Willrich, Maria Alice V.

Abstract Background Electrophoretic methods to detect, characterize and quantify M-proteins play an important role in the management of patients with monoclonal gammopathies (MGs). Significant uncertainty in the quantification and limit of detection (LOD) is documented when M-proteins are <10 g/L. Using spiked sera, we aimed to assess the variability in intact M-protein quantification and LOD across 16 laboratories. Methods Sera with normal, hypo- or hyper-gammaglobulinemia were spiked with daratumumab or elotuzumab, with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Laboratories blindly analyzed samples according to their serum protein electrophoresis (SPEP)/isotyping standard operating procedures. LOD and intra-laboratory percent coefficient of variation (%CV) were calculated and further specified with regard to the method (gel/capillary electrophoresis [CZE]), gating strategy (perpendicular drop [PD]/tangent skimming [TS]), isotyping (immunofixation/immunosubtraction [ISUB]) and manufacturer (Helena/Sebia). Results All M-proteins ≥1 g/L were detected by SPEP. With isotyping the LOD was moderately more sensitive than with SPEP. The intensity of polyclonal background had the biggest negative impact on LOD. Independent of the method used, the intra-laboratory imprecision of M-protein quantification was small (mean CV = 5.0%). Low M-protein concentration and high polyclonal background had the strongest negative impact on intra-laboratory precision. All laboratories were able to follow trend of M-protein concentrations down to 1 g/L. Conclusions In this study, we describe a large variation in the reported LOD for both SPEP and isotyping; overall LOD is most affected by the polyclonal immunoglobulin background. Satisfactory intra-laboratory precision was demonstrated. This indicates that the quantification of small M-proteins to monitor patients over time is appropriate, when subsequent testing is performed within the same laboratory.

Abstract Background Serum protein electrophoresis (SPEP) is used to quantify the serum monoclonal component or M-protein, for diagnosis and monitoring of monoclonal gammopathies. Significant imprecision and inaccuracy pose challenges in reporting small M-proteins. Using therapeutic monoclonal antibody-spiked sera and a pooled beta-migrating M-protein, we aimed to assess SPEP limitations and variability across 16 laboratories in three continents. Methods Sera with normal, hypo- or hypergammaglobulinemia were spiked with daratumumab, Dara (cathodal migrating), or elotuzumab, Elo (central-gamma migrating), with concentrations from 0.125 to 10 g/L (n = 62) along with a beta-migrating sample (n = 9). Provided with total protein (reverse biuret, Siemens), laboratories blindly analyzed samples according to their SPEP and immunofixation (IFE) or immunosubtraction (ISUB) standard operating procedures. Sixteen laboratories reported the perpendicular drop (PD) method of gating the M-protein, while 10 used tangent skimming (TS). A mean percent recovery range of 80%–120% was set as acceptable. The inter-laboratory %CV was calculated. Results Gamma globulin background, migration pattern and concentration all affect the precision and accuracy of quantifying M-proteins by SPEP. As the background increases, imprecision increases and accuracy decreases leading to overestimation of M-protein quantitation especially evident in hypergamma samples, and more prominent with PD. Cathodal migrating M-proteins were associated with less imprecision and higher accuracy compared to central-gamma migrating M-proteins, which is attributed to the increased gamma background contribution in M-proteins migrating in the middle of the gamma fraction. There is greater imprecision and loss of accuracy at lower M-protein concentrations. Conclusions This study suggests that quantifying exceedingly low concentrations of M-proteins, although possible, may not yield adequate accuracy and precision between laboratories.

