Investigation on the potential of liquid biopsies in multiple myeloma and its presymptomatic stages

Multiple myeloma (MM) stands as one of the most prevalent and insidious hematological malignancies, characterized by the abnormal proliferation of malignant plasma cells within the bone marrow. This disease has a multifactorial origin, involving a complex interaction of genetic and environmental events. It evolves from an antecedent pre-malignant state termed "monoclonal gammopathy of undetermined significance" (MGUS), which can progress to MM at a rate of approximately 1% per year, sometimes through the asymptomatic stage termed "smoldering multiple myeloma" (SMM). Since MM is often diagnosed in advanced stages of the disease, there is a critical need to develop more sensitive and non-invasive diagnostic methodologies for early disease detection. Liquid biopsies (LB), represent a promising frontier in MM diagnostics. These enable the isolation and purification of cell-free circulating tumor DNA (ccfDNA), offering a less invasive alternative to traditional bone marrow (BM) biopsies, which have long been considered the gold standard for analyzing the genetic profile of MM. Analyzing ccfDNA through LB offers significant advantages, including the possibility of monitoring the disease progression and treatment response over time. This method could also have the capability to detect genetic mutations associated with MM early on, allowing for timely and personalized therapeutic interventions. Therefore, the objective of this study was to assess the feasibility of LB in the context of MM. Specifically, LB obtained from patients at various stages of MM were compared to those from healthy control groups. The study aimed to investigate potential mutations in ccfDNA among the different cohorts. Next generation sequencing analysis was carried out using Unique Molecular Identifiers and a customized panel comprising 84 recurrent genes associated with MM. This analysis was performed on a cohort consisting of patients with MGUS, SMM, and MM, including individuals exhibiting disease progression, and compared with a control group of healthy subjects. Significant mutational heterogeneity was observed in the analyzed samples, with a notable presence of mutations even in healthy subjects. Furthermore, the most frequently mutated genes within each group differed from those reported in two genomic databases for MM. Persistent mutations have also been identified in samples from patients who have had disease progression. Interestingly, no single nucleotide variants of NRAS and KRAS, which are commonly mutated in MM, were found in MGUS samples. Additionally, our study provided valuable insights into the types of mutations, revealing a significantly unbalanced Insertions/Deletions ratio in healthy subjects, which was subsequently rebalanced as the disease progressed. These findings underscore the dynamic nature of mutational processes in MM and highlight the potential utility of liquid biopsies in elucidating disease progression mechanisms. We also compared different biopsy specimens from the same individual at different time points and disease stages, employing the high-depth digital PCR technique to detect mutations in the NRAS and KRAS genes in a cohort of 85 patients. These results revealed a correlation between the mutations found in BM and LB, with greater concordance in the LB genomic DNA (gDNA) and BM gDNA samples, highlighting the importance and potential of the use of LB. Finally, the effects deriving from the overexpression of the NRAS p.Q61R mutation in MM cell lines were explored through functional studies. Overexpression of NRAS appears to induce reduced metabolic activity and cell proliferation. However, functional tests revealed a greater tendency towards aggregation and adhesion, suggesting a more aggressive phenotypic behavior of MM. Overall, this study opens new avenues for understanding MM and its presymptomatic phases, highlighting the potential and limitations of LBs in identifying key events associated with MM progression.