Next generation sequencing improves the accuracy of KRAS mutation analysis in endoscopic ultrasound fine needle aspiration pancreatic lesions

PLoS One. 2014 Feb 4;9(2):e87651. doi: 10.1371/journal.pone.0087651. eCollection 2014.


The use of endoscopic ultrasonography has allowed for improved detection and pathologic analysis of fine needle aspirate material for pancreatic lesion diagnosis. The molecular analysis of KRAS has further improved the clinical sensitivity of preoperative analysis. For this reason, the use of highly analytical sensitive and specific molecular tests in the analysis of material from fine needle aspirate specimens has become of great importance. In the present study, 60 specimens from endoscopic ultrasonography fine needle aspirate were analyzed for KRAS exon 2 and exon 3 mutations, using three different techniques: Sanger sequencing, allele specific locked nucleic acid PCR and Next Generation sequencing (454 GS-Junior, Roche). Moreover, KRAS was also tested in wild-type samples, starting from DNA obtained from cytological smears after pathological evaluation. Sanger sequencing showed a clinical sensitivity for the detection of the KRAS mutation of 42.1%, allele specific locked nucleic acid of 52.8% and Next Generation of 73.7%. In two wild-type cases the re-sequencing starting from selected material allowed to detect a KRAS mutation, increasing the clinical sensitivity of next generation sequencing to 78.95%. The present study demonstrated that the performance of molecular analysis could be improved by using highly analytical sensitive techniques. The Next Generation Sequencing allowed to increase the clinical sensitivity of the test without decreasing the specificity of the analysis. Moreover we observed that it could be useful to repeat the analysis starting from selectable material, such as cytological smears to avoid false negative results.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Carcinoma, Pancreatic Ductal / diagnosis
  • Carcinoma, Pancreatic Ductal / genetics
  • Carcinoma, Pancreatic Ductal / pathology
  • DNA Mutational Analysis / methods
  • DNA Mutational Analysis / standards
  • Endoscopic Ultrasound-Guided Fine Needle Aspiration
  • Exons
  • Female
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Male
  • Middle Aged
  • Mutation*
  • Pancreatic Neoplasms / diagnosis
  • Pancreatic Neoplasms / genetics*
  • Pancreatic Neoplasms / pathology*
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins p21(ras)
  • Reproducibility of Results
  • Young Adult
  • ras Proteins / genetics*


  • KRAS protein, human
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins

Grant support

This study was supported in part by an Italian Government MURST grant (Grant no.20093XZC57_003) to GT. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.