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.

1st Faculty of Medicine Charles University of Prague Institute of Hygiene and Epidemiology Prague Czechia

Faculty of Health Sciences Palacky University Olomouc Czechia

International Agency for Research on Cancer Genetic Cancer Susceptibility group 150 Cours Albert Thomas 69372 Lyon France

International Agency for Research on Cancer Genetic Cancer Susceptibility group 150 Cours Albert Thomas 69372 Lyon France; Faculty of Chemistry Lomonosov Moscow State University Moscow Russian Federation

International Agency for Research on Cancer Genetic Cancer Susceptibility group 150 Cours Albert Thomas 69372 Lyon France; Santa Casa de Sao Paulo of medical Sciences Sao Paulo Brazil

Masaryk Memorial Cancer Institute and Medical Faculty of Masaryk University Brno Czechia

Regional Authority of Public Health Banska Bystrica and Faculty of Health Catholic University Ružomberok Slovakia

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