Validation of expression patterns for nine miRNAs in 204 lymph-node negative breast cancers

Abstract

Introduction: Although lymph node negative (LN-) breast cancer patients have a good 10-years survival (∼85%), most of them still receive adjuvant therapy, while only some benefit from this. More accurate prognostication of LN- breast cancer patient may reduce over- and under-treatment. Until now proliferation is the strongest prognostic factor for LN- breast cancer patients. The small molecule microRNA (miRNA) has opened a new window for prognostic markers, therapeutic targets and/or therapeutic components. Previously it has been shown that miR-18a/b, miR-25, miR-29c and miR-106b correlate to high proliferation.

Methods: The current study validates nine miRNAs (miR-18a/b miR-25, miR-29c, miR-106b, miR375, miR-424, miR-505 and let-7b) significantly correlated with established prognostic breast cancer biomarkers. Total RNA was isolated from 204 formaldehyde-fixed paraffin embedded (FFPE) LN- breast cancers and analyzed with quantitative real-time Polymerase Chain Reaction (qPCR). Independent T-test was used to detect significant correlation between miRNA expression level and the different clinicopathological features for breast cancer.

Results: Strong and significant associations were observed for high expression of miR-18a/b, miR-106b, miR-25 and miR-505 to high proliferation, oestrogen receptor negativity and cytokeratin 5/6 positivity. High expression of let-7b, miR-29c and miR-375 was detected in more differentiated tumours. Kaplan-Meier survival analysis showed that patients with high miR-106b expression had an 81% survival rate vs. 95% (P = 0.004) for patients with low expression.

Conclusion: High expression of miR-18a/b are strongly associated with basal-like breast cancer features, while miR-106b can identify a group with higher risk for developing distant metastases in the subgroup of Her2 negatives. Furthermore miR-106b can identify a group of patients with 100% survival within the otherwise considered high risk group of patients with high proliferation. Using miR-106b as a biomarker in conjunction to mitotic activity index could thereby possibly save 18% of the patients with high proliferation from overtreatment.

Figure 1. Expression level of miRNAs for different prognostic features.

Independent T-test was used to determined significant relationship. Let-7b is down-regulated in high MAI ≥10 (A), miR-25 (B) and miR-505 (C) are expressed higher in MAI ≥10. MiR-18a (D) and miR-18b (E) are expressed higher in ERα negative patients, while miR-375 (F) is down-regulated in ERα negative breast cancer patients.

Figure 2. Long-term distant metastasis free survival curves according to miR-106b expression level and miR-106b expression in patients with MAI≥10.

Figure 3. Supervised hierarchical clustering for ERα.

Genes were filtered using analysis of variance, p-value ≤0.0001 and absolute correlation for ERα. The heat-map diagram shows the result of the two-way hierarchical clustering of miRNAs and samples. Good prognosis indicates patients with DMFS, while worse prognosis refers to patients with distant metastasis or who have died of distant metastasis. Each row represents a miRNA and each column represents a patient sample. The miRNA clustering tree is shown on the left, and the sample clustering tree appears at the top. The colour scale shown at the bottom illustrates the relative expression level of a miRNA across all samples: red colour represents an expression level above mean, blue colour represents expression lower than the mean. Gray colour means that the specific miRNA has not been successfully detected with qPCR. Numbers for clinicopathological features indicate the following: EOFUS (0 = no distant metastasis, 1 = distant metastasis), MAI10 (0<10, 1≥10), ERα (0<1% positive tumour cells, 1≥1% positive tumour cells), PPH3_13 (0<13, 1≥13), KP_Ki67 (0<10%, 1≥10%), Her2 (0 = 0 or 1+, 1 = 2+ or 3+), TNP (0 = positive for either ERα/PR/Her2, 1 = negative for ERα and PR and Her2), PR (0≤10% positive tumour cells, 1>10% positive tumour cells), CK5/6 (0 = no staining, 1 = any percentage of positive tumour cells), Tsize (Tumour size: 0≤2cm, 1>2 cm) and Nottgrade (Nottingham grade: 1 = grade 1, 2 = grade 2, 3 = grade 3).

Figure 4. An illustration of the miRNAs impact on ERα, based on literature and results presented in this paper.