Low serum miR-19a expression as a novel poor prognostic indicator in multiple myeloma

Abstract

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the clonal expansion of plasma cells. Despite continuing advances, novel biomarkers are needed for diagnosis and prognosis of MM. In our study, we characterized the diagnostic and prognostic potential of circulating microRNAs (miRNAs) in MM. Serum miRNA levels were analyzed in 108 newly diagnosed symptomatic MM patients and 56 healthy donors (HDs). Our analysis identified 95 dysregulated miRNAs in newly diagnosed MM patients. Of the 95 dysregulated miRNAs, dysregulation of miR-19a, miR-92a, miR-214-3p, miR-135b-5p, miR-4254, miR-3658 and miR-33b was confirmed by quantitative reverse transcription PCR (RT-qPCR). Receiver operating characteristic analysis revealed that a combination of miR-19a and miR-4254 can distinguish MM from HD with a sensitivity of 91.7% and specificity of 90.5%. Decreased expression of miR-19a was positively correlated with international staging system advancement, del(13q14) and 1q21 amplification. Furthermore, downregulation of miR-19a resulted in significantly decreased progression-free survival (PFS) and overall survival (OS). Our analysis indicated that the poor prognostic correlation of miR-19a expression was independent of genetic abnormalities in MM. Multivariate analysis revealed that miR-19a was a significant predictor of shortened PFS and OS. Interestingly, although miR-19a levels portend a poor prognosis, patients with low miR-19a levels had an improved response to bortezomib compared to those with high miR-19a profile. Patients with downregulated miR-19a experienced a significantly extended survival upon bortezomib-based therapy. These data demonstrate that the expression patterns of serum microRNAs are altered in MM, and miR-19a levels are a valuable prognostic marker to identify high-risk MM.

Keywords: biomarkers; miR-19a; multiple myeloma; prognostic indicator; serum miRNAs.

Figure 1. CONSORT (Consolidated Standards of Reporting Trails) flow diagram

Among 108 newly diagnosed symptomatic MM patients, fifty-three patients were included in arm A, fifty-five patients were included in arm B. Survival analysis included 103 patients with completed clinical data.

Figure 2. Hierarchical clustering analysis of miRNA expression and Validation of candidate miRNAs using RT-qPCR

(A) The expression of miRNA is hierarchically clustered on the x axis, and MM serum samples or healthy control serum samples are hierarchically clustered on the y axis. The legend indicates the miRNA represented in the corresponding row. Relative miRNA expression is depicted according to the color scale shown: Red indicates up-regulation and blue, down-regulation. Numbers with P indicate MM samples; numbers with H, healthy control samples. (B) Relative expression of 10 miRNAs on a large cohort of 108 newly diagnosed MM patients and 56 HD samples. The significant dysregulation of miRNAs was observed in miR-19a, miR-92a, miR-214-3p, miR-135b-5p, miR-4254, miR-3658 and miR-33b. Statistical significance was determined by a student’s t-test (*p<0.05, **p<0.01, ***p<0.0001, NS: not significant).

Figure 3. The miR-19a combined with miR-4254 offers a powerful diagnostic tool for identification of MM

(A) Receiver operating characteristic analysis was performed for miR-19a and miR-4254, and area under the ROC curve (AUC) was performed for miR-19a or miR-4254 to test the prognostic validity of each miRNA. Furthermore, ROC and AUC were performed for both miR-19a and miR-4254 to determine the sensitivity of a combination of miRNAs as a biomarker. (B) Expression profiling of miR-19a and miR-4254 was performed at various stages (newly diagnosed, remission and relapse) of MM progression. Statistical significance for miRNA expression profiles was determined by a one-way ANOVA (p<0.05).

Figure 4. Prognostic value of miR-19a level in MM patients

(A) Progression free survival and overall survival was determined for MM patients according to expression of miR-19a. Survival analysis was determined via Kaplan–Meier survival analysis, with differences between curves analyzed via a log-rank test. Significance was defined as p<0.05. (B) Patients with low miR-19a expression had significantly greater response to bortezomib than patients with high miR-19a expression. Progression free survival was determined for MM patients treated with a thalidomide-based or a bortezomib-based therapy according to their miR-19a expression profile. Survival analysis was determined using Kaplan–Meier survival analysis, with the differences between curves analyzed via a log-rank test. Significance was defined as p<0.05.