Introduction: Waldenström macroglobulinemia (WM) is clinically heterogeneous with a subset of patients experiencing IgM-driven complications at minimal monoclonal protein levels that are missed or unquantifiable by serum protein electrophoresis with immunofixation (SPEP-IFX). Mass spectrometry (MS), adopted by the IMWG, has demonstrated superior sensitivity in multiple myeloma (Murray et al, 2021). We therefore conducted the first dedicated MS study in WM/IgM MGUS to characterize monoclonal IgM across phenotypes, including familial WM where clonal diversity due to genetic predisposition is suspected.

Methods: We performed mass spectrometry using the EXENT® platform integrating automated immuno-enrichment with high-sensitivity MALDI-TOF to detect and quantify monoclonal peaks (isotype, m/z, concentration) and evaluate light chain glycosylation. 150 plasma samples from 105 patients with monoclonal IgM were analyzed (93 WM, 7 MGUS/MGCS, 5 with overlapping WM and cold agglutinin disease [CAD] features), including 45 with paired baseline and 6-month post-treatment samples. Patient cohorts with various paraprotein related pathologies included: anti-MAG neuropathy (n=24); hyperviscosity and acquired VWD (n=15); cryoglobulinemia (n=9); amyloidosis (n=10); CAD (n=5); familial WM (n=6); renal WM (n=2); WM without IgM-related complications as controls (n=34).

Results: Median age was 66 (range 43-82 years); 37/105 (35%) were female. At sampling, 81/105 (77%) were treatment-naïve; median bone marrow (BM) involvement was 45% (range 0-95%). MYD88 and CXCR4 mutations were present in 89/99 (90%) and 36/96 (38%), respectively. Median IgM levels and BM involvement were highest in HV/VWD (6100 mg/dL; 70%) and WM without IgM complications (3776; 70%), and lowest in CAD (462; 10%) and anti-MAG neuropathy (986; 10%). All 5 CAD were MYD88 wild-type, while CXCR4 mutations were enriched in HV/VWD (9/15, 60%). No significant differences were observed in age, sex, or treatment status.

MS detected monoclonal IgM in 104/105 patients (99%), including 3 with ongoing neuropathy symptoms (anti-MAG titers >70,000 and no detectable M-spike or IgM by SPEP-IFX) and 4 with unquantifiable M-spike by SPEP-IFX. In one case, MS failed to detect an IgM peak within a polyclonal background, due to antibody independent binding. Using the first sample per patient, MS detected 129 monoclonal IgM peaks (median 1, range 0-4) vs 109 by SPEP-IFX (median 1, range 0-2); 21 patients (21%) had ≥2 peaks by MS vs 7 (7%) by SPEP-IFX (p<0.001). Light chain glycosylation was identified in 14 of 104 evaluable cases (13%) and was most frequent in CAD (3/5 cases, 60%; corrected p=0.06), all of whom had fully glycosylated peaks. Percent change in monoclonal IgM by MS after treatment initiation (n=45) correlated strongly with changes in serum nephelometric IgM (r=0.91) and SPEP-IFX (r=0.84). In the lowest baseline IgM quartile (<1500 mg/dL), MS-IgM correlation remained high (r=0.89; p<0.001), MS-SPEP was also strong (r=0.72; p=0.008), and SPEP-IgM was lower (r=0.57; p=0.02).

Familial WM, defined by a patient having at least one first-degree relative with WM, was strongly enriched for multi-clonality by MS (5/6, 83%). In contrast, multi-clonality was observed in 22% of cryoglobulinemia (2/9), 21% of neuropathy (5/24), 20% of amyloidosis (2/10), 20% of CAD (1/5), 12% of WM without IgM complications (4/34), and 7% of HV/VWD (1/15) (corrected p=0.007).

All familial WM peaks were IgM kappa. Four patients had two monoclonal IgM peaks and one had three, with clear intra-patient differences in molecular mass (median m/z difference 254; range 200-271). Individual peak concentrations ranged from 1.2 to 21.7 g/L. Two patients had paired post-treatment samples; in both, each peak persisted at the same m/z, with variable reductions in concentration. In patient 1 (two peaks), post-treatment reductions were 39% and 29%; in patient 2 (three peaks), 50%, 85%, and 31%, respectively, by decreasing baseline abundance.

Conclusion: Mass spectrometry (EXENT®) offers enhanced sensitivity and structural resolution over conventional methods, enabling detection of clinically meaningful monoclonal IgM in low-burden cases and characterization of features such as light chain glycosylation. In familial WM, this increased sensitivity uncovered marked clonal complexity, with frequent bi- or tri-clonal patterns indicating multiple coexisting clones and reinforcing its classification as a biologically distinct entity.

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2025
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