Circulating microRNAs as specific biomarkers for breast cancer detection

Enders K O Ng, Rufina Li, Vivian Y Shin, Hong Chuan Jin, Candy P H Leung, Edmond S K Ma, Roberta Pang, Daniel Chua, Kent-Man Chu, W L Law, Simon Y K Law, Ronnie T P Poon, Ava Kwong, Enders K O Ng, Rufina Li, Vivian Y Shin, Hong Chuan Jin, Candy P H Leung, Edmond S K Ma, Roberta Pang, Daniel Chua, Kent-Man Chu, W L Law, Simon Y K Law, Ronnie T P Poon, Ava Kwong

Abstract

Background: We previously showed microRNAs (miRNAs) in plasma are potential biomarkers for colorectal cancer detection. Here, we aimed to develop specific blood-based miRNA assay for breast cancer detection.

Methodology/principal findings: TaqMan-based miRNA profiling was performed in tumor, adjacent non-tumor, corresponding plasma from breast cancer patients, and plasma from matched healthy controls. All putative markers identified were verified in a training set of breast cancer patients. Selected markers were validated in a case-control cohort of 170 breast cancer patients, 100 controls, and 95 other types of cancers and then blindly validated in an independent set of 70 breast cancer patients and 50 healthy controls. Profiling results showed 8 miRNAs were concordantly up-regulated and 1 miRNA was concordantly down-regulated in both plasma and tumor tissue of breast cancer patients. Of the 8 up-regulated miRNAs, only 3 were significantly elevated (p<0.0001) before surgery and reduced after surgery in the training set. Results from the validation cohort showed that a combination of miR-145 and miR-451 was the best biomarker (p<0.0001) in discriminating breast cancer from healthy controls and all other types of cancers. In the blind validation, these plasma markers yielded Receiver Operating Characteristic (ROC) curve area of 0.931. The positive predictive value was 88% and the negative predictive value was 92%. Altered levels of these miRNAs in plasma have been detected not only in advanced stages but also early stages of tumors. The positive predictive value for ductal carcinoma in situ (DCIS) cases was 96%.

Conclusions: These results suggested that these circulating miRNAs could be a potential specific biomarker for breast cancer screening.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Schematic diagram of the workflow…
Figure 1. Schematic diagram of the workflow of this study.
Figure 2. MicroRNA selection and validation by…
Figure 2. MicroRNA selection and validation by quantitative RT-PCR analysis.
Changes of plasma levels of (A) miR-16, (B) miR-21, (C) miR-451 and (D) miR-145 in breast cancer patients (n = 15) before (pre-Op) and 14 days after (14 day post-Op) surgical removal of the tumor. Expression levels of the miRNAs (Log10 scale at Y-axis) are normalized to RNU6B. Statistically significant differences were determined using Wilcoxon tests.
Figure 3. Large-scale validation of miR-16, miR-21,…
Figure 3. Large-scale validation of miR-16, miR-21, miR-451 and miR-145 on an independent group of plasma samples (n = 270).
Box plot of plasma levels of (A) miR-16 and (B) miR-21 and (C) miR-451 (D) miR-145 in healthy normal (N) subjects (n = 100) and breast cancer patients (n = 170). Expression levels of the miRNAs are normalized to RNU6B. The lines inside the boxes denote the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. Filled circles indicate data points outside the 10th and 90th percentiles. Statistically significant differences were determined using Mann-Whitney tests.
Figure 4. ROC curve analysis using (…
Figure 4. ROC curve analysis using (A) plasma miR-16, (B) plasma miR-21, (C) plasma miR-451 (D) miR-145 for discriminating breast cancer from normal subjects.
Figure 5. Large-scale validation of combined plasma…
Figure 5. Large-scale validation of combined plasma miR-145 and miR451.
(A) Box plot of combined plasma miR-145 and miR-451 levels in patients of breast cancer (BC), healthy normal (N), colorectal cancer (CRC), esophagus cancer (EC), gastric cancer (GC), hepatocellular carcinoma (HCC) and lung cancer (LC). Expression levels of the miRNAs (Log10 scale at Y-axis) are calculated by the equation 2-ΔCt. ΔCt was calculated by subtracting the Ct values of miR-145 from the Ct values of the miR-451. The lines inside the boxes denote the medians. The boxes mark the interval between the 25th and 75th percentiles. The whiskers denote the interval between the 10th and 90th percentiles. Filled circles indicate data points outside the 10th and 90th percentiles. Statistically significant differences were determined using Mann-Whitney tests. (B) ROC analysis using combined plasma miR-451 and miR-145 for discriminating breast cancer from normal subjects and other cancers.

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