A Molecular Signature of Three tRNA-Derived RNA Fragments May Discriminate Smoldering from Symptomatic Multiple Myeloma Patients

Introduction: Multiple myeloma (MM) is the second most common hematological malignancy arising from terminally differentiated plasma cells. The diagnosis of symptomatic ΜΜ is usually based on the presence of end-organ damage, as defined by the CRAB criteria. ΜΜ is part of a spectrum of disorders characterized as monoclonal gammopathies. Smoldering multiple myeloma (SMM) is a plasma cell dyscrasia preceding MM. The risk of sMM progression to active MM is determined by risk stratification models, such as the Mayo Clinic and the Spanish models. The clinical course of MM is quite heterogenous; patients survival is ranging from months to more than a decade. MM risk is determined using the international staging system (ISS) and the revised-ISS (R-ISS), which incorporates LDH and cytogenetics to ISS.

tRNA-derived RNA fragments suggest a class of small non-coding RNAs, which derive from either pre- or mature tRNAs. tRNA fragments have only recently emerged and therefore have not been broadly studied. These fragments are not random degradation products, yet occur through specific enzymatic activity. Two large groups of these molecules are currently known and are distinguished based on their length; tiRNAs or tRNA halves consist of 30-50 nucleotides, while tRNA-derived fragments (tRFs) consist of 16-28 nucleotides. Both categories are further subgrouped according to their site of origin, in 5′-, 3′-, and internal fragments. Although their role is still under evaluation, studies have linked them to pathological situations, such as neurodegenerative diseases, various cancer types and hematological malignancies.

In this study, the clinical significance of 6 of these fragments was evaluated in MM, and three of them showed interesting results. These molecules are 3′-tRFs or internal tRFs (i-tRFs), which occur from the tRNAs bearing leucine, glutamic acid and proline anticondons, namely 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG, respectively.

Methods: CD138+ plasma cells were collected from 80 patients at the time of diagnosis: 65 with MM and 15 with SMM. Total RNA was extracted from CD138+ cells using TRIzol and thereafter was polyadenylated by Escherichia coli poly(A) polymerase. First-strand cDNA synthesis was performed by MMLV transcriptase, using an oligo-dT-adaptor primer. Subsequently, we used and in-house real-time quantitative PCR assay, based on SYBR Green chemistry, in order to quantify tRFs in all samples. Specific forward primers were designed for all tested molecules, along with a common reverse primer, complementary to the adaptor used during reverse transcription. The small nucleolar RNAs RNU43 and RNU48 were used as reference genes, in order to normalize qPCR data. Biostatistical analysis was carried out to assess these results.

Results: Out of 65 MM patients 13 were classified as having ISS I stage, 22 as ISS II stage, and 30 as ISS III stage. Similarly, 13 of them were grouped in R-ISS I stage, 35 as R-ISS II, and 17 as R-ISS III. The Mann Whitney U test revealed that the levels of 3′-tRF-LeuAAG/TAG, itRF-GluCTC, and i-tRF-ProTGG differed significantly between MM and SMM cases (P=0.001, P=0.047 and P=0.033, respectively). Specifically, these molecules had higher levels in the CD138+ cells of the SMM cases. These results were validated by logistic regression analysis, which showed that patients with lower expression levels of these molecules were at a higher risk of suffering from MM (P=0.003 for 3′-tRF-LeuAAG/TAG , P=0.036 for itRF-GluCTC, and P=0.032 for i-tRF-ProTGG). Furthermore, 3′-tRF-LeuAAG/TAG was related with translocation t(4;14).

Conclusions: 3′-tRF-LeuAAG/TAG, i-tRF-GluCTC, and i-tRF-ProTGG may represent novel molecular biomarkers for the differential diagnosis of MM from SMM in ambiguous cases, in which the diagnostic work-up does not provide a clear diagnosis. These novel biomarkers could also play a role in the prediction of sMM cases at high risk of evolution to active multiple myeloma given their overexpression in asymptomatic cases. A possible correlation of these biomarkers to prognosis in MM patients may be indicated by their relationship with the t(4;14) translocation.