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Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates
  1. Dario de Biase1,
  2. Caterina de Luca2,
  3. Gianluca Gragnano2,
  4. Michela Visani3,
  5. Claudio Bellevicine2,
  6. Umberto Malapelle2,
  7. Giovanni Tallini3,
  8. Giancarlo Troncone2
  1. 1Department of Pharmacy and Biotechnology (FaBiT), Molecular Pathology Unit AUSL di Bologna, Bellaria Hospital, University of Bologna, Bologna, Italy
  2. 2Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, Napoli, Italy
  3. 3Department of Medicine (DIMES), Molecular Pathology Unit AUSL di Bologna, Bellaria Hospital, University of Bologna School of Medicine, Bologna, Italy
  1. Correspondence to Professor Giancarlo Troncone, Department of Public Health, Anatomic Pathology Unit, University of Napoli Federico II School of Medicine, via S. Pansini 5, Napoli 80131, Italy; giancarlo.troncone{at}unina.it

Abstract

Aims 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.

  • PANCREATIC CANCER
  • MOLECULAR BIOLOGY
  • CYTOLOGY

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