Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR

Alessandra Mangolini, Manuela Ferracin, Maria Vittoria Zanzi, Elena Saccenti, Sayda Omer Ebnaof, Valentina Vultaggio Poma, Juana M Sanz, Angela Passaro, Massimo Pedriali, Antonio Frassoldati, Patrizia Querzoli, Silvia Sabbioni, Paolo Carcoforo, Alan Hollingsworth, Massimo Negrini, Alessandra Mangolini, Manuela Ferracin, Maria Vittoria Zanzi, Elena Saccenti, Sayda Omer Ebnaof, Valentina Vultaggio Poma, Juana M Sanz, Angela Passaro, Massimo Pedriali, Antonio Frassoldati, Patrizia Querzoli, Silvia Sabbioni, Paolo Carcoforo, Alan Hollingsworth, Massimo Negrini

Abstract

Background: Breast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15-3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique.

Results: We investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis.

Conclusions: The study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs.

Keywords: Breast cancer; Circulating miRNAs; Diagnostic markers; Droplet digital PCR; Prognostic markers.

Figures

Fig. 1
Fig. 1
Levels of miRNAs in sera of two independent cohorts of breast cancer and disease-free patients. The level of each miRNA was measured by the ddPCR technique and expressed in copies per microliter in each sample. Each miRNA displays comparable levels and consistent dysregulation in both cohorts. miR-652-3p and miR-148b-3p exhibited a statistically significant reduction in breast cancer patients in both cohorts. The unpaired t-test with Welch’s correction was performed to assess significance of differences between patient and control groups. P-values of less than 0.05 were deemed to be significant. BRCA breast cancer patients
Fig. 2
Fig. 2
Diagnostic potential of miR-148b and miR-652. Receiver operating characteristic (ROC) curve analyses show that miR-148b and miR-652 exhibit a significant ability to predict breast cancer in both cohorts. Binary logistic regression analysis was performed and ROC curves were generated to evaluate the ability of chosen miRNAs to distinguish cancer patients from controls. AUC area under the curve, CI confidence interval
Fig. 3
Fig. 3
Serum miR-10b-5p increases in patients presenting poor prognostic parameters. a Levels of miR-10b increased progressively according to tumor stage. Levels from patients with stages II-IV cancer exhibit a significant difference in comparison to those from patients with stage I cancer (p = 0.0047) or controls (p = 0.0028). The diagnostic value of miR-10b was assessed by receiver operating characteristic curve analysis. No significant difference was found between stage I and controls. BRCA breast cancer patients, AUC area under the curve. b Higher levels of miR-10b were associated with various clinicopathological parameters: lymph node metastasis (pN) (p = 0.014) and tumor grade (p = 0.026). Albeit not statistically significant, a trend toward increased levels of miR-10b was also detected in cases characterized by increased tumor size (pT) or as being human epidermal growth factor receptor 2 positive (HER2+) or estrogen receptor/progesterone receptor negative (ER-/PR-). “PRE” or “POST” refer to the menopausal status

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