Plasma microRNA panel for minimally invasive detection of breast cancer

Abstract

Over the last few years, circulating microRNAs (miRNAs) have emerged as promising novel and minimally invasive markers for various diseases, including cancer. We already showed that certain miRNAs are deregulated in the plasma of breast cancer patients when compared to healthy women. Herein we have further explored their potential to serve as breast cancer early detection markers in blood plasma. Circulating miR-127-3p, miR-376a and miR-652, selected as candidates from a miRNA array-based screening, were found to be associated with breast cancer for the first time (n = 417). Further we validated our previously reported circulating miRNAs (miR-148b, miR-376c, miR-409-3p and miR-801) in an independent cohort (n = 210) as elevated in the plasma of breast cancer patients compared to healthy women. We described, for the first time in breast cancer, an over-representation of deregulated miRNAs (miR-127-3p, miR-376a, miR-376c and miR-409-3p) originating from the chromosome 14q32 region. The inclusion of patients with benign breast tumors enabled the observation that miR-148b, miR-652 and miR-801 levels are even elevated in the plasma of women with benign tumors when compared to healthy controls. Furthermore, an analysis of samples stratified by cancer stage demonstrated that miR-127-3p, miR-148b, miR-409-3p, miR-652 and miR-801 can detect also stage I or stage II breast cancer thus making them attractive candidates for early detection. Finally, ROC curve analysis showed that a panel of these seven circulating miRNAs has substantial diagnostic potential with an AUC of 0.81 for the detection of benign and malignant breast tumors, which further increased to 0.86 in younger women (up to 50 years of age).

Figure 1. Investigation of three new miRNA marker candidates (miR-127-3p, miR-376a and miR-652) in cohort A.

Circulating miR-127-3p, miR-376a and miR-652 are present at elevated levels in the plasma of breast cancer patients when compared to healthy women. A two-tailed P<0.05 was considered significant (Wilcoxon rank sum test).

Figure 2. Independent validation of seven circulating miRNAs (cohort B).

Circulating miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652 and miR-801 levels were independently validated as being elevated in the plasma of malignant breast cancer patients compared to healthy women. Circulating miR-148b, miR-652 and miR-801 were also elevated in the plasma of women with benign breast tumors when compared to healthy individuals. A two-tailed P<0.05 was considered significant (Wilcoxon rank sum test).

Figure 3. Diagnostic potential of deregulated circulating miRNAs for benign and malignant breast tumors (cohort B).

In ROC curve analysis individual circulating miRNAs were found to have discriminatory accuracy of 0.59–0.75. A panel of seven circulating miRNAs (miR-127-3p, miR-148b, miR-376a, miR-376c, miR-409-3p, miR-652 and miR-801) discriminated between healthy women and those with benign and malignant breast tumors with an AUC of 0.81 and the discriminatory power was superior in younger women (AUC = 0.86).

Figure 4. MiRNA levels in benign and malignant breast tissue.

Levels of miR-127-3p, miR-376a and miR-652 are decreased in malignant primary breast cancer when compared to benign breast tissue. Box and whisker plots show RNU6B normalized relative miRNA levels for miR-127-3p and miR-652. As an exception un-normalized Ct values are presented for miR-376a as the normalization strategy was not applicable for this particular miRNA (due to the rather low miR-376a levels in the investigated tissue samples). A two-tailed P<0.05 was considered significant (Wilcoxon rank sum test).