High Serum miR-19a Levels Are Associated with Inflammatory Breast Cancer and Are Predictive of Favorable Clinical Outcome in Patients with Metastatic HER2⁺ Inflammatory Breast Cancer

<div><p>Introduction</p><p>Altered serum microRNA (miRNA) levels may be correlated with a dysregulated expression pattern in parental tumor tissue and reflect the clinical evolution of disease. The overexpression of miR-21, miR-10b, and miR-19a is associated with the acquisition of malignant characteristics (increased tumor cell proliferation, migration, invasion, dissemination, and metastasis); thus, we determined their utility as serum biomarkers for aggressive breast cancer (HER2-overexpressed or -amplified [HER2⁺] and inflammatory breast cancer [IBC]).</p><p>Experimental Design</p><p>In this prospective study, we measured miR-21, miR-10b, and miR-19a levels using quantitative reverse transcriptase-polymerase chain reaction in the serum of 113 breast cancer patients and determined their association with clinicopathologic factors and clinical outcome. Thirty healthy donors with no history of cancer were enrolled as controls.</p><p>Results</p><p>Patients with non-metastatic HER2⁺ breast cancer had higher serum miR-21 median levels than patients with non-metastatic HER2⁻ disease (p = 0.044); whereas patients with metastatic HER2⁺ breast cancer had higher serum miR-10b median levels than patients with metastatic HER2⁻ disease (p = 0.0004). There were no significant differences in serum miR-19a median levels between HER2⁺ and HER2⁻ groups, regardless of the presence of metastases. High serum miR-19a levels were associated with IBC (p = 0.039). Patients with metastatic IBC had significantly higher serum miR-19a median levels than patients with metastatic non-IBC (p = 0.019). Finally, high serum miR-19a levels were associated with longer progression-free survival time (10.3 vs. 3.2 months; p = 0.022) and longer overall survival time (median not reached vs. 11.2 months; p = 0.003) in patients with metastatic HER2⁺ IBC.</p><p>Conclusion</p><p>High levels of miR-21 and miR-10b were present in the serum of patients with non-metastatic and metastatic HER2⁺ breast cancer, respectively. High levels of serum miR-19a may represent a biomarker for IBC that is predictive for favorable clinical outcome in patients with metastatic HER2⁺ IBC.</p></div

Mean threshold cycle values of miR-192± standard deviation and 95% confidence interval in the serum of breast cancer patients and healthy donors.

<p>Unpaired <i>t</i>-test (Mann-Whitney U test).</p><p>SD: standard deviation; CI: confidence interval; Ct: threshold cycle.</p

Clinical characteristics of breast cancer patients at the beginning of the study and their association with serum miR-21, miR-10b, and miR-19a levels.

<p>Clinical characteristics of breast cancer patients at the beginning of the study and their association with serum miR-21, miR-10b, and miR-19a levels.</p

Mean threshold cycle values of miR-192± standard deviation in the serum of breast cancer patients and healthy donors.

<p>No significant differences in mean threshold cycle (Ct) values of miR-192 were observed among the serum of different groups (Kruskal–Wallis test, p = 0.785).</p

Serum miR-19a levels in patients with MIBC and MNIBC.

<p>The box plots show: a) relative serum miR-19a levels in patients with MNIBC and MIBC; and b) relative serum miR-19a levels in patients with MNIBC HER2⁻, MIBC HER2⁻, MNIBC HER2⁺ and MIBC HER2⁺. Thirty HDs were included as controls. The differences in serum miR-19a levels were evaluated using the Mann-Whitney U test, and the p values are indicated above the plots.</p

Kaplan-Meier plots of breast cancer patients according to serum miR-19a levels.

