Glycosylation of immunoglobulin light chains is highly prevalent in cold agglutinin disease
Authors: Surbhi Sidana, David L. Murray, Surendra Dasari, Ronald S. Go, Eli Muchtar, Maria A. Willrich, Melissa Snyder, Mindy Kohlhagen, John A. Lust, Taxiarchis V. Kourelis, Shaji K. Kumar, Angela Dispenzieri, Morie A. Gertz
Journal: American Journal of Hematology (2020)

Primary cold agglutinin disease (CAD) is a rare disorder and accounts for 15% of all autoimmune hemolytic anemia.1 It is associated with an underlying lymphoproliferative disorder and accompanying circulating monoclonal protein, which is IgM kappa in the majority of patients.1, 2 The IgM protein in CAD acts as an auto-antibody that triggers complement mediated hemolysis through activation of the classical complement pathway. The thermal amplitude at which an individual patient's IgM cold agglutinin binds to red cell antigens can vary, with clinically significant disease seen in patients in whom the thermal amplitude is high. The IgM protein binds to red cell antigens in the acral circulation, where the temperature is lower. This binding activates the classical complement pathway with activation of C1, ultimately resulting in binding of C3b on red blood cells (RBCs). When blood from the acral circulation reaches the central circulation, the IgM dissociates from RBCs. However, the activated and bound C3b is still adherent on red cells, which activates complement receptors on macrophages of the reticuloendothelial system, resulting in extravascular hemolysis. Treatment of CAD includes supportive care with cold avoidance and treating the associated lymphoproliferative disorder which is the source of IgM cold agglutinin, using rituximab or rituximab-based immunochemotherapy.1, 2 Blockade of complement activation through use of C1 inhibitors or eculizumab, a C5 inhibitor has also shown promising results.3 The underlying pathophysiology by which autoimmune hemolysis affects only a small subset of patients with IgM monoclonal gammopathies is poorly understood. It is likely that there are unique structural features of the IgM immunoglobulin in patients with CAD that make the IgM more likely to act as an auto-antibody. Changes in the immunoglobulin structure via post-translational modifications of light chains (LCs), such as glycosylation are enriched in patients with AL amyloidosis, another rare disorder where disease manifestations are driven by the protein structure.4, 5 We hypothesized that the presence of post-translational glycosylation is a potential mechanism allowing the IgM protein to trigger complement mediated hemolysis in patients with CAD. Presence of light chain glycosylation in serum immunoglobulins was analyzed by immuno-enrichment-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX as described in detail previously.4-6 Note, MASS-FIX has been developed and validated at the Mayo Clinic for detection and identification of monoclonal immunoglobulins and is now routinely used in clinical care of patients with monoclonal gammopathies.4, 5 Patients with an IgM monoclonal protein identified by MASS-FIX from 23 July2018 to 14 May2019 were included this study (N = 438). Glycosylated LCs were identified by the shift in their molecular mass due to the added molecular mass of carbohydrates as described previously.4, 5 Prevalence of glycosylation was compared in patients with IgM-associated CAD vs other IgM disorders. Data on clinical characteristics were obtained from patients' electronic medical records. Of the 438 unique patients with an IgM monoclonal protein identified on MASS-FIX during the study period, CAD was present in 3% (n = 14) of patients. The primary underlying diagnoses in the remaining patients with IgM gammopathies were monoclonal gammopathy of undetermined significance (MGUS); 47% (n = 206), lymphoplasmacytic lymphoma (LPL)/Waldenstrom's Macroglobulinemia (WM); 30% (n = 132), non-LPL lymphoma; 6% (n = 28), AL amyloidosis; 4% (n = 19), CLL; 4% (n = 18), cryoglobulinemia 2% (n = 10), multiple myeloma; 2% (n = 9) and other: 0.5% (n = 2). Overall, glycosylation was present in 7% (31/438) of patients with IgM gammopathies. Glycosylation was enriched in patients with CAD, with 64% (9/14) of patients with CAD noted to have LC glycosylation compared to 5% (22/424) of patients with other IgM gammopathies, P < .001. In other words, CAD was present in 29% (9/31) of patients with glycosylation and in only 1% (5/407) of patients without glycosylation, P < .001. All patients with CAD (100%) had an IgM kappa monoclonal protein. Median serum monoclonal protein was 0.5 g/dL (range: 0.01-1.9). The associated lymphoproliferative disorder was IgM MGUS in 50% (n = 7) of patients, LPL/WM in 29% (n = 4), marginal zone lymphoma in 14% (n = 2) and a