Abstract Background Serum free light chain (FLC) assays are used clinically to measure the concentration of κ and λ FLC in patients with suspected or diagnosed plasma cell proliferative disorders. Previous studies have demonstrated a loss of linearity in low concentration ranges of these assays. We hypothesized that this result could be caused by a matrix effect. Methods Recovery studies were performed for κ and λ FLC in both serum and saline using the Freelite assay (Binding Site) on a Cobas c502 system (Roche). Samples were analyzed either at the recommended dilution or undiluted. Follow-up studies were performed in varying matrices ranging from 0% to 100% saline. Retrospective patient data were analyzed to assess the impact on reported κ FLC, λ FLC, and κ/λ ratio. Results FLC in a serum matrix demonstrated underrecovery relative to samples diluted in saline for both κ and λ FLC. Of 255 patient samples with λ FLC measured undiluted (λ FLC &lt;6.0 mg/L), an unexpected gap was observed in patient results between 2.0 and 6.0 mg/L. In addition, 23 patients measured serially with λ FLC between 2.0 and 6.0 mg/L demonstrated dramatic changes in κ/λ ratio, with no changes in κ FLC, likely because of the matrix effect. Conclusions The κ and λ Freelite assays exhibit a matrix effect when samples are tested undiluted, which has the potential to affect the κ/λ ratio. Consequently, our laboratory has stopped reporting λ FLC &lt;6.0 mg/L.

Abstract Background and Aims We aimed to evaluate the relationship of serum adalimumab trough levels (ATL) with disease activity of inflammatory bowel disease (IBD) patients in a large, well-characterized referral center-based cohort. Methods We compared serum ATL between those with clinical, biochemical, or endoscopic/radiologic disease activity and those without. Results A total of 236 patients with IBD were included. Higher cutoff levels were associated with endoscopic and/or radiologic responses (cutoff value: 5.3 mcg/mL, P = 0.003) compared with improvement in C-reactive protein (cutoff value: 4.3 mcg/mL, P = 0.031). Conclusions Higher cutoff ATL was associated with endoscopic and/or radiologic response.

Abstract Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Introduction: Primary cold agglutinin disease (CAD) is a rare disorder and accounts for 15% of all autoimmune hemolytic anemia. It is associated with an underlying monoclonal protein, which is IgM kappa in the majority of patients. The IgM protein triggers complement mediated extravascular hemolysis. The underlying pathophysiology by which autoimmune hemolysis affects only a small subset of patients with IgM monoclonal protein is poorly understood. Changes in the IgM 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. (Kumar S et al Leukemia 2017). We hypothesized that presence of post-translational glycosylation is a potential mechanism allowing the IgM protein to trigger complement mediated hemolysis in CAD. Methods: Serum samples of patients being evaluated for monoclonal protein disorders were analyzed by immuno-enrichment-based matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), termed MASS-FIX. Patients who had an underlying IgM monoclonal protein identified by MASS-FIX from 07/23/2018 to 5/14/2019 were included this study. Glycosylated LCs were identified by the shift in their molecular mass due to the added molecular mass of the carbohydrates. Glycosylation was compared in patients with IgM associated CAD vs. other IgM disorders. Results: 438 unique patients with an IgM monoclonal protein identified on MASS-FIX were included in the study. CAD was present in 3% (n=14) of patients. The primary underlying diagnosis in the remaining patients was monoclonal gammopathy of undetermined significance (MGUS): 47% (n=206), lymphoplasmacytic lymphoma (LPL)/Waldenstrom 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) Glycosylation was present in 7% (31/438) of patients in this cohort. Glycosylation was significantly more prevalent in patients with CAD than other IgM gammopathies (64% vs. 5%, p &lt;0.001). CAD was present in 29% of patients with glycosylation and in only 1% of patients without glycosylation, p&lt;0.001. Disease characteristics of patients with CAD, including patients with and without glycosylated LCs are listed in Table 1. No statistically significant differences were observed in the features of CAD patients with and without glycosylation, though sample size was small. All patients (100%) had IgM kappa monoclonal protein. Median M-spike was 0.5 g/dL. The associated disorder was IgM MGUS in 50% of patients, LPL/WM in 29%, marginal zone lymphoma in 14% and a non-specific B-cell lymphoproliferative disorder in 7% of patients. Median hemoglobin was 11.2 g/dL. 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 (&gt;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 were tested with monospecific DAT had an antiC3 antibody (n=10). One patient also had monospecific anti IgG antibody. Transfusions were needed in 21% of patients. Other clinical manifestations included acrocyanosis in 43% (n=6) and Raynaud's phenomenon in 50% (n=7) of patients, respectively. Treatment for CAD included rituximab with or without steroids in 50% (n=7) of patients, rituximab-based chemotherapy in 29% (n=4) of patients, with observation alone in 21% (n=3) of patients. Conclusions: Modification of the IgM immunoglobulin structure by post-translational glycosylation of LCs was significantly more common in patients with CAD compared with other IgM monoclonal gammopathies (64% vs. 5%, p &lt;0.001). CAD was seen in only 1% of patients without glycosylation, while one-third of patients with glycosylation had CAD. This is a novel finding. As MASS FIX is now routinely done for detection of monoclonal proteins at our institution, the presence of LC glycosylation can provide diagnostic clues and can be a potential therapeutic target in the future. Future studies in larger, independent cohorts of patients are needed to confirm these findings. *SS & DLM contributed equally Disclosures Murray: The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Dasari:The Binding Site: Patents & Royalties: US Patent Rights on Mass Spectroscopy Licensing agreement with The Binding Site, Research Funding. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding. Dispenzieri:Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding; Celgene: Research Funding; Alnylam: Research Funding; Intellia: Consultancy; Akcea: Consultancy. Gertz:Spectrum: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria; Prothena: Honoraria; Ionis: Honoraria; Alnylam: Honoraria.

