Abstract
Background: Liquid biopsy (LB) provides an examination of the peripheral blood of cancer patients for circulating tumor cells, cell-free nucleic acids and microRNAs (miRNAs) and is an established tool of precision medicine. Unlike most previous LB studies that focused on advanced metastatic colorectal cancer (CRC), we assessed miRNA dysregulation in blood samples obtained on the day of surgery from patients with primary CRC lesions but no clinical evidence of extra-colonic diffusion. In this study, plasma preparation included miRNAs associated to exosomes, but excluded large macrovesicles from the preparation. Methods: The miRNA profile in plasma isolated from a cohort of 35 CRC patients at the day of surgery was analyzed by Next Generation Sequencing (NGS) and further confirmed by droplet digital RT-PCR (dd-RT-PCR). Results: A miR-141-3p/miR-221-3p/miR-222-3p upregulation signature previously described in advanced CRC did not discriminate the analyzed early-CRC cohort from six tumor-free donors (Tf-D). In contrast, NGS-based miRNome analysis of a training cohort of five CRC and three tumor-free donors identified a novel, distinct nine miRNA signature comprising five up-regulated and four down-regulated miRNAs, six of which could be confirmed in the full CRC and tumor-free donor validation dataset by dd-RT-PCR. Additionally, a KRAS (Kirsten Rat Sarcoma Viral Oncogene Homolog) mutant status was correlated with the plasma content of three identified miRNAs. Conclusions: When the data obtained were comparatively evaluated, at least one of the miRNAs belonging to the signature list was found to be dysregulated in 34/35 (97.1%) of our early-CRC plasma samples. The miRNA list provides diagnostic markers as well as possible molecular targets for protocols focusing on “microRNA therapeutics”.
Highlights
Colorectal cancer (CRC) is the third most common malignancy and the fourth leading cause of cancer death worldwide [1,2,3,4]
We show that three blood miRNAs, long known to be upregulated in advanced colorectal cancer (CRC), are weakly associated with early CRC
Spearman’s correlation coefficient was used to detect association between miRNA plasma levels. To our knowledge this is the first report, with the only noted exception of miR-483-5p [52], that provides evidence for the discrimination ability (CRC vs. tumor-free donors) of the nine miRNAs making up our signature
Summary
Colorectal cancer (CRC) is the third most common malignancy and the fourth leading cause of cancer death worldwide [1,2,3,4]. Stool and blood tests to identify methylated DNA are among the preferred methods for the screening of pre-symptomatic and pauci-symptomatic CRC patients [7]. They are far from optimal, opening several issues, such as proband compliance, costs, standardization, and false negatives/positives. Unlike most previous LB studies that focused on advanced metastatic colorectal cancer (CRC), we assessed miRNA dysregulation in blood samples obtained on the day of surgery from patients with primary CRC lesions but no clinical evidence of extra-colonic diffusion. Results: A miR-141-3p/miR-221-3p/miR-222-3p upregulation signature previously described in advanced CRC did not discriminate the analyzed early-CRC cohort from six tumor-free donors (Tf-D). Conclusions: When the data obtained were comparatively evaluated, at least one of the miRNAs belonging to the signature list was found to be dysregulated in
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