Next Generation Sequencing Improves the Accuracy of KRAS Mutation Analysis in Endoscopic Ultrasound Fine Needle Aspiration Pancreatic Lesions

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.

Fully automated PCR detection of KRAS mutations on pancreatic endoscopic ultrasound fine-needle aspirates

“…In this retrospective study, the clinical performance of the Idylla KRAS test was assessed on archival DNA from a well-characterised series of EUS-FNAs that had already been tested for KRAS mutational status in a prior study by using three different techniques—Sanger sequencing, Allele Specific Locked Nucleic Acid PCR (ASLNAqPCR) and 454 Next Generation Sequencing (454-NGS)—as previously reported 13. Details regarding the modality of EUS-FNA sample collection and specimen handling and preparation for microscopic observation have been described 13.…”

“…To date, real-time PCR (RT-PCR) assays have mostly been designed to target only exon 2 ‘hot-spot’ mutations 9 13. Conversely, next-generation sequencing (NGS) ensures analytical sensitivity similar to that of mutation-specific assays, allowing for the detection of common and uncommon mutations, including those of KRAS exons 3 and 4 13. However, the NGS procedure requires a complex validation procedure,14 being cost-effective only in large-volume centralised laboratories 15…”

Lymph Node Fine-Needle Cytology: Beyond Flow Cytometry

“…Therefore, different NGS platforms use FNC to harvest cells from different tumors [92,93,94,95,96,97,98,99,100]. These studies demonstrated that NGS-based mutational profiling can be performed with a few nanograms of DNA (∼40 ng/μl), which can be easily obtained from FNC in different tumors [93,94,95,96,97,98]. Therefore, NGS may enhance the molecular FNC potential, providing mutational information on genes with high diagnostic and predictive relevance, such as EGFR, BRAF, K/N/HRAS, KIT, PTEN, CDKN2A, just to mention some, thus contributing to personalized therapies.…”

Variant Profiling of Candidate Genes in Pancreatic Ductal Adenocarcinoma

“…This validation demonstrates the reliability of the AMP-based assay for detection of a broad range of variants. A previous study of KRAS mutations that used fine needle aspiration material from various pancreatic lesions showed that the NGSbased method had higher sensitivity than Sanger sequencing and allele-specific locked nucleic acid PCR (16 ). The ability of NGS-based assays to use small amounts of input material, e.g., from fine needle aspiration and cyst fluid, while simultaneously testing a panel of important genes, makes this method potentially useful for preoperative clinical management of pancreatic lesions.…”