NGS in Purified CD138+ Plasma Cells Reveals Distinct Mutational Signatures. a Comparative Study of Newly Diagnosed Hyperdiploid Vs t(11;14) Multiple Myeloma
Authors: Piñero Paula , Gonzalez Rocio , Diaz Vanesa , Marin Elena , Gonzalez Marina , Manresa Pablo , Lopez Javier , Tarin Fabian 
Published: December 2024

Introduction

Multiple Myeloma (MM) is a complex hematological malignancy characterized by a range of well-known cytogenetic alterations. Despite the last advances in the treatment of MM, it remains to be a challenging disease with a high variability of responses to therapy. The presence of specific genetic alterations, such as those associated with hyperdiploidy, t(11;14), 1q21 amplifications, and p53 deletions, contributes to the variability in disease progression and treatment outcomes.

Aims:

To elucidate the molecular alterations specific to different MM cytogenetic subtypes and to understand their implications for disease pathogenesis and progression.

To evaluate whether mutational analysis of the CD138+ cell population is an effective approach for molecular characterization of mutations with very low Variant Allele Frequency (VAF) in MM.

Methods:

Next-Generation Sequencing (NGS) was performed on a cohort of 64 newly diagnosed Multiple Myeloma (MM) patients. This cohort included 39 individuals with hyperdiploidy, 25 with t(11;14), 24 with 1q21 amplifications, and 7 with p53 deletions. The percentage of aberrant plasma cells was assessed by flow cytometry using the standardized EuroFlow consortium panel for plasma cell disorders. Plasma cells were isolated from bone marrow aspirate samples using magnetic separation with anti-CD138 beads (MACs Miltenyi). Cytogenetic alterations were identified using Fluorescence in Situ Hybridization (FISH). NGS libraries were synthetized using the SOPHIA Custom Multiple Myeloma NGS panel, which targets 43 genes (1500X coverage), and analyzed in the Sophia DDM software. The minimum VAF considered was 1% in selected CD138+ population.

Results

The most notable alterations detected were involved in 5 major pathways: cell cycle, transcriptional regulation, signal transduction pathways, apoptosis and immune response. Interestingly, alterations were not found in the same regions, but affected different genes in each myeloma subtype. A significative proportion of mutations (17,5%) were subclonal (VAF= 1-5%). The most frequently mutated genes were p53, KRAS, BRAF, FGFR3 and FMN2. In hyperdiploid MM (HDMM), the MAPK/ERK signaling pathway was affected by BRAF mutations (18.5%), as well as KRAS (22.4%) and NRAS mutations (14.3%). Remarkably, BRAF mutations were only observed in HDMM. Transcriptional process was also affected, including mutations in EGR1 and TENT5C (13.8% and 10.3% of cases respectively). Regarding apoptosis and cell survival, we identified mutations in CYLD and BIRC2 in 14.8% of cases. Finally, alterations in SP140, implicated in the immune regulation, were found in 10.3% of HDMM cases compared to 4.5% in t(11;14), (p&lt;0.05). On the contrary, MM with t(11;14) were characterized by alterations in cell cycle, mainly in RB1 (18.2%) and CCND1 (45.5%). Attending to signaling pathways mutations affected mostly FGFR3 (27.3%) and FAT3 (22.7%), while the MAPK/ERK pathway was altered by mutations in RASA2 (9.1%) and NF1 (10.2%). DIS3 was the most frequently altered gene (18.2%) involved in transcriptional regulation. Finally, in this group, apoptosis was impaired by mutations in PPM1D (9.1% of cases). TP53 lesions were detected in 16.3% of cases across both subtypes, with lesions occurring in cases with (29.4%) and without deletion (41.2%). Similarly, KRAS mutations were found in 22.4% of all cases, while NRAS mutations were present in 14.3%. NRAS and KRAS mutations were mutually exclusive. Notably, NRAS mutations were strongly associated with 1q21 amplification (50% vs. 18% without amplification, p&lt;0,05), whereas KRAS mutations were absent in cases with 1q21 amplification. Additionally, we identified mutations with potential contribution to metastatic capacity in FMN2 (28.6% of all cases).

Conclusions Comparative analysis of t(11;14) and hyperdiploid MM revealed significant differences in critical pathways. The high frequency of genetic events within the same molecular pathways (such as CCND1, RAS, or p53/RB) suggests potential synergistic effects that may contribute to drug resistance and disease progression. The sequencing of purified CD138+ cells allows the detection of low VAF mutations, offering crucial data for risk stratification and optimal management of the disease. The detection of specific mutations in the different MM subtype is the first step for future advances in the development of personalized therapy.