Combination of serum microRNA-320a and microRNA-320b as a marker for Waldenström macroglobulinemia
Authors: Lenka Kubiczkova Besse, Lenka Sedlarikova, Fedor Kryukov, Jana Nekvindova, Lenka Radova, Martina Almasi, Jana Pelcova, Jiri Minarik, Tomas Pika, Zuzana Pikalova, Vlastimil Scudla, Marta Krejci, Zdenek Adam, Ludek Pour, Roman Hajek, Sabina Sevcikova
Journal: American Journal of Hematology (2014)

IgM monoclonal gammopathies are a group of diseases characterized by increased level of IgM immunoglobulin produced by one clone of B cells. These diseases range from benign (monoclonal gammopathy of undetermined significance, MGUS) to malignant, such as Waldenström macroglobulinemia (WM) or to a lesser extent multiple myeloma (MM) 1, 2. The criteria that differentiate WM from IgM-MGUS are based on the extent of bone marrow (BM) involvement, amount of serum concentration of the M-protein, presence or absence of symptomatic disease or more recently, MYD88 (L265P) or CXCR4 mutations 3-6. Despite that, new criteria for the differential diagnosis between these conditions are still needed, circulating microRNAs (miRNAs) being one of them. Circulating miRNAs are present in different body fluids; they reflect physiological or pathological conditions and can be used for patient classification [7, 8]. Thus, we aimed to investigate the ability of serum miRNAs to distinguish WM from IgM-MGUS as well as IgM-MM patients and healthy donors (HD). For this purpose, circulating miRNAs were isolated from serum samples and screening of 667 miRNAs using TaqMan Low Density arrays was performed on five WM patients, five IgM-MGUS, five IgM-MM patients, and five HD samples to identify differently expressed circulating miRNAs in WM (Supporting Information Fig. S1). Out of deregulated miRNAs, miR-320b, miR-320a, miR-151-5P, and let-7a were further validated by quantitative real-time PCR (qPCR) on a larger cohort of 21 WM, 15 IgM-MGUS, 10 IgM-MM, and 18 HD serum samples (Supporting Information Table S1), as they were present at the top of the list of deregulated miRNAs between IgM-MGUS, HD, and WM and showed highest fold change and most favorable expression (Ct < 30). In addition, some of the miRNA levels were correlated with clinically important parameters and MYD88 (L265P) mutation status. MiR-320a and miR-320b showed different expression between WM and all other groups of samples (P < 0.05). Let-7a and miR-151-5P were significantly decreased in WM samples as compared with HD (all P < 0.05) and IgM-MGUS samples (all P < 0.05) but not with IgM-MM (P = 0.285 and P = 0.286, respectively). As only miR-320a and miR-320b remained statistically significant, only these two miRNAs were chosen for further analyses (Supporting Information Table S2). Receiver Operating Characteristic curves (ROC) were used to evaluate diagnostic effectiveness of miR-320b and miR-320a and to estimate the appropriate cutoff (Supporting Information Table S3). MiR-320b was more potent than miR-320a to distinguish WM from HD with sensitivity of 85.7% and specificity of 94.4% using cutoff value of 1,072 copies per 1 ng of miRNA/RNA. However, combination of miR-320b with miR-320a improved sensitivity up to 90.5% with specificity of 94.4% using cutoff value −0.6253 obtained from nominal logistic regression model (Fig. 1A). More importantly, miR-320b discriminated WM from IgM-MGUS with specificity of 73.3% and sensitivity of 85.7% using cutoff value of 1,072 copies per 1 ng of total miRNA/RNA, and combination of miR-320b with miR-320a reached specificity of 73.3% and increased sensitivity up to 90.5% with cutoff defined as −0.2373 (Fig. 1B). Furthermore, miR-320b distinguished also WM from IgM-MM with sensitivity of 71.4% and specificity of 80.0% with cutoff 904 copies per 1 ng of total miRNA/RNA, and together with miR-320a, the two-miRNA based combination yielded sensitivity of 81.0% and specificity of 100.0% with cutoff value defined as −1.4322 (Fig. 1C). Additionally, associations between miR-320a and miR-320b expression levels and important disease parameters of IgM-MGUS, WM, and IgM-MM were investigated. In IgM-MGUS group, a positive correlation was found between levels of miR-320b and albumin (P < 0.05; rs = 0.521) and moderate positive correlation