Circulating cell-free miRNAs as biomarker for triple-negative breast cancer

Breast cancer is the most common cause of cancer mortality in women worldwide (GLOBOCAN 2012, http://globocan.iarc.fr/Pages/fact_sheets_population.aspx). Triple negative-breast cancer (TNBC) is characterised by lack of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). This subtype accounts for 15–20% of all breast cancers (Boyle, 2012). It is a heterogeneous disease that partake distinct histopathological features and clinical behaviour. Epidemiological studies revealed that TNBC is associated with young age (<40) and more frequently in African–American and black ethnicity (Boyle, 2012). TNBC tumours represent the most aggressive phenotype with relatively high recurrence rates (Dent et al, 2007). Patients with TNBC do not respond to endocrine therapy or HER2 targeted therapy, and treatment are based on a combination of commonly used breast cancer therapies including surgery, radiation and chemotherapy regimens. Clinical trials conducted on TNBC using cisplatin-based regimes as neoadjuvant therapy found that the response rate to single-agent cisplatin was only 21%. An improved response rate was seen using a combination of paclitaxel/fluorouracil, doxorubicin and cyclophosphamide but this was still only 45% (Silver et al, 2010). TNBC and BRCA mutations associated tumour share common pathologies. Poly (ADP-ribose) polymerase (PARP) inhibitor is a class of anti-tumour agents which targets tumours with BRCA mutations but could only benefit a small group of patients (Metzger-Filho et al, 2012). Combining inhibitors of PARP and PI3K were shown to be effective in primary tumour xenografts where tumours had no BRCA mutations, and the clinical outcome of this trial is awaiting (Ibrahim et al, 2012). To date, the current treatment options for TNBC do not demonstrate promising clinical outcomes and often results in poor treatment response and overall survival rate. This is also partly due to the lack of biomarkers to envisage which groups of patients are likely to respond to specific chemotherapeutic and targeted therapies nor are there reliable biomarkers which have been identified to be used as a screening marker. Hence, there is a substantial need for discovering a predictive biomarker for diagnosis that could identify patients with this aggressive and fast growing tumour at an earlier stage to improve the prognosis. With limited targeted therapy currently available for TNBC, it is also important to have a marker for disease monitoring and evaluate the need to change therapy if the treatment response is poor. MicroRNA (miRNA) has a critical regulatory role in gene expression and is involved in breast carcinogenesis (Serpico et al, 2014). Currently, there are over 1800 miRNAs identified in human (miRBase release 20) and these regulate >50% of the genes. Several lines of evidence have shown that miRNAs are differentially expressed in cancerous tissues when compared with adjacent non-tumour counterparts (Calin and Croce, 2006; Volinia et al, 2006). Expression profiles of miRNAs have been reported to form more defined cluster with similar tumour types than mRNA expression. Numerous studies suggested that miRNAs are highly stable and can be detected in the circulation (Mitchell et al, 2008). It has also been reported that miRNA patterns are unique in the cancers of breast (Ng et al, 2013), colon (Ng et al, 2009), gastric (Tsujiura et al, 2010), ovary (Shapira et al, 2014) and prostate (Brase et al, 2011). The recognition of miRNAs in the circulation marks the milestone in cancer diagnosis and therapeutic application. Detection of circulating miRNAs can serve as non-invasive and cost-effective markers to identify high-risk patients, which are beneficial in clinical setting in terms of diagnosis, prognosis and treatment response. Published data reported that the levels of miR-30a were lower in patients with breast cancer, and showed improved sensitivity and specificity compared with conventional circulating tumour markers (CEA and CA153) (Zeng et al, 2013). Furthermore, high level of miR-155 was associated with better clinical outcome in TNBC patients (Gasparini et al, 2014). Our group has established a robust platform to profile miRNA in various diseases (Ng et al, 2009; Lai et al, 2013; Ng et al, 2013), and have identified a panel of breast cancer-associated miRNAs from plasma, which can potentially serve as screening tool for breast cancer patients (Ng et al, 2013). In this study, we aim to uncover TNBC-associated miRNAs in the circulation based on our established platform. The profiles of miRNAs in TNBC were compared with those in non-TNBC, as well as healthy controls. Candidate miRNAs were selected based on the differential expression between these groups, validated in an independent cohort, and correlated with clinical outcomes.