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

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

Author information

1
Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy ; Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
2
Department of Pharmacology and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.
3
Department of Medicine (DIMES) - Anatomic Pathology Unit, Bellaria Hospital, University of Bologna, Bologna, Italy.
4
Unit of Gastroenterology, Azienda Unità Sanitaria Locale di Bologna - Bellaria Hospital, Bologna, Italy.
5
School of Gastroenterology, University of Ferrara, Ferrara, Italy.
6
Anatomic Pathology Unit, Azienda Unità Sanitaria Locale di Bologna - Maggiore Hospital, Bologna, Italy.
7
Indiana University, Bloomington, Indiana, United States of America.
8
Unit of General Surgery, Azienda Unità Sanitaria Locale di Bologna - Maggiore Hospital, Bologna, Italy.

Abstract

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.

PMID:
24504548
PMCID:
PMC3913642
DOI:
10.1371/journal.pone.0087651
[Indexed for MEDLINE]
Free PMC Article

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