P3-03-02: Higher Expression Levels of Circulating miR-21, miR-19a and miR-10b Are Associated with High Risk Features in Breast Cancer.
Authors: Anfossi S , Giordano A , Cohen EN , Gao H , Cristofanilli M , Valero V , Alvarez RH , Hortobagyi GN , Woodward W , Ueno NT , Lee B-N , Reuben JM 
Published: June 2012

Background: MicroRNAs (miRs) have oncogenic and tumor-suppressor functions. MiR-21, miR-19a and miR-10b are overexpressed in breast cancer and regulate tumor cell migration, invasion and angiogenesis. We assessed the levels of miR-21, -19a, and -10b in sera of breast cancer patients and their association with the stage, histological type, hormonal receptor (HR) status and HER2 amplification in the primary tumor. Since circulating tumor cells (CTC) detected by CellSearch® are an independent and strong predictor of overall survival in metastatic breast cancer (MBC), we assessed the relationship between circulating miRs and CTCs.

Methods: The study consisted of 30 healthy donors (HD) and 95 breast cancer (BC) patients. Patients’ sera were collected before starting a new line of treatment. Total RNA was isolated, reverse transcribed to cDNA and then subjected to qRT-PCR for the detection of miR-21, -19a, -10b and -192 using the TaqMan MicroRNA Assay (Applied Biosystems, Foster City, CA). Mir-192 was used to normalize the expression levels of the other miRs. Fold-changes in expression of miRs were calculated using the 2−DCt method, where DCt= mean CTtarget-miRNA -mean CTmiR-192. CTCs were enumerated using CellSearch™ (Veridex LLC, Warren, NJ). Mann-Whitney U test was used to determine differences in serum miR expression levels between patients and HD.

Results: Of the 95 BC patients, 39 were non-MBC and 56 MBC. Patients grouped according to the receptor expression by immunohistochemical staining consisted of 27 HR+HER2−, 30 HR+HER2+, 20 HRHER2+, and 18 HRHER2− triple negative BC (TNBC). MiR-21 and miR-19a were higher in non-MBC patients than in HD (p=.001, p&lt;.001, respectively). MiR-21, miR-19a and miR-10b levels were higher in metastatic patients than in HD (p&lt;.001, p&lt;.001, p=.038, respectively). MBC patients had a higher median level of miR-21 than that of non-MBC patients (p=.02). Patients with (HR+HER2+, HR−HER2+, TNBC) had significantly higher median levels of both miR-21 (p=.018; p=.009; p=.045) and miR-10b (p=.011; p=.014; p=.03) compared with HR+HER2− BC. HER2+ patients had higher median levels of both miR-21 and miR-10b than those of HER2− BC (p=.033; p=.01) and HD (p&lt;.001; p=.009). Further, median miR-19a expression was higher in IBC patients than in non-IBC patients (p=.025). Finally, patients with &lt;5 CTCs had a higher median expression level of miR-10b than that of patients with ≥5 CTCs (p=.042).

Discussion: High expression levels of miR-21, miR-19a and miR-10b in sera are observed in breast cancer patients, especially with advanced disease. HER2+ BC patients had higher serum levels of miR-21 and miR-10b than HER2−. IBC patients had a higher serum level of miR-19a than non-IBC patients. Moreover, patients with &lt;5 CTCs had high serum levels of miR-10b that can be induced by Twist1 during the epithelial-mesenchymal transition (EMT) and, in part, explain the inability of CellSearch® to detect CTCs undergoing EMT.

Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-03-02.