Dissertation committee:
Μελέτιος-Αθανάσιος Δημόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευάγγελος Τέρπος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Φλώρα Ζαγουρή, Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Χρήστος Κοντός, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Μιχάηλ Λιόντος, Επίκουρος Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μαρία Γαβριατοπούλου, Αναπληρώτρια Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Summary:
Background: Multiple myeloma (MM) is the second most common hematologic malignancy arising from the clonal proliferation of malignant plasma cells. Multiple myeloma bone disease (MMBD) constitutes a common and severe complication of MM. miRNAs are small non-coding RNAs that participate in the post-transcriptional regulation of gene expression. tRNA-derived RNA fragments (tRFs) constitute a class of small non-coding RNAs, deriving from tRNAs. Herein, we have studied the clinical utility of miRNAs and tRFs in improving patients’ risk stratification and prognosis, as well as their role as biomarkers able to predict the presence of MMBD.
Materials and Methods: The study was conducted in three stages. For the first stage, CD138+ plasma cells were selected from bone marrow aspirates from MM (n=76) and smoldering MM (sMM) patients (n=18). Total RNA was extracted and in vitro polyadenylated. First-strand cDNA synthesis was performed, using an oligo-dT-adapter primer. For the relative quantification of the investigated miRNAs and tRFs, an in-house real-time quantitative PCR (qPCR) assay was developed. During the second stage, miRNA-seq was performed in CD138+ plasma cells of MM, sMM, and monoclonal gammopathy of undetermined significance (MGUS) patients. The MM cohort consisted of 138 patients. Quantification was performed by RT-qPCR. Regarding the third stage, miRNAs were isolated from the plasma of 62 MM patients with or without MMBD. First-strand cDNA was synthesized, and relative quantification was performed using qPCR. Lastly, we carried out extensive biostatistical analysis.
Results: miR-16-5p (p=0.036), miR-155-5p (p=0.045) and 3’-tRF-LeuAAG/TAG (p = 0.041) expression was significantly lower in the CD138+ plasma cells of MM patients compared to sMM patients. Furthermore, lower levels of miR-15a-5p, miR-16-5p, miR-222-3p, and i-tRF-GlyGCC (p=0.037, p=0.035, p=0.037 and p = 0.047 respectively) were observed in the CD138+ plasma cells of MM patients with MMBD, compared to those without. miR-125b-5p levels were higher in the CD138+ plasma cells of MM patients presenting with skeletal-related events (SREs) (p = 0.005). Our results showed that elevated levels of itRF-ProTGG, i-tRF-GluCTC, i-tRF-HisGTG, i-tRF-PheGAA, 3’-tRF-LeuAAG/TAG, and miR-223-3p are indicators of prolonged overall survival (OS) compared to lower levels of these molecules (p = 0.024, 0.014, 0.003, 0.040, 0.039, and p=0.046 respectively). i-tRF-GlyGCC and 3’-tRF-LeuAAG/TAG overexpression is associated with significantly longer progression-free survival intervals (PFS) regarding first-line treatment (p = 0.016 and 0.013 respectively). miRNA-seq highlighted miR-181a to be upregulated in MM vs. sMM/MGUS, and R-ISS III vs. I patients. miR-181a overexpression was associated with significantly higher risk for short-term progression (p= 0.001) and worse survival (p < 0.001). Multivariate models integrating miR-181a with disease established markers led to superior risk stratification and clinical benefit for MM prognosis. Circulating levels of let-7b-5p (p = 0.034), miR-143-3p (p = 0.021), miR-17-5p (p = 0.025), miR-214-3p 3p (p = 0.004), and miR-335-5p (p = 0.022), were significantly higher in the plasma of MM patients with MMBD compared to those without. Receiver operating characteristic curve and logistic regression analyses showed that these miRNAs could accurately predict MMBD, either standalone or in a multi-miRNA–based logistic regression model.
Conclusions: In conclusion, we propose a miRNA and tRF signature with putative clinical utility in MM. We also propose miR-181a as a biomarker strongly correlated with inferior disease outcome and survival, which is independent of the established prognostic biomarkers and can enhance their prognostic ability. Lastly, our study proposes a circulating miRNA signature to facilitate MMBD diagnosis
Keywords:
Multiple myeloma, MGUS, Biomarkers, miRNA, tRF