Abstract Background: Current laboratory methods for detection of monoclonal gammopathies include a panel of serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain quantitation (FLC). Our group has recently described a new assay based on matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) which is capable of detecting, isotyping and quantitating M-proteins in a single assay. The basic principle of the method involves leveraging the unique mass resulting from light chain (LC) Ig gene rearrangement in B-cells. In a similar fashion to SPEP, the mass distribution of LCs after dissociation from their heavy chains can be examined for the presence of an over expressed light chain. In this study we retrospectively compared the MALDI-TOF MS method to SPEP, IFE and Hevylite ratios for monitoring patients with monoclonal gammopathies. Methods: Serum from patients who had at least 1 diagnostic and 4 serial follow-up samples was enriched for IgG, IgA, IgM, kappa and lambda using nanobodies. After disassociating the heavy and light chains by reduction, the five purified samples were spotted onto a Bruker Microflex MALDI plate. Automated acquisition (~10 seconds/sample) was performed and the five LC mass spectra from each enrichment were overlaid. M-proteins were detected, quantitated and isotyped by the presence of distinct peaks (m-spike) within LC mass to charge regions. The serial patient samples were then measured by MALDI-TOF and a comparison of results was made to previous aquired data. The results were then analyze in light of clinical and lab data in order to evaluate the ability of the MALDI-TOF MS method to monitor patients with monoclonal gammopathies. Results: In M-protein positive patient samples, serial dilution revealed MALDI-TOF MS to have ~10-times lower limit of detection than IFE. M-protein isotype in the cohort by MALDI-TOF MS was 100 percent concordant with the isotype from IFE. The MALDI TOF M-protein quantitation was linear over a clinically acceptable range (0.1 to 6 g/dL) and had improved linearity over SPEP at lower M-protein levels. M-proteins quantitation by MALDI-TOF MS compared well with SPEP with a slope of 1.16 (R2 -0.93). Hevylite Ig ratios for each isotype were statistically similar to those from MALDI-TOF MS demonstrating the ability of the MALDI to measure isotype suppression. For some patients, the MALDI method was able to detect M-proteins in patients with normal Hevylite ratios. Finally, the results for each patient were then compared over the course of monitoring. The time evolution change in M-protein concentration was similar among the three methods with the exception of a few samples in which the MALDI-TOF detected residual M-proteins in treated patients not detected by the other methods. Conclusion: This preliminary study demonstrates that MALDI-TOF MS method can provide detection, quantitation and isotyping data suitable for monitoring patients. This is a significant finding since the MALDI-TOF method is amendable to automation, is rapid and in its current format is cost-competitive with current clinical assays. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed. Mills:Mayo Clinci: Patents & Royalties: Patent Application Filed. Barnidge:Mayo Clinic: Patents & Royalties: Patent Application Filed.

