Evaluation of microRNA-10b prognostic significance in a prospective cohort of breast cancer patients

Abstract

Background: MicroRNA-10b (miR-10b) has a prominent role in regulating tumor invasion and metastasis by targeting the HOXD10 transcriptional repressor and has been found up-regulated in several tumor types.

Methods: We evaluated the expression of miR-10b in paired tumor and normal specimens obtained from a prospective cohort of breast cancer patients with at least 36 months follow-up enrolled according to the REMARK guidelines (n = 150). RNA quality was measured and only samples with RNA Integrity Number (RIN) ≥7.0 were analyzed.

Results: The relative expression of miR-10b in tumor as compared to its normal counterpart (RER) was determined by RT-qPCR. miR-10b RERs were higher in the subgroup of patients with synchronous metastases (n = 11, Median 0.25; IQR 0.11-1.02) as compared with patients without metastases (n = 90, Median 0.09; IQR 0.04-0.29) (p = 0.028). In the subgroup of patients without synchronous metastases (n = 90), higher miR-10b RERs were associated with increased risk of disease progression and death in both univariable (HR 1.16, p = 0.021 and HR 1.20, p = 0.015 respectively for 0.10 unitary increase of miR-10b RERs levels) and multivariable (HR1.30, p < 0.001, and HR 1.31, p = 0.003 respectively for 0.10 unitary increase of miR-10b RERs levels) Cox regression models. The addition of miR-10b RERs to the Nottingham Prognostic Index (NPI) provided an improvement in discrimination power and risk reclassification abilities for the clinical outcomes at 36 months. Survival C-indices significantly increased from 0.849 to 0.889 (p = 0.009) for OS and from 0.735 to 0.767 (p = 0.050) for DFS.

Conclusions: Our results provide evidences that the addition of miR-10b RERs to the prognostic factors used in clinical routine could improve the prediction abilities for both overall mortality and disease progression in breast cancer patients.

Figure 1

HOXD10 protein expression by immunohistochemistry. a) representative image of normal breast epithelium from an healthy individual (HBS1): HOXD10 was constitutively expressed in normal ductal and lobular epithelium and therefore were used as internal positive control. b) representative image of breast cancer case BC4 developing distant metastases (brain, bone, liver) during follow-up: miR-10b RERs were 0.78 and HOXD10 protein was expressed in 20% of cancer cells; c) and d) representative images of BC6 and BC7 non-metastatic breast cancer cases. HOXD10 showed a diffuse staining, miR-10b RERs were 0.01 and 0.42 respectively and percentage of stained cells were 70% and 100% respectively. Original magnification: 100X a, b, c, d images; 400X squared area of an image.

Figure 2

Diagram showing cases selection and RNA quality evaluation.