Combined evaluation of a panel of protein and miRNA serum‐exosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity

Abstract

Late diagnosis contributes to pancreatic cancer (PaCa) dismal prognosis, urging for reliable, early detection. Serum-exosome protein and/or miRNA markers might be suitable candidates, which we controlled for patients with PaCa. Protein markers were selected according to expression in exosomes of PaCa cell line culture supernatants, but not healthy donors' serumexosomes. miRNA was selected according to abundant recovery in microarrays of patients with PaCa, but not healthy donors' serum-exosomes and exosome-depleted serum. According to these preselections, serum-exosomes were tested by flow cytometry for the PaCa-initiating cell (PaCIC) markers CD44v6, Tspan8, EpCAM, MET and CD104. Serum-exosomes and exosomedepleted serum was tested for miR-1246, miR-4644, miR-3976 and miR-4306 recovery by qRT-PCR. The majority (95%) of patients with PaCa (131) and patients with nonPa-malignancies reacted with a panel of anti-CD44v6, -Tspan8, -EpCAM and -CD104. Serum-exosomes of healthy donors' and patients with nonmalignant diseases were not reactive. Recovery was tumor grading and staging independent including early stages. The selected miR-1246, miR-4644, miR-3976 and miR-4306 were significantly upregulated in 83% of PaCa serum-exosomes, but rarely in control groups. These miRNA were also elevated in exosome-depleted serum of patients with PaCa, but at a low level. Concomitant evaluation of PaCIC and miRNA serumexosome marker panels significantly improved sensitivity (1.00, CI: 0.95-1) with a specificity of 0.80 (CI: 0.67-0.90) for PaCa versus all others groups and of 0.93 (CI: 0.81-0.98) excluding nonPa-malignancies. Thus, the concomitant evaluation of PaCIC and PaCa-related miRNA marker panels awaits retrospective analyses of larger cohorts, as it should allow for a highly sensitive, minimally-invasive PaCa diagnostics.Pancreatic adenocarcinoma (PaCa), ranking fourth in cancerrelated mortality, remains the deadliest cancer. Chemotherapy-, and radiation-resistance, early spread and late diagnosis prohibiting resection account for nonsatisfactory therapeutic progress. 1 Serum markers like CA19-9 lack specificity and require additional diagnostic tools. 2 Recently, two noninvasive diagnostics attracted attention. Tumor-exosomes are readily detected in body fluids. Their protein, mRNA and miRNA profiles might serve for diagnosis. 3 It also was reported that serum miRNA differs between inflammation, benign and malignant tumors. 4

Carcinogenic activity of PbS quantum dots screened using exosomal biomarkers secreted from HEK293 cells

“…Intensification of miRNA expression signals has been reported in several recent studies. [73][74][75] However, the exosomal intensification effect on protein biomarkers was not as significant as its effect on miRNAs in this study. Further study is needed on the mechanism of miRNA and protein compartmentation into exosomes to explain the significant reduction of signal intensification of proteins compared to that of miRNAs.…”

Involvement of Mesenchymal Stem Cells in Cancer Progression and Metastases

“…Both types of EV therefore carry, in their membranes or in their soluble fractions, some characteristics of the plasma membrane of the cell from which they originate. An in depth description is beyond the scope of the current review, however, an example of this in cancer is the transmembrane molecule CD44v6 expressed on metastasizing cancer cells [43,64,65]. In the case of pancreatic adenocarcinoma, CD44v6 carried by tumor exosomes in their soluble matrix appears to play a role in premetastatic niche preparation by modulating cells of the organ in provision for tumor cell arrival and embedding [44,64].…”

Versatile roles of extracellular vesicles in cancer

“…Importantly, miRNAs can be readily detected in small-volume samples using specific and sensitive quantitative real-time PCR (qRT-PCR) (68). Additionally, EV-associated miRNAs have been used as diagnostic and prognostic markers (20,(69)(70)(71)(72)(73)(74)(75)(76)(77)(78)(79). Microarray analyses of miRNAs in EV-enriched fractions of serum from 88 patients with primary colorectal cancer (CRC) and 11 healthy controls revealed that that the levels of 7 miRNAs were significantly higher in the serum EVs of CRC patients compared with serum EVs from healthy donors (74).…”