Circulating miRNAs in Untreated Breast Cancer: An Exploratory Multimodality Morpho-Functional Study

Abstract

The aim of this study was to identify new disease-related circulating miRNAs with high diagnostic accuracy for breast cancer (BC) and to correlate their deregulation with the morpho-functional characteristics of the tumour, as assessed in vivo by positron emission tomography/magnetic resonance (PET/MR) imaging. A total of 77 untreated female BC patients underwent same-day PET/MR and blood collection, and 78 healthy donors were recruited as negative controls. The expression profile of 84 human miRNAs was screened by using miRNA PCR arrays and validated by real-time PCR. The validated miRNAs were correlated with the quantitative imaging parameters extracted from the primary BC samples. Circulating miR-125b-5p and miR-143-3p were upregulated in BC plasma and able to discriminate BC patients from healthy subjects (miR-125-5p area under the receiver operating characteristic ROC curve (AUC) = 0.85 and miR-143-3p AUC = 0.80). Circulating CA15-3, a soluble form of the transmembrane glycoprotein Mucin 1 (MUC-1) that is upregulated in epithelial cancer cells of different origins, was combined with miR-125b-5p and improved the diagnostic accuracy from 70% (CA15-3 alone) to 89% (CA15-3 plus miR-125b-5p). MiR-143-3p showed a strong and significant correlation with the stage of the disease, apparent diffusion coefficient (ADC_mean), reverse efflux volume transfer constant (Kep_mean) and maximum standardized uptake value (SUV_max), and it might represent a biomarker of tumour aggressiveness. Similarly, miR-125b-5p was correlated with stage and grade 2 but inversely correlated with the forward volume transfer constant (Ktrans_mean) and proliferation index (Ki67), suggesting a potential role as a biomarker of a relatively more favourable prognosis. In situ hybridization (ISH) experiments revealed that miR-143-3p was expressed in endothelial tumour cells, miR-125-5p in cancer-associated fibroblasts, and neither in epithelial tumour cells. Our results suggested that miR-125-5p and miR-143-3p are potential biomarkers for the risk stratification of BC, and Kaplan-Maier plots confirmed this hypothesis. In addition, the combined use of miR-125-b-5p and CA15-3 enhanced the diagnostic accuracy up to 89%. This is the first study that correlates circulating miRNAs with in vivo quantified tumour biology through PET/MR biomarkers. This integration elucidates the link between the plasmatic increase in these two potential circulating biomarkers and the biology of untreated BC. In conclusion, while miR-143-3b and miR-125b-5p provide valuable information for prognosis, a combination of miR-125b-5p with the tumour marker CA15-3 improves sensitivity for BC detection, which warrants consideration by further validation studies.

Keywords: PET/MRI; biomarkers; breast cancer; circulating miRNAs; imaging parameters.

Figure 1

Study design for the identification of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). * miRNAs significantly upregulated (p < 0.05) in BC patients vs. healthy donors (C,D).

Figure 1

Study design for the identification of circulating miRNAs and protein biomarkers able to discriminate breast cancer (BC) patients from healthy donors (A). miRNA expression analysis in a set population of 14 healthy donors (CTR) and 27 BC subjects. Expression analysis of 84 miRNAs by using a 96-well miScript miRNA PCR Array (B). * miRNAs significantly upregulated (p < 0.05) in BC patients vs. healthy donors (C,D).

Figure 2

Validation II. Relative expression of miRNAs selected in a study population of 78 healthy donors (CTR) and 77 breast cancer (BC) subjects. Box plot analyses performed to show the relative expression of miR-125b-5p (A) and miR-143-3p (B) in the plasma samples of healthy donors vs. BC patients. Logistic stepwise regression analysis (C). * miRNAs that significantly (p < 0.05) estimate the probability of having BC.

Figure 3

ROC curve analyses and AUC values of miR-125-5p (A), miR-143-3p (B), CA15-3 (C), and CEA (D) for discriminating BC patients from healthy controls in 76 BC patients and 78 healthy donors. ROC: Receiver operating characteristic; AUC: area under the ROC curve; CEA: carcinoembryonic antigen.

Figure 4

Correlation studies. Co-expression analyses of miR-125b-5p, miR-143-3p and miR-145-5p (A). Expression levels of miR-125b-5p vs. Ki67 (low <20%; high ≥20%) (B), and grade (C). Expression levels of miR-125b-5p (D), miR-143-3p (E), and CA15-3 (F) vs. stage. Statistical significance (G). Non-significant results are reported in bold.

Figure 5

Kaplan-Meier plots, which are based on breast cancer data from the METABRIC database, illustrate the survival probability for patients with low or high miR-125b (upper panel) and miR-143 (lower panel) expression levels in breast cancer. Hazard ratios (HRs) and p-values (log-rank test) were generated.

Figure 6

In situ hybridization staining patterns in benign and malignant breast tissues. Scrambled miR negative control probe and U6 positive control probe (A). miR-143-3p in normal (left) and BC tissues (right) (B). miR-145-5p in normal (left) and BC tissues (right) (C). miR-125b-5p in normal (left) and BC tissues (right) (D). miR-125b-5p in stromal tumour tissue (E). BC: breast cancer.

Figure 7

ROC curve analyses and AUC values combining the profiles of miR-125b-5p and CA15-3 (A) and miR-125-5p and miR-143-3p (B) for discriminating BC patients from healthy patients. ROC: Receiver operating characteristic; AUC: area under the ROC curve; BC: breast cancer.