non-specific B-cell lymphoproliferative disorder in 7% (n = 1) of patients. Median hemoglobin was 11.2 g/dL (range: 6.5-15.5). All patients had characteristic RBC agglutination on peripheral smear. Elevated LDH and undetectable haptoglobin were seen in 100% of patients. Cold agglutinin titer was positive (≥1:64) in 12 of 13 patients (85%) in whom it was tested. The remaining patient had a titer of 1:8. Polyspecific Coombs test/direct antiglobin test (DAT) was positive in 100% of patients (n = 12 tested) and all patients who underwent monospecific DAT testing were found to have a monospecific anti-C3 antibody (n = 10). One patient also had monospecific anti IgG antibody. RBC transfusions were needed in 21% (n = 3) of patients. Other clinical manifestations in patients with CAD included acrocyanosis in 43% (n = 6) and Raynaud's phenomenon in 50% (n = 7) of patients, respectively. Disease characteristics of patients with CAD, including patients with and without glycosylated LCs are listed in Table 1. No significant differences were observed in the CAD clinical features in patients with and without glycosylation, though sample size for comparison was small. Treatment for CAD included rituximab (with or without steroids) in 50% (n = 7) of patients, rituximab-based immunochemotherapy in 29% (n = 4) of patients and observation alone in 21% (n = 3) of patients. Overall, modification of the IgM immunoglobulin structure by post-translational glycosylation of LCs was significantly more common in patients with CAD compared to patients with other IgM monoclonal gammopathies (64% vs 5%, P < .001). So, CAD was seen in only 1% of patients without glycosylation, while one-third of patients with glycosylation had CAD. This is a novel finding and may explain why IgM monoclonal proteins act as autoantibodies in some patients, triggering complement mediated hemolysis. MASS-FIX can detect post-translational modifications of the immunoglobulin structure as described. Moreover, it has a higher sensitivity for detecting monoclonal proteins compared to serum immunofixation, and can also distinguish monoclonal proteins from therapeutic monoclonal antibodies.5, 7 Now, MASS FIX is available for clinical care, and similar mass spectrometry-based testing is being developed at other referral centers.8 As this testing becomes more widely available, the presence of LC glycosylation can provide diagnostic clues for CAD. LC glycosylation may also be a potential therapeutic target in the future. Higher prevalence of LC glycosylation has also been observed in patients with AL amyloidosis, with glycosylation seen in one-third of patients with κ-AL amyloidosis and 10% of patients with λ-AL amyloidosis.4 It is likely that glycosylation predisposes light chains to misfolding and deposition in amyloidosis. Glycosylation is detectable in patients with AL amyloidosis several years prior to diagnosis (ie, when they are still in the MGUS state).9 We now recommend that patients with MGUS who have glycosylation be clinically monitored for AL amyloidosis. It is possible that MGUS patients with glycosylation also have a higher likelihood of developing CAD with time. This needs to be evaluated prospectively. Changes in glycosylation of IgG antibodies have also been observed in other autoimmune cytopenias that are not directly associated with a monoclonal gammopathy, including immune thrombocytopenia.10 Our study is limited by the lack of a validation cohort. A population-based study of glycosylation in patients with monoclonal gammopathies in Olmsted County is ongoing, which is expected to provide validation data. In summary, glycosylation of light chains is enriched in patients with CAD and can provide diagnostic clues to suspect CAD in patients with IgM gammopathies. Validation in future studies is awaited. None. S.S.: Consultancy: Janssen; D.L.M.: Intellectual property rights on Mass-Fix assay; S.D.: Intellectual property rights on Mass-Fix assay. A.D.: Research Funding from Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, G.S.K.; S.K.K.: Research Funding and membership on an entity's Board of Directors or advisory committees: AbbVie, Celgene, Janssen, KITE, Merck. Membership on an entity's Board of Directors or advisory committees: Oncopeptides, Takeda. Research funding from Novartis and Roche.; M.A.G.: Honoraria/consultancy from Ionis, Alnylam, Prothena, Celgene, Janssen, Specytrum, Annexon, Apellis, Amgen, Medscape, Abbvie, Research to Practice, Physcians Education Resource and Teva. S.S., D.L.M., M.A.G. designed the study, collected the data, performed the analysis and wrote the first draft of the manuscript. D.L.M., T.V.K., M.K., M.S., M.W., A.D., J.A.L. analyzed the serum samples on MASS-FIX. All authors critically reviewed the manuscript.