Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates.
OBJECTIVE
In cystic and solid pancreatic lesions, KRAS mutational status refines the diagnosis of uncertain endoscopic ultrasound (EUS) aspirates. This test should have a fast turnaround time and ideally be performed at the centre where the patient is diagnosed. The Idylla KRAS Mutation Test enables standardisation even in units without molecular expertise.


METHODS
The Idylla test was designed for use with formalin-fixed paraffin-embedded (FFPE) sections. However, we directly pipetted 3 µL (corresponding to 1/10th of a DNA preparation from the aspirate sample) in the cartridge, which was automatically run as if an FFPE sample had been inserted. The performance was compared with Sanger sequencing, Allele Specific Locked Nucleic Acid PCR (ASLNAqPCR), and 454 Next Generation Sequencing (454-NGS) in light of clinicopathological end points.


RESULTS
Idylla yielded valid results in 49/52 (94.2%) cases, in 2 h. A total of 18/49 cases showed mutation either in KRAS exon 2 (14/18) or in exon 3 (4/18). Idylla KRAS test had 100% specificity and a sensitivity (55.1%) higher than Sanger sequencing (41.3%) and identical to ASLNAqPCR (55.1%). When the low-abundant mutant allele (<5%) cases were excluded from the analysis, the Idylla KRAS Mutation Test clinical sensitivity increased to 61.9% approaching that of 454-NGS (66.6%).


CONCLUSIONS
This is the first study that applied the novel Idylla KRAS test to the clinical setting of pancreatic cancer. In particular, this system can be easily implemented in the routine assessment of pancreatic EUS-fine-needle aspiration-derived DNA samples to quickly provide information on KRAS mutational status to supplement cytological evaluation.
Department of Pharmacy and Biotechnology (FaBiT), Molecular Pathology Unit AUSL di Bologna, Bellaria Hospital, University of Bologna, Bologna, Italy.
Dario
D
de Biase
Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy.
Caterina
C
de Luca
Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy.
Gianluca
G
Gragnano
Department of Medicine (DIMES), Molecular Pathology Unit AUSL di Bologna, Bellaria Hospital, University of Bologna School of Medicine, Bologna, Italy.
Michela
M
Visani
Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy.
Claudio
http://orcid.org/0000-0002-7479-6457
C
Bellevicine
Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy.
Umberto
U
Malapelle
Department of Medicine (DIMES), Molecular Pathology Unit AUSL di Bologna, Bellaria Hospital, University of Bologna School of Medicine, Bologna, Italy.
Giovanni
G
Tallini
Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy.
Giancarlo
G
Troncone
CYTOLOGY; MOLECULAR BIOLOGY; PANCREATIC CANCER