between levels of miR-320b and calcium (P < 0.06; rs = 0.509). In WM group, levels of miR-320a negatively correlated with β2-microglobulin (P < 0.05; rs = −0.468) and with percentage of lymphoplasmacytic cells infiltration in the BM (P < 0.05; rs = −0.573). Next, in IgM-MM group, miR-320b and miR-320a negatively correlated with levels of M-Ig (P < 0.05; rs = −0.782 and rs = −0.636, respectively), and there was a positive correlation between levels of miR-320a and lactate dehydrogenase (P < 0.05; rs = 0.818). All associations are present in Supporting Information Table S4. We also evaluated mutation status of MYD88 (L265P) in all available PB samples (Supporting Information Fig. S2). Levels of miR-320a were significantly lower in MYD88 (L265P) positive patients (P = 0.032), and there was an identical trend for miR-320b, although not significant (P = 0.079). Both miR-320a and miR-320b were observed in higher concentrations in cellular fractions in comparison with cell-free fractions, and interestingly, more copies of these microRNAs were observed in the CD19-fraction. Additionally, both miR-320a and miR-320b were present in exosomes as well as in exosome-depleted samples; however, their levels tend to be increased in exosomal fractions (Supporting Information Fig. S3). The investigation of molecular features of IgM-monoclonal gammopathies is essential to identify specific risk markers for disease development. Routinely, differentiation of IgM-MGUS from WM is possible only by trepanobiopsy, as neither BM aspiration nor flow-cytometry provide sufficient data for diagnosis. Therefore, in this study, we focused on the role of circulating serum miRNAs as biomarkers of WM. Combination of miR-320a and miR-320b served as the best indicator for WM as it was able to distinguish WM from HD, but more importantly WM from premalignant IgM-MGUS and malignant IgM-MM. Our data suggest that such miRNAs combination might be a novel effective tool for WM discrimination which, however, needs further validation and study. Although the amount of patients with mutant MYD88 (L265P) was small, it can be hypothesized that mutation in MYD88 might be connected to lower miRNA levels; however, underlying biology again needs to be elucidated. It still remains an open question, if studied miRNAs are actively or passively secreted from tumor cells; nevertheless, it is plausible to assume that they are actively transported in vesicles, as they are present primarily in exosomes. Association of low levels of these miRNAs with WM diagnosis suggests their involvement in the disease; however, the origin of such miRNAs in circulation still remains unclear. Nonetheless, considering the minimally invasive character of serum sampling, reproducibility, and easy detection of circulating miRNA, they may provide a convenient and inexpensive method to establish the diagnosis of WM or to predict the evolution of IgM-MGUS. The authors would like to thank the patients involved in this study and their caregivers. The authors would also like to thank Barbora Sablikova, Bozena Hanakova, and Marcela Stouracova for technical support. John B. Smith proofread the manuscript. Lenka Kubiczkova Besse1,2, Lenka Sedlarikova1,2, Fedor Kryukov1,3, Jana Nekvindova4Lenka Radova5, Martina Almasi1,2, Jana Pelcova6, Jiri Minarik7, Tomas Pika7Zuzana Pikalova7, Vlastimil Scudla7, Marta Krejci6, Zdenek Adam6Ludek Pour6, Roman Hajek1,2,3 and Sabina Sevcikova1,2* 1 Babak Myeloma Group, Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic; 2Department of Clinical Hematology, University Hospital Brno, Brno, Czech Republic; 3Department of Hematooncology, University Hospital Ostrava and Faculty of Medicine, University of Ostrava, Ostrava, Czech Republic; 4Institute for Clinical Biochemistry and Diagnostics, University Hospital Hradec Kralove, Czech Republic; 5Central European Institute of Technology, Masaryk University, Brno, Czech Republic; 6Department of Internal Medicine - Hematooncology, University Hospital Brno, Brno, Czech Republic; 7Department of Hematooncology, University Hospital Olomouc, Olomouc, Czech Republic Additional Supporting Information may be found in the online version of this article. 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