Abstract BACKGROUND: Complement-mediated thrombotic microangiopathies (TMAs) are a subset of TMAs in which the thrombocytopenia, microangiopathic hemolytic anemia, and end organ damage are caused by mutations in genes encoding the alternative complement pathway. Numerous complement genes have been implicated in complement-mediated TMAs including complement factor H (CFH), CD46 (MCP), complement factor I (CFI), complement component 3 (C3), complement factor B (CFB), factor H related 5 (CFHR5), and thrombomodulin (THBD) among others. Genetic analysis typically yields a mutation in 60% of patients whose TMA is presumed to be mediated by complement dysregulation. However, the description of novel disease-associated variants may increase this proportion. METHODS: A retrospective study of patients with TMAs diagnosed between 2000 and 2014 was performed. TMA diagnosis was made based on thrombocytopenia and evidence of microangiopathic hemolytic anemia. Analysis was performed with Alamut¨ software with additional in silico prediction tools (SIFT, MutationTaster, and Polyphen) for classification of gene variants. Variants of unknown significance (VUS) and likely pathogenic variants were further assessed using several mutation databases, including HGMD, ClinVar, and Factor H database RESULTS: Of patients diagnosed with a TMA, genetic analysis was performed in only a 10% of patients. Of the 29 patients with genetic studies performed, mutations were identified in 18 patients (62%). The majority of the mutations had been described previously in the literature, but four novel variants were identified: three missense and one splice-site. The table below summarizes these variants as well as laboratory findings on presentation. These were two variants of CFH, one variant of CFHR5, and one variant of CFI. In silico modeling of these variants revealed two polymorphisms likely to be pathogenic, one polymorphism likely benign given the lack of predicted splicing changes, and one VUS. Table 1. Protein Mutation Classification Age Sex Hemoglobin (g/dL) Platelets (thousands) Creatinine (mg/dL) CFH c.245-10_245-9dup Likely benign 61 F 11.6 51 6.7 CFH c.476G>A, p.Ser159Asn Likely pathogenic 43 F 9.1 101 9.1 CFHR5 c.1412G>A p.Gly471Glu Likely pathogenic 30 F 9.8 129 4.9 CFI c.1190T>A p.Val397Glu Unknown significance 51 M 9.4 125 6.1 DISCUSSION: With therapy available to target the alternative complement pathway, genetic analysis to identify genetic variants capable of causing complement mediated TMAs is an essential part of the evaluation. This genetic data must be interpreted and correlated with functional analysis and clinical phenotype. The reporting of novel variants in clinical databases, with inclusion of relevant clinical findings, is necessary to accurately classify and verify variants as pathologic mutations or benign polymorphisms. The full understanding of this diverse disease requires a more complete understanding of its genetics. While complement pathway-directed therapies are available, their rational use requires thorough interpretation of laboratory data, including genetic analysis. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed.

