Impact of Delayed Whole Blood Processing Time on Plasma Levels of miR- 1 and miR-423-5p up to 24 Hours.

BACKGROUND: Circulating cell-free miRNAs hold great promise as a new class of biomarkers due to their high stability in body fluids and association with disease stages. However, even using sensitive and specific methods, technical challenges are associated with miRNA analysis in body fluids. A major source of variation in plasma and serum is the potential cell-derived miRNA contamination from hemolysis. OBJECTIVES: The study aimed to evaluate the effect of the delayed whole blood processing time on the concentrations of miR-1 and -423-5p. METHODS: Ten blood samples were incubated for 0, 3 and 24 hours at room temperature prior to processing into plasma. For each time point, hemolysis was assessed in plasma by UV spectrophotometry at 414nm wavelength (λ414). Circulating levels of miR-1 and -423-5p were measured by RT-qPCR; miR-23a and -451 were also analyzed as controls. RESULTS: A significant hemolysis was observed only after 24h (λ414 0.3 ± 0.02, p < 0.001). However, only small changes in miR-1 and -423-5p levels were observed up to 24h of storage at room temperature (Ct 31.5 ± 0.5 to 31.8 ± 0.6for miR-1, p = 0.989; and 29.01 ± 0.3 to 29.04 ± 0.3, p = 0.614 for - 423-5p). No correlation was observed between hemolysis and the levels of miR-1 and -423-5p. CONCLUSION: Our data indicate that the storage of whole blood samples at room temperature for up to 24h prior to their processing into plasma does not appear to have a significant impact on miR-1 and - 423-5p concentrations.

Circulating miR-1 as a potential biomarker of doxorubicin-induced cardiotoxicity in breast cancer patients.

Cardiotoxicity is associated with the chronic use of doxorubicin leading to cardiomyopathy and heart failure. Identification of cardiotoxicity-specific miRNA biomarkers could provide clinicians with a valuable prognostic tool. The aim of the study was to evaluate circulating levels of miRNAs in breast cancer patients receiving doxorubicin treatment and to correlate with cardiac function. This is an ancillary study from "Carvedilol Effect on Chemotherapy-induced Cardiotoxicity" (CECCY trial), which included 56 female patients (49.9±3.3 years of age) from the placebo arm. Enrolled patients were treated with doxorubicin followed by taxanes. cTnI, LVEF, and miRNAs were measured periodically. Circulating levels of miR-1, -133b, -146a, and -423-5p increased during the treatment whereas miR-208a and -208b were undetectable. cTnI increased from 6.6±0.3 to 46.7±5.5 pg/mL (p<0.001), while overall LVEF tended to decrease from 65.3±0.5 to 63.8±0.9 (p=0.053) over 12 months. Ten patients (17.9%) developed cardiotoxicity showing a decrease in LVEF from 67.2±1.0 to 58.8±2.7 (p=0.005). miR-1 was associated with changes in LVEF (r=-0.531, p<0.001). In a ROC curve analysis miR-1 showed an AUC greater than cTnI to discriminate between patients who did and did not develop cardiotoxicity (AUC = 0.851 and 0.544, p= 0.0016). Our data suggest that circulating miR-1 might be a potential new biomarker of doxorubicin-induced cardiotoxicity in breast cancer patients.