Abstract 3137: NGS-based detection of KRAS hotspot mutations in plasma cell-free DNA of pancreatic cancer cases

• Published in: Cancer research (Chicago, Ill.) Ill.), 2016-07, Vol.76 (14_Supplement), p.3137-3137
• Main Authors: Le Calvez-Kelm, Florence, Foll, Matthieu, Wozniak, Magdalena B., Durand, Geoffroy, Chopard, Priscilia, Pertesi, Maroulio, Delhomme, Tiffany, Holcatova, Ivana, Foretova, Lenka, Janout, Vladimir, Fabianova, Eleonora, Vallée, Maxime P., Brennan, Paul, McKay, James D., Byrnes, Graham, Scélo, Ghislaine
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.AM2016-3137

Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by hotspot mutations in the KRAS gene (codons 12, 13 or 61) in 85-90% of cases. Codon 12 KRAS mutations have been detected in pancreatic juice, blood and stool samples from pancreatic cancer cases and represent promising biomarkers for early detection. However, the proportion of tumor-derived KRAS mutations in cell-free DNA fragments (cfDNA) has shown large variations, probably because of the heterogeneity in biosamples and assays tested. Deep sequencing technologies (NGS) allow the identification of low-abundance somatic variants, but have not previously been applied to the detection of KRAS hotspot mutations in cfDNA of PDAC cases. Moreover, variant calling methods have rarely been tested against cancer-free individuals so the proportion of false positives is unknown. We investigated whether deep sequencing of KRAS mutations at codons 12, 13 and 61 in plasma samples could represent a robust assay to distinguish pancreatic cancer from chronic pancreatitis and healthy controls. Methods: We developed an Ion Torrent-based NGS KRAS assay (partial exons 2 and 3, totalling 208bp) to screen cfDNA from plasma samples of 461 PDAC cases, 154 individuals with chronic pancreatitis and 421 healthy controls. cfDNA extraction (>4ng) and sequencing (>1000x coverage on average, and absence of systematic high sequencing error rates on the 208bp) performed well on 431 (93%) PDAC cases, 138 (90%) chronic pancreatitis, and 388 (95%) controls. We fit a robust negative-binomial regression to estimate the distribution of the sequencing errors at each DNA bp position and identified outlying samples, which were considered as KRAS positive when q-value