De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer

Abstract

Background: MicroRNAs (miRNAs) have been recently detected in the circulation of cancer patients, where they are associated with clinical parameters. Discovery profiling of circulating small RNAs has not been reported in breast cancer (BC), and was carried out in this study to identify blood-based small RNA markers of BC clinical outcome.

Methods: The pre-treatment sera of 42 stage II-III locally advanced and inflammatory BC patients who received neoadjuvant chemotherapy (NCT) followed by surgical tumor resection were analyzed for marker identification by deep sequencing all circulating small RNAs. An independent validation cohort of 26 stage II-III BC patients was used to assess the power of identified miRNA markers.

Results: More than 800 miRNA species were detected in the circulation, and observed patterns showed association with histopathological profiles of BC. Groups of circulating miRNAs differentially associated with ER/PR/HER2 status and inflammatory BC were identified. The relative levels of selected miRNAs measured by PCR showed consistency with their abundance determined by deep sequencing. Two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with clinical outcomes, including NCT response and relapse with metastatic disease. In the validation cohort, higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II-III BC patients.

Conclusions: Our study indicates that certain miRNAs can serve as potential blood-based biomarkers for NCT response, and that miR-122 prevalence in the circulation predicts BC metastasis in early-stage patients. These results may allow optimized chemotherapy treatments and preventive anti-metastasis interventions in future clinical applications.

Figure 1

MiRNAs detected in circulation. A. Unsupervised hierarchical clustering over all detected miRNAs. Colors in the heatmap represent a sample's higher (red) or lower (green) level in relation to the mean of all samples. The status of each clinical parameter was represented by colored boxes as indicated in the figure. B. The top 100 miRNAs with the highest quantities in circulation. For each miRNA shown, the range of normalized counts was indicated by a column, and the mean value was indicated by a line. Arrows indicate the positions of miR-122 and miR-375 in the order of abundance.

Figure 2

PCR-determined levels of miR-375 and miR-122 are associated with clinical outcome. Levels of miR-375 and miR-122 were measured by PCR and normalized to the level of miR-16 in each sample. Comparisons were carried out between the two groups of relapsed vs. non-relapsed (A) or pCR vs. non-pCR (B). For each group, mean and the standard error of the mean (SEM) are indicated. T-tests were performed between the two groups and P values are indicated.

Figure 3

Circulating miR-122 predicts metastatic relapse in an independent BC cohort. Levels of serum miR-122 in an independent validation cohort of 26 stage II-III BC patients were measured by PCR and normalized to the level of miR-16 in each sample.