Mass Spectrometry (MS)-Based Exent® Solution Reveals High Prevalence of Multiple M-Proteins in African American Patients with Multiple Myeloma (MM) and Precursor Conditions

Background: Identifying the monoclonal immunoglobulin (M protein) components, typically produced by the proliferating plasma cell clone, is central to the diagnosis and monitoring of plasma cell disorders (PCD). Biclonal gammopathies, defined by more than one type of M protein, are rare, representing 1-6% of PCD cases. Tracking both clones in biclonal MM is crucial as one clone may respond while the other may not, potentially representing an MGUS state. Standard serum electrophoretic methods, such as serum protein electrophoresis (SPEP) and immunofixation (IFE), lack the analytical sensitivity needed to detect minor additional M proteins or sufficient resolution to differentiate between two M spikes, especially if they share the same isotype. In a previous report including predominantly White individuals, the second M protein by MS was noted in 22% PCD cases. Here, we used the MS data acquired with the Immunoglobulin Isotypes (GAM) for the EXENT® Analyzer (The Binding Site, part of Thermo Fisher Scientific) to evaluate the prevalence of multiple M proteins in African American (AA) patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed MM.

Methods: Diagnostic serum samples collected from patients under an IRB-approved protocol were analyzed using an EXENT iP-500 mass spectrometer, processed via EXENT iQ software, and reported in terms of presence/absence, isotype, mass to charge ratio (m/z), and quantity of M protein. Quantification of M protein relied on measurements of total IgG, IgA, and IgM by Optilite®. A second M protein was considered distinct from the primary M protein if it differed in heavy chain (HC) or light chain (LC), or if it shared the same isotype but had a different molecular mass (m/z). Samples with glycosylated light chains, producing additional peaks, were excluded from biclonal categorization. A ≥0.02g/dL threshold was applied for M protein, approximating the limit of detection for standard methods. High molecular weight (HMW) LC signal was defined as λ peaks with m/z > 2 standard deviation . Patients' demographic and baseline disease characteristics were collected.

Results. A total of 109 samples collected from AA patients diagnosed with MGUS (44), SMM (33), and newly diagnosed MM (32) were analyzed. The median age was 64 (34-84) years, with females comprising 48.6%. By conventional diagnostics, free LC disease was present in 20 (18%) patients and biclonal disease in 14 (12.8%) patients. The average M-spike detected by EXENT was 1.19g/dL (range 0.02-8.4) compared with 1.16g/dL (range 0.1-6.4) with SPEP. Thirty-five (32.1%) samples had a second M protein detected by EXENT including 2 IgA, 12 IgG, 3 IgM and 18 κ or λ LC. The second M protein was of the same HC and LC isotype as the primary peak in 9 of 35 cases. Additionally, a third M protein was identified in 8 (7.3%) patients including 4 IgG, 1 IgM and 3 LC. The prevalence of multiple M proteins was highest in the MGUS group (38.6%) compared to SMM (27.3%) and MM (27.3%).

Overall, the EXENT GAM assay has detected monoclonal LC in 30 patients (34.9%), totaling 38 LC (14 κ, 24 λ) either as the main or secondary M protein; 4 patients had a glycosylated LC. Fifteen patients also had a HMW LC signal (m/z 12,025 ± 1.3) detected in the λ LC spectra, which may have been due to interference from C1q complement protein. The HMW LC signal was present predominantly among patients with MGUS (86.7%) vs SMM/MM (13.3%). MGUS patients with a HMW LC signal were younger (61 ± 9.4 vs. 73±8.7 years, p<0.05), had better renal function (creatinine 1.89 ± 2.6 vs 2.8 ± 2.0 mg/dL, p<0.05), and showed a trend toward lower M-spike levels (0.33 ± 0.28 vs 0.59 ± 0.33 g/dL, p=0.126) compared to those with normal m/z LC.

Conclusions. The frequency of two or more M proteins by MS was notably higher in our AA cohort compared to previous reports using traditional technologies. In contrast to previously published reports, primarily composed of white individuals, our study revealed a higher prevalence of multiple M proteins among AA patients with PCDs. The clinical significance of HMW LC signal and the potential for C1q-interference needs further investigation. Additional studies comparing White and AA cohorts are essential to understand potential differences in the prevalence of multiple M proteins, their functional role in the pathogenesis of PCDs, and their evolution over the course of the disease.

Foureau:Astrazeneca: Research Funding. Berlanga:The Binding Site, part of Thermo Fisher Scientific: Current Employment. Varga:Janssen: Consultancy, Research Funding; LavaTherapeutics: Research Funding. Paul:AbbVie Inc: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding. Atrash:GSK: Research Funding; Amgen: Research Funding; Karyopharm: Research Funding; Janssen: Honoraria. Ferreri:Affimed Therapeutics.: Current equity holder in private company; Sanofi: Consultancy; Janssen: Consultancy. Lakos:Christopher Ferreri: Current Employment. Voorhees:Lava Therapeutics: Consultancy; Regeneron: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy. Bhutani:Janssen: Research Funding; BMS: Research Funding; Caribou Biosciences: Research Funding; Amgen: Research Funding; Takeda: Research Funding; Abvvie: Research Funding.