Tissue and Serum miRNA Profile in Locally Advanced Breast Cancer (LABC) in Response to Neo-Adjuvant Chemotherapy (NAC) Treatment

<div><p>Introduction</p><p>MicroRNAs (miRNAs) are small non-coding RNA that plays a vital role in cancer progression. Neo-adjuvant chemotherapy (NAC) has become the standard of care for locally advanced breast cancer. The aim of this study was to evaluate miRNA alterations during NAC using multiple samples of tissue and serum to correlate miRNA expression with clinico-pathological features and patient outcomes.</p><p>Methods</p><p>Tissue and serum samples were collected from patients with locally advanced breast cancer undergoing NAC at four time points: time of diagnosis, after the first and fourth cycle of doxorubicin/cyclophosphamide treatment, and after the fourth cycle of docetaxel administration. First, we evaluated the miRNA expression profiles in tissue and correlated expression with clinico-pathological features. Then, a panel of four miRNAs (miR-451, miR-3200, miR-21, and miR-205) in serum samples was further validated using quantitative reverse-transcription polymerase chain reaction (RT-qPCR). The alterations in serum levels of miRNA, associations with clinical and pathological responses, correlation with clinico-pathological features, and survival outcomes were studied using Friedman, Mann-Whitney U, and Spearman, Wilcoxon signed-ranks tests. <i>P</i>≤0.05 was considered statistically significant.</p><p>Results</p><p>We analyzed 72 tissue samples and 108 serum samples from 9 patients and 27 patients, respectively. MicroRNA expression profiling of tumor versus normal tissue revealed more than 100 differentially expressed miRNAs. Serum miR-451 levels were significantly decreased during treatment, and higher serum levels were associated with improved clinical and pathological responses and disease-free survival. This is one of the early reports on miR-3200 in response to treatment in breast cancer, as serum levels of miR-3200 found to decline during NAC, and higher serum levels were associated with lower residual breast cancer burden and relapse rates at time of diagnosis.</p><p>Conclusion</p><p>Variations in serum miRNA levels during NAC treatment may be therapeutically significant for predicting response and survival outcomes.</p></div

Fold change in miR-451 and miR-3200 expression during treatment in relation to relapse status.

<p>Boxplots of (A) miR-451 and (B) miR-3200 expression over time comparing relapsed patients versus non-relapsed. *<i>P</i><0.05 using the Wilcoxon sign-rank test for the non-relapsing patients.</p

Median fold change expression changes in miR-451 in clinical responders (data shown for the clinical responders only).

<p>*Significant <i>p</i> values of <i>p</i><0.05 Wilcoxon sign rank test (n = 27).</p

Differential expression of some randomly selected miRNAs between normal and breast cancer tumor tissue samples with various fold changes.

<p>Differential expression of some randomly selected miRNAs between normal and breast cancer tumor tissue samples with various fold changes.</p

Disease-free survival and miR-451 expression.

<p>Kaplan-Meier estimates of disease-free survival based on low versus high expression of miR-451 at the time of diagnosis. Log-rank <i>P</i> = 0.046.</p

Cancer staging, clinical responses, and pathological responses before and after treatment.

<p>Cancer staging, clinical responses, and pathological responses before and after treatment.</p

Hierarchal clustering of healthy versus tumor breast cancer tissue samples.

<p>Over 100 detected miRNAs that were differentially expressed. Heat map colors represent miRNA expression as indicated in the color key where tumor samples are from the all patients whereas normal tissue from the full follow-up patients.</p

Correlation between miRNA expression and skin involvement.

<p>Boxplot of median miR-451 expression comparing patients with skin involvement versus patients with no skin involvement at different time points throughout chemotherapy (n = 27), Spearman correlation (<i>r</i> = -0.48, <i>P</i> = 0.028).</p

Differential expression of miRNAs in relation to clinico-pathological features using SAM analysis.

<p>(A) Tissue miRNAs significantly associated with Estrogen receptor (ER+) expression (<i>p</i>≤0.05). (B) Tissue miRNAs significantly associated with relapse (<i>p</i>≤0.05). (C) Tissue miRNAs significantly associated with pathological stage (<i>p</i>≤0.05). (D) Tissue miRNAs significantly associated with Basal-like (triple negative) (<i>p</i>≤0.05).</p

Schematic representation of the sample collection and study design.

<p>Schematic representation of the sample collection and study design.</p