pancreatic cysts on MDCT. Am J Roentgenol. 2008;191:802–7.CrossRefGoogle Scholar
  24. 24.
    Lee KS, Sekhar A, Rofsky NM, et al. Prevalence of incidental pancreatic cysts in the adult population on MR imaging. Am J Gastroenterol. 2010;105:2079–84.CrossRefPubMedGoogle Scholar
  25. 25.
    Brugge WR, Lewandrowski K, Lee-Lewandrowski E, et al. The diagnosis of pancreatic cystic neoplasms: a report of the cooperative pancreatic cyst (CPC) study. Gastroenterology. 2004;126:1330–6.CrossRefPubMedGoogle Scholar
  26. 26.
    Cizginer S, Turner B, Bilge AR, et al. Cyst fluid carcinoembryonic antigen is an accurate diagnostic marker of pancreatic mucinous cysts. Pancreas. 2013;40:1024–8.CrossRefGoogle Scholar
  27. 27.
    Jimenez RE, Warshaw AL, Z’graggen K, et al. Sequential accumulation of KRAS mutations and p53 overexpression in the progression of pancreatic mucinous cystic neoplasms to malignancy. Ann Surg. 1999;230:501–9.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Z’graggen K, Rivera JA, Compton CC, et al. Prevalence of activating KRAS mutations in the evolutionary stages of neoplasia in intraductal papillary mucinous tumors of the pancreas. Ann Surg. 1997;226:498–500.Google Scholar
  29. 29.
    Biankin AV, Biankin SA, Kench JG, et al. Aberrant p16(INK4A) and DPC4/Smad4 expression in intraductal papillary mucinous tumors of the pancreas is associated with invasive ductal adenocarcinoma. Gut. 2002;50:861–8.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Iacobuzio-Donahue CA, Wilentz RE, Argani P, et al. Dpc4 protein in mucinous cystic neoplasms of the pancreas: frequent loss of expression in invasive carcinomas suggests a role in genetic progression. Am J Surg Pathol. 2000;24:1544–8.CrossRefPubMedGoogle Scholar
  31. 31.
    Lapkus O, Gologan O, Liu Y, et al. Determination of sequential mutation accumulation in pancreas and bile duct brushing cytology. Mod Pathol. 2006;19:907–13.CrossRefPubMedGoogle Scholar
  32. 32.
    Caldas C, Hahn SA, da Costa L, et al. Frequent somatic mutations and homozygous deletions of the p16 (MTS1) gene in pancreatic adenocarcinoma. Nat Genet. 1994;8:27–32.CrossRefPubMedGoogle Scholar
  33. 33.
    Shao J, Zhang L, Gao J, et al. Aberrant expression of PTCH (patched gene) and SMO (smoothened gene) in human pancreatic cancerous tissues and its association with hyperglycemia. Pancreas. 2006;33:38–44.CrossRefPubMedGoogle Scholar
  34. 34.
    Khalid A, Zahid M, Finkelstein SD, et al. Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study. Gastrointest Endosc. 2009;69:1095–102.CrossRefGoogle Scholar
  35. 35.
    Macgregor-Das AM, Iacobuzio-Donahue CA. Molecular pathways in pancreatic carcinogenesis. J Surg Oncol. 2013;107:8–14.CrossRefPubMedGoogle Scholar
  36. 36.
    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 U S A. 2011;27:21188–93.CrossRefGoogle Scholar
  37. 37.
    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.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Ryu JK, Matthaei H, dal Molin M, et al. Elevated microRNA miR-21 levels in pancreatic cyst fluid are predictive of mucinous precursor lesions of ductal adenocarcinoma. Pancreatology. 2011;11:343–50.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Matthaei H, Wylie D, Lloyd MB, et al. miRNA biomarkers in cyst fluid augment the diagnosis and management of pancreatic cysts. Clin Cancer Res. 2012;17:4713–24.CrossRefGoogle Scholar
  40. 40.
    Bellizzi AM, Stelow EB. Pancreatic cytopathology: a practical approach and review. Arch Pathol Lab Med. 2009;133:388–404.PubMedGoogle Scholar
  41. 41.
    Bournet B, Buscail C, Muscari F, et al. Targeting KRAS for diagnosis, prognosis, and treatment of pancreatic cancer: Hopes and realities. Eur J Cancer. 2016;54:75–83.CrossRefPubMedGoogle Scholar
  42. 42.
    Zagouri F, Sergentanis TN, Chrysikos D, et al. Molecularly targeted therapies in metastatic pancreatic cancer: a systematic review. Pancreas. 2013;42:760–73.CrossRefPubMedGoogle Scholar
  43. 43.
    Pitman MB, Layfield LJ. Guidelines for pancreaticobiliary cytology from the Papanicolaou Society of Cytopathology: a review. Cancer Cytopathol. 2014;122:399–411.CrossRefPubMedGoogle Scholar
  44. 44.
    Pitman MB, Centeno BA, Ali SZ, et al. Standardized terminology and nomenclature for pancreatobiliary cytology: the Papanicolaou Society of Cytopathology guidelines. Diagn Cytopathol. 2014;42:338–50.CrossRefPubMedGoogle Scholar
  45. 45.
    Layfield LJ, Ehya H, Filie AC, et al. Utilization of ancillary studies in the cytologic diagnosis of biliary and pancreatic lesions: the Papanicolaou Society of Cytopathology guidelines. Cytojournal. 2014;11(suppl 1):4.PubMedPubMedCentralGoogle Scholar
  46. 46.
    Gillis A, Cipollone I, Cousins G, et al. Does EUS-FNA molecular analysis carry additional value when compared to cytology in the diagnosis of pancreatic cystic neoplasm? A systematic review. HPB. 2015;17:377–86.CrossRefPubMedGoogle Scholar
  47. 47.
