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Multicenter Study
. 2016 Nov 29;7(48):78827-78840.
doi: 10.18632/oncotarget.12386.

KRAS Mutations in Blood Circulating Cell-Free DNA: A Pancreatic Cancer Case-Control

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Free PMC article
Multicenter Study

KRAS Mutations in Blood Circulating Cell-Free DNA: A Pancreatic Cancer Case-Control

Florence Le Calvez-Kelm et al. Oncotarget. .
Free PMC article

Abstract

The utility of KRAS mutations in plasma circulating cell-free DNA (cfDNA) samples as non-invasive biomarkers for the detection of pancreatic cancer has never been evaluated in a large case-control series. We applied a KRAS amplicon-based deep sequencing strategy combined with analytical pipeline specifically designed for the detection of low-abundance mutations to screen plasma samples of 437 pancreatic cancer cases, 141 chronic pancreatitis subjects, and 394 healthy controls. We detected mutations in 21.1% (N=92) of cases, of whom 82 (89.1%) carried at least one mutation at hotspot codons 12, 13 or 61, with mutant allelic fractions from 0.08% to 79%. Advanced stages were associated with an increased proportion of detection, with KRAS cfDNA mutations detected in 10.3%, 17,5% and 33.3% of cases with local, regional and systemic stages, respectively. We also detected KRAS cfDNA mutations in 3.7% (N=14) of healthy controls and in 4.3% (N=6) of subjects with chronic pancreatitis, but at significantly lower allelic fractions than in cases. Combining cfDNA KRAS mutations and CA19-9 plasma levels on a limited set of case-control samples did not improve the overall performance of the biomarkers as compared to CA19-9 alone. Whether the limited sensitivity and specificity observed in our series of KRAS mutations in plasma cfDNA as biomarkers for pancreatic cancer detection are attributable to methodological limitations or to the biology of cfDNA should be further assessed in large case-control series.

Keywords: KRAS mutations; cell-free DNA; pancreatic cancer detection; plasma.

Conflict of interest statement

CONFLICTS OF INTEREST

None.

Figures

Figure 1
Figure 1. Mutation detection of KRAS c.35G>T; p.G12V in serial dilution and cfDNA samples using the Needlestack approach
Negative-binomial regression plot at KRAS c.35G>T; p.G12V displaying the total number of reads (coverage, DP) and the number of reads matching the candidate variant (AO). Black solid line: Estimated error rate (e) at the c.35 position for this G>T base change. Blue dashed line: Detection limit at q-values <10−3; >30 in Phred scale (QVAL). Dots above the blue dashed line: Outliers of the regression (QVAL≥30), declared as mutant KRAS samples (c.35G>T; p.G12V). Dots below the blue dashed line: Inliers (QVAL<30) declared unmutated at this position for specified base change. A. Serial dilution of SW480 cell-lines in duplicates (N=28) and cfDNA from the pilot series (N=96) sequenced on a Ion Torrent PGM 316 Chip (e= 4.2×10−4); B. Serial dilution of SW480 cell-lines in duplicates (N=28) and cfDNA from the validation series (N=903) sequenced on Ion Torrent PGM 318 chips (e=1.4×10−4).
Figure 2
Figure 2. Distribution of KRAS mutations detected in plasma samples from pancreatic cases, chronic pancreatitis and healthy controls compared to somatic KRAS mutations reported in ICGC and COSMIC database
A. Comparison of cfDNA KRAS mutation location; B. Comparison of KRAS mutation spectrum at hotspot codons (12, 13 and 61). N= Number of KRAS mutations.

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