<p>The Kaplan-Meier plots show the survival time of breast cancer patients according to serum miR-19a levels. In a) patients with MIBC HER2⁺ and high serum miR-19a levels had longer PFS time (10.3 vs. 3.2 months; p = 0.022) and OS time (median not reached vs. 11.2 months; p = 0.003) than patients with MIBC HER2⁺ and with low serum miR-19a levels; in b) patients with MNIBC HER2⁺ and high serum miR-19a levels had longer PFS time (7.7 vs. 5.1 months; p = 0.061) and OS time (32.9 vs. 13.3 months; p = 0.015) than patients with MNIBC HER2⁺ and low serum miR-19a levels. High and low serum miR-19a levels were defined according to a cut-off corresponding to the mean values of miR-19a in the serum of HDs plus 2 standard deviations. A log-rank test was used to analyze the differences in the survival times between patients with high and low serum miR-19a levels. Characteristics of patients with MIBC HER2⁺ and high serum miR-19a levels: median age (48.3); trastuzumab-treated (12/17 = 70.6%); treatment-naïve (3/17 = 17.6%); no trastuzumab-treated (2/17 = 11.8%). Characteristics of patients with MIBC HER2⁺ and low serum miR-19a levels: median age (53.2); trastuzumab-treated (5/10 = 50.0%); treatment-naïve (0/10 = 0.0%); no trastuzumab-treated (5/10 = 50.0%). Characteristics of patients with MNIBC HER2⁺ and high serum miR-19a levels: median age (53.8); trastuzumab-treated (5/10 = 50.0%); treatment-naïve (2/10 = 20.0%), no trastuzumab-treated (3/10 = 30.0%). Characteristics of patients with MNIBC HER2⁺ and low serum miR-19a levels: median age (54.1); trastuzumab-treated (11/14 = 78.6%); treatment-naïve (1/14 = 7.1%); no trastuzumab-treated (2/14 = 14.3%).</p

Immune conditioned media induces migration and adhesion in IBC cells.

<p>(a) Conditioned media (CM) was collected from healthy donor peripheral blood mononuclear cells stimulated for 48 hours with LPS (LPS-CM), through the T-cell receptor (TCR-CM) or left unstimulated (US-CM). CM were added to established SUM149 cultures at 25% of media volume and incubated an additional 48 hours prior assaying. (b) SUM149 were grown on an xCelligence E-plate and exposed to TCR-CM, LPS-CM and US-CM at time 0. Cell index was measured at 15-minute intervals. Robust changes are observed at 9 hours after treatment. (c) Migration towards fetal bovine serum (FBS) is enhanced by immune CM. TCR-CM (blue line) induces rapid migration of SUM149 cells. LPS-CM enhancement of migration (green line) is noted from 7 to ~36 hours, but is not significantly different from controls (US-CM, Media, no FBS; red, pink and light blue) at later time points.</p

Analysis of multiple cell lines by Fluidigm.

<p>Breast cancer cell lines were incubated with immune CM for 48 hours and mRNA was analyzed by qRT-PCR. Media control appears as a solid black line at 1 in the center of each plot, points falling outside the circle represent increased relative expression and lines inside are decreased. TCR-CM anti-CD3 induced EMT-related factors to varying degrees in all cell lines. Following treatment with TCR-CM, IBC cell lines with the exception of KPL4, had increased expression of E-cadherin.</p

Conditioned media from activated healthy donor PBMC induces EMT in IBC.

<p>(a) Morphological changes visualized by bright field in IBC cell lines consistent with stress and EMT were observed following 48-hour incubation with CM. (b) Immunohistochemical stains of paraffin cell blocks: the percentage of positive cells is listed in each image and shown in the bar graph at right. Pancytokeratin expression is shown for both percent of cells with membrane localization (top number) and cytoplasmic localization (bottom number). MCF-7 control cells show a characteristic epithelial phenotype with high E-cadherin, low vimentin, low keratin 5/6 expression and strong membrane and cytoplasmic localization of cytokeratins, MDA-231 control cells are mostly mesenchymal with low E-cadherin, high vimentin and decreased cytokeratins. Following exposure to TCR-CM, SUM149 cells show increased expression of E-cadherin, vimentin, keratin 5/6 staining and decreased pan cytokeratin staining. (c) Expression levels of EMT-related transcription factors SNAIL, ZEB1 and TG2 were quantified by Taq-Man qRT-PCR. TCR-CM and to a lesser extent LPS-CM, induced large increases in ZEB1 and TG2.</p