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Background: The DNA released into the bloodstream by malignant tumours· called circulating tumour DNA (ctDNA), is often a small fraction of total cell-free DNA shed predominantly by hematopoietic cells and is therefore challenging to detect. Understanding the biological properties of ctDNA is key to the investigation of its clinical relevance as a non-invasive marker for cancer detection and monitoring., Methods: We selected 40 plasma DNA samples of pancreatic cancer cases previously reported to carry a KRAS mutation at the 'hotspot' codon 12 and re-screened the cell-free DNA using a 4-size amplicons strategy (57 bp, 79 bp, 167 bp and 218 bp) combined with ultra-deep sequencing in order to investigate whether amplicon lengths could impact on the capacity of detection of ctDNA, which in turn could provide inference of ctDNA and non-malignant cell-free DNA size distribution., Findings: Higher KRAS amplicon size (167 bp and 218 bp) was associated with lower detectable cell-free DNA mutant allelic fractions (p < 0·0001), with up to 4·6-fold (95% CI: 2·6-8·1) difference on average when comparing the 218bp- and the 57bp-amplicons. The proportion of cases with detectable KRAS mutations was also hampered with increased amplicon lengths, with only half of the cases having detectable ctDNA using the 218 bp assay relative to those detected with amplicons less than 80 bp., Interpretation: Tumour-derived mutations are carried by shorter cell-free DNA fragments than fragments of wild-type allele. Targeting short amplicons increases the sensitivity of cell-free DNA assays for pancreatic cancer and should be taken into account for optimized assay design and for evaluating their clinical performance., Funding: IARC; MH CZ - DRO; MH SK; exchange program between IARC and Sao Paulo medical Sciences; French Cancer League., Competing Interests: Declaration of Competing Interest We declare no competing interests., (Copyright © 2019. Published by Elsevier B.V.)

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.