Breast cancer (BC) is the most common cancer among females and a leading cause of cancer death worldwide . Current clinical decision-making for BC treatment relies on tumor characteristics and therapeutic targets including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Chemotherapy is given in the neoadjuvant and adjuvant settings to patients with locally advanced/high-risk primary BC as treatment for metastatic BC leading to life-threatening parenchymal disease, or to treat endocrine resistant (ER/PR-negative) metastatic BC [2, 3]. Neoadjuvant chemotherapy (NCT) is increasingly being used for initial treatment of locally advanced and inflammatory BCs. Pathologic complete response (pCR), defined as the preoperative eradication of tumors from the breast and axillary lymph nodes , is associated with optimal clinical outcome, including improved disease-free and overall survival [5, 6]. However, conventional NCT regimens result in pCR in only 10-30% of treated BC patients . In patients with residual invasive disease after NCT, a substantial proportion experience disease progression to metastatic stage within a few years after surgical resection. Patients with both de novo and recurrent metastatic BC face poor prognosis, with a median survival of 1-2 years and a 5-year survival rate < 20% [7–9]. Development of early diagnostic markers capable of predicting a patient's response to therapy and recurrence with metastatic BC is therefore critical to advancing more effective, personalized treatment. In this study, we explored the use of circulating miRNAs as blood-based, minimally invasive biomarkers for NCT response and disease relapse in locally advanced and inflammatory BC patients.
MiRNAs are naturally-occurring, non-coding small RNA molecules of 21-24 nucleotides (nts) that form partially complementary base pairs within the 3' untranslated regions of protein-encoding mRNAs, resulting in mRNA destabilization and/or translational inhibition . To date, approximately 1000 miRNAs have been identified in humans. Compared to the large number of mRNA genes (~30, 000 mRNAs per cell), this relatively small number of miRNAs allows for large-scale evaluation for individualized diagnosis and treatment with higher efficiency and lower cost. Increasingly, reports demonstrate applications of miRNAs as tissue-based markers for the classification and prognosis of several human cancers, including BC [11–14]. A number of miRNAs have been found differentially expressed between BC and normal tissues, with significant up- (e.g., miR-21 and miR-155) or down-regulation (e.g., miR-10b and miR-145) in cancerous tissues [12, 15, 16]. Expression of certain BC-related miRNAs has been correlated with specific biopathologic features, such as ER and PR expression, tumor stage, vascular invasion, and proliferation index [12, 14–18].
MiRNAs are stably present in whole blood, serum, and plasma [19, 20]. Therefore, assessment of circulating miRNA profiles from cancer patients allowing for correlations between tumor traits (e.g., treatment response and metastatic potential) and cancer-released miRNAs are of great clinical interest. Using PCR assessments of selected miRNAs that are reportedly dysregulated in BC, several recent studies indicate associations of different circulating miRNAs with primary BC, metastatic disease, and ER status [21–25]. Microarray-based profiling has also been carried out in pilot studies, in which certain circulating miRNAs exhibit promising role as BC diagnostic markers [26, 27]. Results from these previous studies, however, share limited consistency, possibly due to the different sensitivity and specificity of the detection approaches, as well as different patient numbers and compositions in the study cohorts.
Because some miRNAs may exclusively exist in the circulation with low or undetectable levels in cancer cells, powerful discovery approaches, such as deep sequencing analysis, may be more likely to identify diagnostic miRNA markers in the blood. Accordingly, we set out to explore the potential of deep sequencing in the current study to comprehensively analyze miRNAs that can serve as blood-based markers for NCT response and relapse with metastasis. As the first exploration to profile circulating miRNAs in BC patients using a de
novo sequencing strategy, our study revealed global patterns of circulating miRNAs, and led to the identification of miRNA markers that can predict clinical outcome in primary stage II- III BC.