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

Abstract

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.

Diagnostic and Prognostic Utility of the Extracellular Vesicles Subpopulations Present in Pleural Effusion

“…In order to comprehensively separate the vesicles and soluble proteins associated with BE, MPM, and AD pleural effusion, we collected samples from 27 patients and subjected them to differential centrifugation [ 31 ]. Due to the distinct sizes and densities of these components, this allowed us to separate the cells, apoptotic bodies, microvesicles, and exosomes from the soluble proteins within the effusion.…”

Circulating extracellular vesicles in lung cancer patients are not enriched in tumor-derived DNA fragments as revealed by whole genome sequencing

“…Prior reports indicate that mutations could be more easily detected in exosomes or EV from plasma samples than in cfDNA [12, 13, 22–24]. The observed variation in the ctDNA fractions and detection rates reported in prior reports can be explained by the differences in the EV isolation methods, non-standardized EV characterization post-fractionation, the diversity of methods used for detecting tumor signal, or issues related to the stochastic noise for detecting molecules with mutations in low DNA concentrations.…”

“…Approaches based on mutation testing (e.g. digital PCR) have identified either increased or decreased tumor fraction in DNA from EV fraction [12][13][14].…”

Estimating the number of diseases – the concept of rare, ultra-rare, and hyper-rare

“…As genome-wide analyses of tumors have increased profoundly over the last few years ( Degasperi et al., 2022 ), they have already led to an improved appreciation of distinct malignant diseases ( Calderaro et al., 2017 ). Additionally, liquid biopsy analysis has the potential to revolutionize our understanding of tumor evolution by providing information in real-time, though research into the biology of blood-borne tumor material is still in its infancy ( Hagey et al., 2021 ). To this end, we believe that many more studies are needed before it is possible to reach any consensus on how many tumor subtypes exist and to what extent they represent different entities.…”