Extracellular vesicles are the primary source of blood-borne tumour-derived mutant KRAS DNA early in pancreatic cancer

Up to now, the field of liquid biopsies has focused on circulating tumour DNA and cells, though extracellular vesicles (EVs) have been of increasing interest in recent years. Thus, reported sources of tumour-derived nucleic acids include leukocytes, platelets and apoptotic bodies (AB), as well as large (LEV) and small (SEV) EVs. Despite these competing claims, there has yet to be a standardized comparison of the tumour-derived DNA associated with different components of blood. To address this issue, we collected twenty-three blood samples from seventeen patients with pancreatic cancers of known mutant KRAS G12 genotype, and divided them into two groups based on the time of patient survival following sampling. After collecting red and white blood cells, we subjected 1 ml aliquots of platelet rich plasma to differential centrifugation in order to separate the platelets, ABs, LEVs, SEVs and soluble proteins (SP) present. We then confirmed the enrichment of specific blood components in each differential centrifugation fraction using electron microscopy, Western blotting, nanoparticle tracking analysis and bead-based multiplex flow cytometry assays. By targeting wild type and tumour-specific mutant KRAS alleles using digital PCR, we found that the levels of mutant KRAS DNA were highest in association with LEVs and SEVs early, and with SEVs and SP late in disease progression. Importantly, we established that SEVs were the most enriched in tumour-derived DNA throughout disease progression, and verified this association using size exclusion chromatography. This work provides important direction for the rapidly expanding field of liquid biopsies by supporting an increased focus on EVs as a source of tumour-derived DNA.