Circulating microRNAs as blood-based biomarkers for breast cancer

Background: Currently, there is a lack of non-invasive tumour biomarkers with appropriate sensitivity and specificity to be used in routine clinical testing. The use of circulating microRNAs (miRNAs) as cancer biomarkers has been hypothesised based on their presence and stability in the circulation. Promising initial results for these tiny RNAs has been obtained in the field of breast cancer diagnostics. However, the accurate quantification of circulating miRNAs is more challenging than expected. In particular, several pre- and analytical variables have an impact on their final quantification, including the quantification method. Recently, a new droplet digital PCR (ddPCR) system that can be also used for microRNA quantification has been developed and proved to be very promising in liquid biopsy applications. Experimental design and findings: In order to develop a precise and accurate technique for miRNA quantification, we tested and compared the feasibility of quantifying circulating miRNAs with the new BioRad ddPCR system when used with EvaGreen dye– and TaqMan probe–based assays. In plasma and serum of patients with cancer and healthy controls, two circulating miRNAs and one added exogenous miRNA were quantified with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. The EvaGreen-based assay was precise, reproducible and sensitive. In comparison with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma. EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs. Afterwards, we selected a panel of six miRNAs (miR-10b-5p, miR-145-5p, miR-181a-5p, miR-148b-3p, miR-425-5p, miR-6523p) derived from microarray experiments or described in literature as potential circulating biomarkers for breast cancer. Then, we assessed their absolute levels in two independent cohorts of breast cancer patients and disease-free controls (Italy; n = 56, and USA; n = 94) using EvaGreen-based ddPCR. MiR-148b-3p, miR-181a-5p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than in controls in both cohorts. For these three miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analysis. Higher levels of serum miR-10b-5p were associated with clinico-pathological features of poor prognosis. These results confirmed the significant discrimination between breast cancer patients and healthy controls and the direction of down regulation. Conclusion: This study establishes the basis for using on a ddPCR for quantifying circulating miRNA biomarkers. Both study cohorts revealed very good agreement in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients. This study finally powers the use of circulating miRNAs as cancer biomarkers and proposes miR181a-5p and miR-652-3p as diagnostic biomarker and miR10b-5p as prognostic biomarkers of breast cancer.