Circulating mir‐208a fails as a biomarker of doxorubicin‐induced cardiotoxicity in breast cancer patients

Doxorubicin (DOX) is the first-line drug in the treatment of breast cancer. Despite its beneficial therapeutic effects, cardiomyopathy and heart failure are observed when DOX is chronically administered for several weeks (Octavia et al. 2012). Although several cardioprotective therapies have been proposed, cardiotoxicity remains a major concern of oncologists in cancer therapeutic practice (Fadillioğlu et al. 2003; Fadillioglu et al. 2004; Alpsoy et al. 2013a, 2013b). Among all biomarkers of myocardial injury, cardiac troponins are the most used for its good sensitivity and integration in hospital routine. However, cardiac troponins are released in plasma only after membrane and tissue damage has occurred. Therefore, the development of new approaches to detect the cardiotoxic effects of DOX in the earlier stages is still required. Recent studies have suggested that circulating miR-208a (a heartspecific microRNA) may serve as useful earlier biomarker of heart injury and drug-induced cardiotoxicity (Oliveira-Carvalho et al. 2013; Liu et al. 2014; Xiao et al. 2014; Nishimura et al. 2015). However, the potential of miR-208a as a biomarker of DOX-induced cardiotoxicity in clinical samples was not yet been elucidated. In brief, 59 female patients with breast cancer under the first round of chemotherapy with doxorubicin were enrolled in this study. Patients received a cumulative dose of doxorubicin (60mgm; Bergamo), cyclophosphamide (600mgm; Baxter) and paclitaxel (80mgm; Blau) or docetaxel (75mgm; Glenmark) during 3, 6, 9 and 12weeks of treatment. Radiotherapy was indicated only after the end of the treatment; information about the surgery is not available. Cardiac troponin I (cTnI; assessed by Centaur Advia), left ventricle ejection fraction (LVEF; assessed by echocardiography) and plasmatic levels of miR-208a were measured in serial samples (0, 3, 6, 9 and 12weeks). Patients that developed cardiotoxicity were grouped and compared with patients that did not. The study protocol was approved by the ethics committee at Faculdade de Medicina da Universidade de São Paulo and complies with the Declaration of Helsinki. Informed consent was obtained from each patient. Peripheral blood was collected in 4-ml tubes with EDTA and centrifuged at 2000 g for 15min at 4 °C. Plasma samples were transferred to 1.5-ml microtubes and stored at –70 °C. Small RNAs (>200nt) were isolated from 200μl of plasma by a miRNeasy Plasma/Serum kit (Qiagen). Cel-miR-39 was spiked in prior RNA isolation for data normalization. Reverse transcription was performed with a TaqMan MicroRNA Reverse Transcription Kit (Life Technologies) using a fixed volume of 5μl of RNA sample as an input. Circulating levels of miR-208a were measured by RT-qPCR in triplicate using TaqMan MicroRNA Assays (Life Technologies) inputting 1.33μl of cDNA. RT-qPCR reactions were plotted in plates of 384 wells and measured in QuantStudio 12 K Flex (Life Technologies). A heart tissue sample was also included as a positive control. All reactions were performed according to the manufacturer’s protocol. As a result, seven patients (11, 86%) developed cardiotoxicity. Serum levels of cTnI were increased from 6.3 (±0.2) to 31pgml (±3.2) in the non-cardiotoxicity group whereas from 8.3 (±1.9) to