CIRCULATING MIR-34A AND MIR-130A AS BIOMARKERS OF EXTRAMEDULLARY DISEASE

ABSSUB-3660

Background: MicroRNAs (miRNA), short non-coding regulatory RNAs, are implicated in deregulation of critical pathways involved in multiple myeloma (MM) and extramedullary form of MM (EM). Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible body fluids; thus, they have the potential to be useful diagnostic biomarkers, as was shown in our previous study on MM. Here, we have identified a specific serum miRNA profile in patients with extramedullary disease and correlated it with clinically important parameters.

Aims: The goal of this study was to identify circulating miRNA signature using Taqman Low Density Arrays and assay specific quantitative PCR (qPCR) on a cohort of patients with extramedullary disease, MM patients and healthy controls, and to compare miRNA levels with clinical parameters.

Methods: One hundred serum samples obtained from relapsed EM patients, newly diagnosed MM patients and healthy donors (HD) were evaluated for this study. Screening analysis of 667 miRNAs was performed on 5 EM samples, 5 MM samples and 6 HD samples using TaqMan Low Density Arrays (TLDA). Levels of 4 differentially expressed miRNAs from TLDA (p<0.05) between EM vs MM, and EM vs HD were confirmed by qPCR using absolute quantification approach on 35 EM, 35 MM and 30 HD serum samples. Receiver Operating Characteristic (ROC) analysis was used to calculate specificity and sensitivity of each miRNA and their combination. Biochemical characteristics were also available for EM and MM patients. P values <0.05 were considered as significant.

Results: MiRNA TLDA profiling revealed 14 deregulated miRNAs (all p<0.05, adjusted p<0.41) between MM patients and EM. Further, 20 miRNAs were on the top of the list of deregulated miRNAs between EM and HD serum samples (all p<0.05, adjusted p<0.40). MiR-222, miR-130a, miR-34a and miR-195 were further verified on a bigger cohort of EM, MM and HD samples. MiR-130a was significantly down-regulated, miR-222 and miR-34a were up-regulated in EM samples when compared with HD (all p<0.005); moreover, miR-130a was down-regulated and miR-34a up-regulated also in EM when compared with MM sera (p<0.06). To discriminate EM from other groups, ROC curve was calculated.  To distinguish EM from HD, it revealed highest sensitivity of 74.3%, specificity of 90.0% and area under the curve (AUC) = 0.879 for the combination of miR-130a and miR-34a. Further, when EM vs MM were compared, this combination of miRNA revealed sensitivity of 54.3% and specificity of 80% with AUC = 0.675. In the cohort of EM patients, miR-130a significantly correlated with most of clinically relevant parameters; there was a positive correlation with level of hemoglobin and thrombocytes count (r_s=0.397 and 0.439, all p<0.05) and a negative correlation with levels of monoclonal immunoglobulin, β₂-microglobulin and C-reactive protein (r_s=-0.398, -0.427 and -0.488, all p<0.05). This miRNA was also associated with higher ISS stage (p=0.017). Further, miR-222 correlated positively with lactate dehydrogenase (r_s=0.417, p<0.05); miR-222 and miR-34a were associated positively with percentage of plasma cell infiltration in the bone marrow (r_s=0.435 and 0.562, p<0.05).

Summary/Conclusion: Altogether, our first observations demonstrate that circulating miR-130a and miR-34a may be promising biomarkers for patients with extramedullary disease and prompt further studies in this field.

Grants support: NT14575, NT12130, MUNI/11/InGA17/2012, CZ.1.07/2.3.00/20.0046 and CZ.1.07/2.3.00/20.0019 of the Ministry of Education of the Czech Republic

Keywords: Multiple myeloma, Quantitative RT-PCR

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