INTRODUCTION: There are multiple assays for infliximab (IFX) trough level (IFX-TL) and antibody (ATI) measurement. The aims of this study are to examine the correlation and outcomes of IFX-TL and ATI measured with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and electrochemiluminescence (ECLIA) in inflammatory bowel disease (IBD) patients. METHODS: This was a prospective study of IFX-treated IBD patients undergoing drug monitoring with an ECLIA panel (Esoterix Laboratories). Residual serum samples were used to simultaneously quantify IFX-TL via LC-MS/MS and ATI via an in-house ECLIA (iECLIA) (Mayo Clinic). Both LC-MS/MS and iECLIA have been previously validated. Primary outcomes: IFX-TL and ATI comparison between the two assays. Secondary outcomes: association of IFX-TL and ATI with clinical remission, mucosal healing (MH) (absence of ulcers in Crohn's disease and Mayo endoscopic score ≤1 for ulcerative colitis) and normal serum C-reactive protein (CRP ≤8 mg/L). RESULTS: A total of 151 patients were included (median age 32 years (12-84), 45.7% female) (Table 1). Median IFX-TL was 7 mcg/mL (IQR: 1.3, 19.4) and 6 mcg/mL (IQR: 0.9, 20) via LC-MS/MS and ECLIA, respectively (Spearman correlation coefficient r = 0.975). Median IFX-TL via LC-MS/MS was not significantly different between patients in clinical remission, response and no response (5.7 vs. 8.4 vs. 2.3 mcg/mL, respectively; P = 0.46), but significantly higher in patients who had a normal CRP (11.1 vs. 1.4 mcg/mL; P < 0.001) and in those who achieved MH (13.1 mcg/mL vs. 3.9 mcg/mL; P = 0.01). ATI was detected in 13/142 (9.2%) via iECLIA and 39/151 (25.8%) via ECLIA. The frequency of ATI detection via iECLIA was lower in patients in clinical remission or response than no response (7.3% vs. 9.3% vs. 15.4%; P = 0.68). On the contrary, the frequency of ATI detection via ECLIA was higher in patients with clinical remission (35.7%) than response (18.7%) and comparable to patients who had no clinical response (40%) ( P = 0.05) (Table 2). The frequency of ATI was not significantly different between patients who did and did not achieve MH or had normal CRP using both assays (Table 2). CONCLUSION: IFX-TL was comparable between LC-MS/MS and ECLIA assays. IFX-TL correlated with MH and normal CRP but not clinical remission. Frequency of ATI detection was not significantly associated with outcomes via both methods. ATI detected via iECLIA showed a trend towards correlation with clinical outcomes compared to ECLIA.

INTRODUCTION: Combination immunomodulator (IMM) with infliximab (IFX) has been shown to be superior to IFX monotherapy therapy in the management of inflammatory bowel disease (IBD). It is unclear if this is related to IMM solely or higher IFX trough level (IFX-TL). The aims of this study are to examine the correlation of IFX-TL with clinical outcomes of IBD patients and to evaluate whether IFX-TL or combination IMM therapy correlate best with mucosal healing (MH). METHODS: This is a retrospective study of consecutive IFX treated IBD patients undergoing drug monitoring with liquid chromatography-tandem mass spectrometry (LC-MS/MS). IMM included either thiopurines or methotrexate. Primary analyses included: comparing IFX-TL with clinical remission, MH (absence of mucosal ulcers in Crohn's disease (CD) and Mayo endoscopic score ≤1 for ulcerative colitis (UC)) and normal c-reactive protein (CRP ≤8 mg/L). Secondary analyses included determining an IFX-TL cut off to achieve MH and evaluating the predictors of MH (IMM vs. IFX-TL). RESULTS: A total of 151 patients were included (67% CD, 24% UC, 6% indeterminate colitis, 3% pouchitis) (Table 1). The median IFX-TL was 7 mcg/mL (IQR: 1.3, 19.4). The distribution of IFX-TL was not significantly different between patients in clinical remission, response and no response (median: 5.7 vs. 8.4 vs. 2.3 mcg/mL, respectively; P = 0.46). The median IFX-TL was significantly higher in patients who had a normal CRP (11.1 vs. 1.4 mcg/mL; P < 0.001) and in those who achieved MH (13.1 mcg/mL vs. 3.9 mcg/mL; P = 0.01). An IFX-TL ≥7.5 mcg/mL predicted MH with a sensitivity of 67% and specificity of 64% (AUC = 0.67; Figure 1). Combination therapy was associated with higher IFX-TL (median: 15.8 vs. 4.6 mcg/mL; P < 0.001). In a multivariable model to predict MH based on IFX-TL (continuously), combination therapy (yes vs no), dosing frequency (≥8 vs <8 weeks), and albumin (<3.5 vs ≥3.5), higher IFX-TL was associated with higher likelihood of MH (OR for 5-mcg/mL increase: 1.18, 95% CI 1.01-1.39; P = 0.04). In this model, IMM combination was not significant (OR: 0.79, P = 0.66), neither was dosing frequency (OR: 1.44, P = 0.48). Low albumin showed lower likelihood of MH, but this was not statistically significant (OR 0.14, 95% CI: 0.02-1.16, P = 0.07). CONCLUSION: IFX-TL was associated with normal CRP and MH but not clinical remission in this cohort of IBD patients. IFX-TL and not combination IMM therapy was independently associated with MH.