Monitoring the M-protein of multiple myeloma patients treated with a combination of monoclonal antibodies: the laboratory solution to eliminate interference

Objectives The therapeutic monoclonal antibody (t-mAb) daratumumab, used to treat multiple myeloma (MM) patients, interferes with routine, electrophoretic based M-protein diagnostics. Electrophoretic response assessment becomes increasingly difficult when multiple t-mAbs are combined for use in a single patient. This is the first study to address the analytical challenges of M-protein monitoring when multiple t-mAbs are combined. Methods In this proof-of-principle study we evaluate two different methods to monitor M-protein responses in three MM patients, who receive both daratumumab and nivolumab. The double hydrashift assay aims to resolve t-mAb interference on immunofixation. The MS-MRD (mass spectrometry minimal residual disease) assay measures clonotypic peptides to quantitate both M-protein and t-mAb concentrations. Results After exposure to daratumumab and nivolumab, both t-mAbs become visible on immunofixation electrophoresis (IFE) as two IgG-kappa bands that migrate close to each other at the cathodal end of the γ-region. In case the M-protein co-migrates with these t-mAbs, the observed interference was completely abolished with the double IFE hydrashift assay. In all three patients the MS-MRD assay was also able to distinguish the M-protein from the t-mAbs. Additional advantage of the MS-MRD assay is that this multiplex assay is more sensitive and allows quantitative M-protein-, daratumumab- and nivolumab-monitoring. Conclusions Daratumumab and nivolumab interfere with electrophoretic M-protein diagnostics. However, the M-protein can be distinguished from both t-mAbs by use of a double hydrashift assay. The MS-MRD assay provides an alternative method that allows sensitive and simultaneous quantitative monitoring of both the M-protein and t-mAbs.

External quality assessment of M-protein diagnostics: a realistic impression of the accuracy and precision of M-protein quantification

Objectives Studies that investigate the accuracy and precision of M-protein quantification are scarce. These studies are prone to give a biased view, since they are exclusively performed by institutions with international top-expertise on M-protein diagnostics. To obtain a realistic impression of the accuracy and precision of M-protein quantification, we studied results of 73 laboratories participating in the Dutch External Quality Assessment (EQA) program for M-protein diagnostics. Methods To measure accuracy, healthy serum was spiked with respectively 1 and 5 g/L human IgG-kappa monoclonal antibody daratumumab. To measure precision, five sera were selected to be repeatedly send to all blinded EQA-participants. Results The reported concentrations for the EQA-sample spiked with 5 g/L daratumumab ranged from 2.6 to 8.0 g/L (mean 4.9 g/L, between-laboratory CV = 23%). 98% of the participants detected and correctly characterized the 1 g/L daratumumab band. Both the accuracy (mean 1.7 g/L) and precision (between-laboratory CV = 46%) of this 1 g/L M-protein was poor. In the five EQA-samples that were repeatedly send to the same 73 participating laboratories, between-laboratory precision (mean CV = 25%) was significantly different than the within-laboratory precision (mean CV = 12%). Relatively poor precision was observed in sera with small M-proteins. Conclusions The EQA-data reveal a large variation in reported M-protein concentrations between different laboratories. In contrast, a satisfactory within-laboratory precision was observed when the same sample was repeatedly analyzed. The M-protein concentration is correlated with both accuracy and precision. These data indicate that M-protein quantification to monitor patients is appropriate, when subsequent testing is performed within the same laboratory.

Analytical validation of the Hevylite assays for M-protein quantification

Background: The heavy/light chain (HLC) immunoassay quantifies the different heavy chain/light chain combinations of each immunoglobulin (Ig) class. This makes the HLC assay suited to quantify monoclonal immunoglobulins (M-protein) and for monitoring of patients with monoclonal gammopathies. This method is particularly advantageous for those samples in which electrophoretic quantification of the M-protein is not possible. Methods: In this study we tested the analytical performance of the HLC assay in 166 routine clinical samples and in 27 samples derived from the Dutch external quality assessment (EQA) for M-protein diagnostics (74 participating laboratories). Analytical accuracy was assessed by verification that the sum of the HLC-pairs equaled total Ig concentration. Sensitivity of the HLC assay was determined in a direct method comparison with immunofixation electrophoresis (IFE). Results: Comparison of HLC data with routine Ig diagnostics in 27 EQA samples showed very good correlation for both the quantification of polyclonal and monoclonal IgG, IgA and IgM (Pearson correlations [r] were 0.94, 0.99 and 0.99, respectively; slopes were 0.94, 1.07 and 0.98, respectively). The overall concordance between IFE and the HLC ratio was high (93%) with a Cohen κ coefficient of 0.84. Discrepancies between both assays were mainly caused by the higher sensitivity of IFE to detect monoclonality. Conclusions: We conclude that the HLC assay is an accurate method to quantify M-proteins that can improve monitoring of M-proteins in the beta fraction that cannot be quantified using electrophoretic techniques.

A myopic perspective on the future of protein diagnostics

Plasma proteome analyses of the future promise invaluable insights into states of health, not only by measuring proteins whose role it is to ensure blood homeostasis, but increasingly also as a window into the health of practically any tissue in the body via so-called leakage protein biomarkers. Realizing more of this vast potential will require progress along many lines. Here we discuss the main ones, such as optimal selection of target proteins, affinity reagents, immunoassay formats, samples, and applications, with a view from ongoing work in our laboratory.