    Panarelli NC, Sela R, Schreiner AM, et al. Commercial molecular panels are of limited utility in the classification of pancreatic cystic lesions. Am J Surg Pathol. 2012;36:1434–43.CrossRefPubMedGoogle Scholar
  48. 48.
    Weber A, Schmid RA, Prinz C. Diagnostic approaches to cholangiocarcinoma. World J Gastroenterol. 2008;14:4131–6.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Sethi R, Singh K, Warner B, et al. The impact of brush cytology from endoscopic retrograde cholangiopancreatography (ERCP) on patient management at a UK teaching hospital. Frontline Gastroenterol. 2016;7:97–101.CrossRefPubMedGoogle Scholar
  50. 50.
    Brugge W, Dewitt J, Klapman JB, et al. Papanicolaou Society of Cytopathology. Techniques for cytologic sampling of pancreatic and bile duct lesions. Diagn Cytopathol. 2014;42:333–7.CrossRefPubMedGoogle Scholar
  51. 51.
    Stewart CJR, Mills PR, Carter R, et al. Brush cytology in the assessment of pancreatico-biliary strictures: a review of 406 cases. J Clin Pathol. 2001;54:449–55.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Govil H, Reddy V, Kluskens L, et al. Brush cytology of the biliary tract: retrospective study of 278 cases with histopathologic correlation. Diagn Cytopathol. 2002;26:273–7.CrossRefPubMedGoogle Scholar
  53. 53.
    Eiholm S, Thielsen P, Kromann-Andersen H. Endoscopic brush cytology from biliary duct system is still valuable. Dan Med J. 2013;60:A4656.PubMedGoogle Scholar
  54. 54.
    Mehmood S, Loya A, Yusuf MA. Biliary brush cytology revisited. Acta Cytol. 2016;60:167–72.CrossRefPubMedGoogle Scholar
  55. 55.
    Layfield LJ, Cramer H. Primary sclerosing cholangitis as a cause of false positive bile duct brushing cytology: report of two cases. Diagn Cytopathol. 2005;32:119–24.CrossRefPubMedGoogle Scholar
  56. 56.
    Logrono R, Kurtycz DF, Molina CP, et al. Analysis of false-negative diagnoses on endoscopic brush cytology of biliary and pancreatic duct strictures: the experience at 2 university hospitals. Arch Pathol Lab Med. 2000;124:387–92.PubMedGoogle Scholar
  57. 57.
    Kipp BR, Barr Fritcher EG, Pettengill JE, et al. Improving the accuracy of pancreatobiliary tract cytology with fluorescence in situ hybridization: a molecular test with proven clinical success. Cancer Cytopathol. 2013;121:610–9.CrossRefPubMedGoogle Scholar
  58. 58.
    Gonda TA, Glick MP, Sethi A, et al. Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures. Gastrointest Endosc. 2012;75:74–9.CrossRefPubMedGoogle Scholar
  59. 59.
    Vlajnic T, Somaini G, Savic S, et al. Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology. Cancer Cytopathol. 2014;122:627–34.CrossRefPubMedGoogle Scholar
  60. 60.
    Kipp BR, Stadheim LM, Halling SA, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am J Gastroenterol. 2004;99:1675–81.CrossRefPubMedGoogle Scholar
  61. 61.
    Barr Fritcher EG, Kipp BR, Halling KC, et al. A multivariable model using advanced cytologic methods for the evaluation of indeterminate pancreaticobiliary strictures. Gastroenterology. 2009;136:2180–6.CrossRefGoogle Scholar
  62. 62.
    Barr Fritcher EG, Voss JS, Jenkins SM, et al. Primary sclerosing cholangitis with equivocal cytology: fluorescence in situ hybridization and serum CA 19-9 predict risk of malignancy. Cancer Cytopathol. 2013;121:708–17.CrossRefPubMedGoogle Scholar
  63. 63.
    Sturm PD, Rauws EA, Hruban RH, et al. Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis. Clin Cancer Res. 1999;5:629–35.PubMedGoogle Scholar
  64. 64.
    van Heek NT, Clayton SJ, Sturm PD, et al. Comparison of the novel quantitative ARMS assay and an enriched PCR-ASO assay for K-ras mutations with conventional cytology on endobiliary brush cytology from 312 consecutive extrahepatic biliary stenoses. J Clin Pathol. 2005;58:1315–20.CrossRefPubMedPubMedCentralGoogle Scholar
  65. 65.
    Cai G, Mahooti S, Lipata FM, et al. Diagnostic value of K-ras mutation analysis for pancreaticobiliary cytology specimens with indeterminate diagnosis. Cancer Cytopathol. 2012;120:313–8.CrossRefPubMedGoogle Scholar
  66. 66.
    Kipp BR, Fritcher EG, Clayton AC, et al. Comparison of KRAS mutation analysis and FISH for detecting pancreatobiliary tract cancer in cytology specimens collected during endoscopic retrograde cholangiopancreatography. J Mol Diagn. 2010;12(6):780.CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Dudley JC, Zheng Z, McDonald T, et al. Next-generating sequencing and fluorescence in situ hybridization have comparable performance characteristics in the analysis of pancreaticobiliary brushings in malignancy. J Mol Diagn. 2016;18:124–30.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Service d’Anatomie Pathologique, Département de Médecine, Laboratoire National de SantéDudelangeLuxemburg
  2. 2.Department of Pathology and Laboratory MedicineHospital of the University of Pennsylvania, Perelman School of Medicine, University of PennsylvaniaPhiladelphiaUSA

Personalised recommendations