Absolute quantification of cell-free microRNAs in cancer patients
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Manuela Ferracin1,2, Laura Lupini1, Irene Salamon1, Elena Saccenti3,7, Maria Vittoria Zanzi4, Andrea Rocchi6, Lucia Da Ros6, Barbara Zagatti1,2, Gentian Musa1, Cristian Bassi1, Alessandra Mangolini1, Giorgio Cavallesco1,5, Antonio Frassoldati6, Stefano Volpato7, Paolo Carcoforo1,4, Alan B. Hollingsworth8, Massimo Negrini1,2
1Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy
2Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy
3Section of Hematology, S. Anna University Hospital, Ferrara, Italy
4Breast Unit, S. Anna University Hospital, Ferrara, Italy
5Section of General and Thoracic Surgery, S. Anna University Hospital, Ferrara, Italy
6Clinical Oncology Unit, S. Anna University Hospital, Ferrara, Italy
7Department of Medical Sciences, University of Ferrara, Ferrara, Italy
8Department of Surgery, Mercy Hospital – OKC, Oklahoma City, OK, USA
Manuela Ferracin, e-mail: [email protected]
Massimo Negrini, e-mail: [email protected]
Keywords: cell-free microRNA, droplet digital PCR, breast cancer, cancer biomarkers
Received: February 20, 2015 Accepted: April 23, 2015 Published: May 02, 2015
The hypothesis to use microRNAs (miRNAs) circulating in the blood as cancer biomarkers was formulated some years ago based on promising initial results. After some exciting discoveries, however, it became evident that the accurate quantification of cell-free miRNAs was more challenging than expected. Difficulties were linked to the strong impact that many, if not all, pre- and post- analytical variables have on the final results. In this study, we used currently available high-throughput technologies to identify miRNAs present in plasma and serum of patients with breast, colorectal, lung, thyroid and melanoma tumors, and healthy controls. Then, we assessed the absolute level of nine different miRNAs (miR-320a, miR-21-5p, miR-378a-3p, miR-181a-5p, miR-3156-5p, miR-2110, miR-125a-5p, miR-425-5p, miR-766-3p) in 207 samples from healthy controls and cancer patients using droplet digital PCR (ddPCR) technology. We identified miRNAs specifically modulated in one or more cancer types, according to tissue source. The significant reduction of miR-181a-5p levels in breast cancer patients serum was further validated using two independent cohorts, one from Italy (n = 70) and one from US (n = 90), with AUC 0.66 and 0.73 respectively. This study finally powers the use of cell-free miRNAs as cancer biomarkers and propose miR-181a-5p as a diagnostic breast cancer biomarker.
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