Summary:
Multiple myeloma (MM), although a rare disease, is the second most common hematological malignancy, which is in the spectrum of plasma cell dyscrasias. In several cases it starts from a benign stage, monoclonal gammopathy of undetermined significance (MGUS) and progress through an intermediate stage characterized as smoldering myeloma (sMM). The progression of the disease from the benign precursor stages to the fatal malignant MM depends on a complex of factors that are not yet fully understood. The hallmark of this condition is the uncontrolled proliferation of plasma cells in the bone marrow, resulting in the production and secretion of non-functional immunoglobulins. In the last decades improvements in the treatment of MM, beginning with the use of Autologous Stem Cell transplantation and followed by the availability of new therapies such as immunomodulatory drugs (IMIDs) and proteasome inhibitors (PIs), have transformed the history of the disease leading to greater survival times. Despite these substantive advancements, the highly heterogenous treatment response and the high relapse levels hinders reliable prognosis and tailored therapeutics, highlighting the urgent need for the delineation of innovative prognostic markers to be incorporated into clinical care. In this context, high-throughput techniques have unveiled microRNAs (microRNAs), as potent post-transcriptional regulators of gene expression with critical implications in cancer onset and progression. The emergence of these techniques revealed that miRNAs are deregulated in all human tumors and highlighted their utility as potential prognostic indicators. Herein, based on these we decided for the purpose of this thesis to investigate the expression levels and clinical significance of microRNAs in multiple myeloma. The molecule in question are small non-coding RNA molecules, 19-25 nucleotides long that regulate gene expression by causing the inhibition of translation or the complete degradation of their target mRNAs through their base complementarity. They bind to 3’-untrasnlated region of their target mRNAs through a sequence of 6-8 nucleotides (seed region) located close to the 5’-end of the microRNA.
In this context, an extensive literature research was performed for microRNA molecules that have been shown to be actively involved in cancer progression and target potent crucial genes of cell cycle. Specifically, mir-34a-5p, miR-106b-5p, miR-221-3P and miR-222-3p were selected to develop an in-house qPCR protocol, by which we would specifically amplify these molecules and they could be quantitatively detected. Although all 4 molecules were optimized, miR-221-3p and miR-222-3p were selected for screening our cohort. A statistically satisfactory number of samples (n=167) were used, of which with MM (n=130), with smoldering multiple myeloma (n=28) and with monoclonal gammopathy of undetermined significance (n=9). Mononuclear cells were isolated from bone marrow using Ficoll-Paque, while CD138+ plasma cells were positively selected using magnetic beads with anti-CD138 mAbs. Then total RNA isolation, polyadenylation at its 3’end and reverse transcription using oligo (dT)-adapter was held. Subsequently the levels of mir-221-3p and miR-222-3p were quantified using an in-house qPCR methodology. To analyze survival and post-treatment follow-up of patients we used Overall Survival, Progression-free survival and Cancer-related Survival. The result we obtained from the statistical analysis were of particular interest. First, miR-221-3p levels were lower in patients with MM, in contrast with them, who have been diagnosed with sMM or MGUS (p=0,030). This factor indicate that this molecule can potentially function as a potential diagnostic marker to distinguish between sMM/MGUS and MM patients. Synchronously, the low levels of this molecule seem to be related with the upregulation of the levels of b2m (beta-2-microglobulin) (p=0,059), a prognostic marker for MM. Concurrently miR-221-3p levels were significantly reduced in patients at the stage of RISS III compared to those in RISS I/II (p=0,052). Furthermore, the downregulation of this microRNA could be an indicator for bad prognosis for MM. Low levels were negatively associated with total survival (p=0,040) and with the progression of the disease after a therapeutic protocol (p=0,003). The strong predictive value of miR-221-3p levels was also confirmed by Cox regression analysis. Along the same lines, we obtained significantly reduced levels of miR-222-3p in patients with high b2m values (p=0,091) and in MM patients in RISS III stage versus them in stages RISS I/II (p=0,036). In addition, survival analysis through Kaplan-Meier curves highlighted the statistically significant association of low levels of the miR-222-3p with overall patient survival (p=0,015), higher risk of short-term disease progression (p=0,004) and deaths from the specific type of cancer (p=0,022). The strong predictive value of miR-222-3p, in terms of disease progression and overall survival, was also confirmed by Cox regression analysis.
In conclusion, we identified the loss of miR-221-3p and miR-222-3p in CD138+ plasma cells as a powerful indicator of adverse disease outcome, ameliorating tailored patient’s management.
Keywords:
multiple myeloma, cancer, non coding RNA, miRNA, miR-221, miR-222
