N-glycosylation of monoclonal immunoglobulins across plasma cell dyscrasias in routine care: A cohort study using EXENT® mass spectrometry

Abstract Background: N-glycosylation of monoclonal immunoglobulin light chains is increasingly recognised as a potential biomarker of disease biology in plasma cell disorders. Somatic hypermutation can introduce N-glycosylation motifs into the variable regions of immunoglobulins, a phenomenon linked to clonal evolution. Prior studies have associated paraprotein glycosylation with increased risk of MGUS progression to multiple myeloma (MM) (Dispenzieri et al., 2020), and higher prevalence has also been reported in AL amyloidosis (Mellors et al., 2021) and cold agglutinin disease (Juskewitch et al., 2021). However, its role in progression risk and disease severity is still not well defined. We have previously shown that glycosylation of the primary paraprotein remains stable in MGUS patients over time. Here, we hypothesised that the prevalence of M-protein glycosylation increases across the disease spectrum, from MGUS to smouldering multiple myeloma (SMM) to multiple myeloma (MM), reflecting the increased risk of progression and underlying aggressive disease biology. Methods: Baseline sequential diagnostic serum samples from routine patients with MGUS (n=123), SMM (n=37), and MM (n=174) were analysed using the EXENT® mass spectrometry system (The Binding Site Group Ltd, UK). Glycosylation of the primary paraprotein was identified using mass shifted light chain (MSL) flagging for N-glycan-associated light chain mass shifts by the EXENT software. Glycosylation was considered present if detected on the primary paraprotein (heavy and/or light chain) and flagged by the software as an MSL. Data were stratified by diagnostic category, and subgroup analysis was performed on myeloma subtype and IMWG myeloma response definitions. Clinically aggressive disease was either progressive disease or clinical relapse as defined by the IMWG criteria (Kumar et al. 2016). Results: N-glycosylation of the primary paraprotein was observed in:11% of MGUS patients (14/124) (Primary paraprotein subtypes in which N-glycosylation was observed: IgG=8 IgA= 1 IgM=5)16% of SMM patients (6/37) (Primary paraprotein subtypes in which N-glycosylation was observed: IgG = 5, IgA = 1)17% of MM patients (22/174) (Primary paraprotein subtypes in which N-glycosylation was observed: Kappa only = 3, Lambda only = 1, IgG = 10, IgA = 5, IgM = 3) Within the MM cohort:Patients with light chain myeloma had a higher glycosylation frequency of 25% (4/16)Among a subset of MM patients with clinically aggressive disease, glycosylation was observed in 26% of cases (7/27) Isotype distribution among N-glycosylated paraproteins revealed additional patterns of interest. In MGUS, 5 of 14 glycosylated cases (36%) were IgM, despite IgM accounting for a minority of MGUS cases overall. Similarly, in MM, 3 of 22 glycosylated cases (14%) were IgM. These findings show a modest stepwise increase in glycosylation frequency from MGUS to SMM to MM, with higher rates seen in specific high-risk myeloma subgroups. Conclusion: Our data suggest that N-glycosylation of M-protein light chains becomes more prevalent across the spectrum of disease from MGUS to MM and may associate with more aggressive clinical phenotypes of myeloma, including light chain only disease. Although absolute differences are modest, glycosylation status may offer additional biological or prognostic insight, particularly in light chain and high-risk disease. Further studies are warranted to explore the role of N-glycosylation in risk stratification, disease monitoring and correlation with clinical phenotypes.