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Original article
Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia
  1. Aatur D Singhi1,
  2. Kevin McGrath2,
  3. Randall E Brand2,
  4. Asif Khalid2,
  5. Herbert J Zeh3,
  6. Jennifer S Chennat2,
  7. Kenneth E Fasanella2,
  8. Georgios I Papachristou2,
  9. Adam Slivka2,
  10. David L Bartlett3,
  11. Anil K Dasyam4,
  12. Melissa Hogg3,
  13. Kenneth K Lee3,
  14. James Wallis Marsh3,
  15. Sara E Monaco1,
  16. N Paul Ohori1,
  17. James F Pingpank3,
  18. Allan Tsung3,
  19. Amer H Zureikat3,
  20. Abigail I Wald1,
  21. Marina N Nikiforova1
  1. 1 Department of Pathology, University of Pittsburgh Medical Center Health System, Pittsburgh, Pennsylvania, USA
  2. 2 Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  3. 3 Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA
  4. 4 Department of Radiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
  1. Correspondence to Dr Aatur D Singhi, Department of Pathology, University of Pittsburgh Medical Center Health System, Pittsburgh, Pennsylvania, PA 15213, USA; singhiad{at}


Objective DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.

Design Over 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing.

Results KRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).

Conclusions In contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.

  • pancreatic cancer
  • pancreatic pathology
  • pancreatic epidemiology
  • pancreato-biliary disorders

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  • Contributors ADS, KM, REB, AK, HJZ, AS, AIW and MNN: study concept and design. ADS, KG, REB, AK, HJZ, JSC, KEF, GIP, AS, DLB, AKD, MH, KKL, JWM, SEM, NPO, JFP, AT, AHZ, AIW and MNN: acquisition of data. ADS, AIW and MNN: analysis and interpretation of data. ADS, KM, HJZ and MNN: drafting of the manuscript.

  • Funding This study was supported in part by grants from the Pancreatic Cancer Action Network (Translational Research Grant), National Pancreas Foundation, Western Pennsylvania Chapter and the University of Pittsburgh (to AD Singhi).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval University of Pittsburgh Institutional Review Board.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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