MicroRNA expression patterns in medullary and extramedullary plasmacytoma

Plasmacytomas are localized tumors of monoclonal plasma cells without evidence of significant bone marrow infiltration or systemic myeloma, comprising B2–10% of all plasma cell neoplasms. Medullary plasmacytomas classically occur in the bone of the vertebrae or skull, whereas extramedullary plasmacytomas occur in the soft tissues, most frequently in the head and neck region involving the sinuses or rhinopharynx. Both medullary and extramedullary plasmacytomas occur in males more often than in females with a median age at diagnosis of 54 years. Furthermore, plasmacytomas in general are fairly radiosensitive, such that the current treatment of choice is localized radiation therapy except in cases of neurologic compromise or bone instability that require surgical resection. There is no evidence thus far that adjuvant systemic chemotherapy offers additional clinical benefit. Despite these similarities, medullary and extramedullary plasmacytomas are considered distinct clinical entities owing to differences in their predisposition to evolve into multiple myeloma (MM). Medullary plasmacytomas are traditionally thought to have a higher risk of progression, also with a lower 5-year survival rate of 33% once they have progressed to MM (as opposed to 5-year survival rate of 100% in extramedullary plasmacytomas that progress to MM). Yet, very little is known regarding genetic or molecular factors that distinguish these two groups of plasmacytomas and influence their clinical outcome. MicroRNAs (miRNAs) are small non-coding RNAs of B22 nt in length, which regulate target genes through posttranscriptional degradation or translational repression. Downstream programs impact cell cycle progression, apoptosis, proliferation and angiogenesis among others, and through these mechanisms, contribute to hematopoiesis and human cancers. Multiple recent studies have linked deregulated miRNA expression to the pathogenesis and progression of MM. In one study, miRNA expression profiling of CD138þ bone marrow plasma cells from MM patients, normal donors and those with monoclonal gammopathy of undetermined significance (MGUS) identified selective upregulation of miRNAs including miR-19a and -19b in MM patient samples. miR-19a and -19b were shown to suppress the expression of SOCS-1, a negative regulator of IL-6/STAT3 pathway. Another study proposed a ‘MM-specific microRNA signature’ defined by upregulation of miR-222, -221, -382, -181a and -181b, and downregulation of miR-15a and -16, in which the latter functionally inhibit AKT3, ribosomal protein S6 as well as angiogenic VEGF (vascular endothelial growth factor). These miRNA–mRNA–protein networks add another layer of complexity to the pathogenesis of MM. To date, no published study has examined the global miRNA expression differences between plasmacytoma and MM, or between the intraversus extramedullary plasmacytomas, at least in part due to the limited availability of plasmacytoma patient samples. Here, we analyzed 15 patients with plasmacytomas to gain insight into miRNA expression pattern differences between medullary and extramedullary plasmacytomas. On the basis of the diagnostic criteria for localized plasmacytoma, 11 patients were classified as having medullary plasmacytoma and 4 patients as extramedullary. Patient characteristics are summarized in Supplementary Table 1. The median age at diagnosis was 61 and 57 years in the medullary and extramedullary groups, respectively, largely consistent with the reported age range in the literature. The patient population consisted of nine males and six females (with male-to-female ratio of 8:3 in the medullary and 1:3 in the extramedullary groups). Although limited in some cases by loss to follow-up, at least 4 of the 11 medullary plasmacytoma patients definitively progressed to develop MM, whereas only 1 of the 4 extramedullary plasmacytoma patients progressed. For our miRNA microarray analysis, miRNAs derived from the above patient population were profiled using the TaqMan Human MicroRNA Low Density Arrays Version 2.0 (Applied Biosystems, Grand Island, NY, USA), consisting of a total of 667 unique assays against human miRNAs. Comparative marker selection analysis (signal-to-noise ratio score cutoff of ±0.5) of the miRNA microarray data set after missing value imputation and normalization revealed seven miRNAs that were significantly upregulated in the extramedullary versus medullary plasmacytomas: miR-141, miR-200c, miR-200b, miR-135b, miR-1393p, miR-376a and miR-31 (Figure 1a; Supplementary Table 2). In addition, one miRNA was identified as significantly downregulated in the extramedullary versus medullary plasmacytoma: miR-135a. All miRNAs had a fold change of at least 1.5 and a q-value cutoff of 0.05 (Supplementary Figure 1). Notably, hierarchical clustering of our patient samples using these eight candidate miRNAs and Pearson correlation showed very clear linkage segregation between the two phenotypes of plasmacytomas (Figure 1b). This demonstrates that our eight miRNAs could represent a candidate miRNA signature to define the plasmacytoma classes. Indeed, unsupervised hierarchical clustering using all miRNAs analyzed in our data set also showed segregation between medullary and extramedullary plasmacytomas (data not shown). We next predicted target genes for the above candidate miRNAs using the in silico algorithm TargetScan. The list of predicted targets was subsequently entered as input to the PANTHER gene ontogeny database to query for gene pathways enriched in this target gene list. As a control, the same number of miRNAs were selected at random using a computational random number generator (miR-125b,miR-138,miR-185,miR-224,miR-330-3p, miR-487b, miR-520c-3p and miR-766), and their targets and pathways were analyzed in parallel. Several pathways were more significantly enriched in the candidate miRNA target gene list (Table 1; Supplementary Table 3), including FGF, TGF-beta and angiogenesis pathways. Angiogenesis (which in turn is regulated by FGF and TGF-beta pathways, among others) is known to have a pivotal role in MM pathogenesis, and more specifically, in mediating the progression of plasmacytoma to MM. Our finding proposes the notion that differential regulation of angiogenesis pathways may further be biologically relevant to the variant propensity of medullary versus extramedullary plasmacytomas to progress to MM, similar to their role in regulating the progression of MGUS to MM. Our analysis also discerns PI3 kinase and apoptosis pathways to be more highly Citation: Blood Cancer Journal (2014) 4, e223; doi:10.1038/bcj.2014.41 & 2014 Macmillan Publishers Limited All rights reserved 2044-5385/14