Extracellular vesicles from plasma have higher tumour RNA fraction than platelets

Abstract

In addition to Circulating Tumour Cells (CTCs), cell-free DNA (cfDNA) and Extracellular Vesicles (EVs), the notion of "Tumour-Educated Platelets" (TEP) has recently emerged as a potential source of tumour-derived biomarkers accessible through blood liquid biopsies. Here we sought to confirm the suitability of the platelet blood fraction for biomarker detection in comparison to their corresponding EV fraction. As publications have claimed that tumour RNA and other tumour-derived material are transferred from tumour cells to the platelets and that tumourderived transcripts can be detected in platelets, we chose to focus on RNA carrying a mutation as being of bona fide tumour origin. After informed consent, we collected prospective blood samples from a cohort of 12 melanoma patients with tissue-confirmed BRAF V600E mutation. Each blood specimen was processed immediately post collection applying two published standard protocols in parallel selecting for EVs and platelets, respectively. The RNA of each fraction was analysed by a highly sensitive ARMS RT-qPCR enabling the quantification of the mutant allele fraction (%MAF) of BRAF V600E down to 0.01%. In a direct comparative analysis, the EV fraction contained detectable BRAF V600E in 10 out of 12 patients, whereas none of the patient platelet fractions resulted in a mutant allele signal. The platelet fraction of all 12 patients contained high amounts of wild-type BRAF signal, but no mutation signal above background was detectable in any of the samples. Our observations suggest that the phenomenon of tumour RNA transfer to platelets occurs below detection limit since even a very sensitive qPCR assay did not allow for a reliable detection of BRAF V600E in the platelet fraction. In contrast, EV fractions derived from the same patients allowed for detection of BRAF V600E in 10 of 12 blood specimens.

Detecting therapy-guiding RNA aberrations in platelets of non-small cell lung cancer patients

“…However, none of the BRAF mutations were detected in platelet-derived RNA samples of these 12 patients while high amounts of wild-type BRAF transcripts were detected. This study concluded that BRAF p.(V600E) mutant transcripts are too rare in the platelet RNA fraction to be reliably detected [41]. So, these findings are similar to our findings for KRAS and both suggest low abundance of tumor derived transcripts in platelets.…”

“…The high number of wild type transcripts in platelet samples preclude reliable detection of tumor derived mutant transcripts, which are present at very low copy numbers. A comparative study detected BRAF p.(V600E) in 10 of 12 RNA samples from extracellular vesicles in melanoma patients [41]. However, none of the BRAF mutations were detected in platelet-derived RNA samples of these 12 patients while high amounts of wild-type BRAF transcripts were detected.…”

The role of exosomes in liquid biopsy for cancer diagnosis and prognosis prediction

“…As an important biological component of liquid biopsy, exosomes have several advantages compared to TEPs, CTCs and ctDNA. The RNA content of TEPs can likely be affected by therapeutic factors, pathological conditions and activated immune system 60 . CTCs and ctDNA are unstable, fragile and have short half‐lives, leading to the requirement of quick processing after sample collection 9 .…”

Diagnostic and Therapeutic Potential of Extracellular Vesicles in B-Cell Malignancies

“…Beyond miRNA content, RNA packed in plasma EV has the potential to be used to define tumor mutation landscape, such as BRAF V600E for melanoma, with the aim to detect early-stage disease or define the best clinical approach for certain patients (34). This strategy may be transposed to mutated RNA contained in circulating EV from B-cell malignancies, with the attempt to perform a personalized patient follow-up and therapy.…”