B04 implementing m-protein diagnostics in multiple myeloma patients using ultra-sensitive targeted mass spectrometry data and an off-the-shelf calibrator

Introduction: Minimal residual disease (MRD) detection in Multiple Myeloma (MM) patients currently relies on bone marrow samples. These tests provide important information, but are invasive and not suitable for frequent monitoring. However, current blood-based diagnostics, lack the sensitivity to detect MRD. Our group has published a targeted mass spectrometry-based MRD blood-test (MS-MRD) that detects clonotypic peptides originating from the variable region of the M-protein. This assay provides a patient-specific test solution that is more sensitive compared to currently used M-protein detection methods. For quantification of the M-protein Stable Isotope Labeled (SIL) peptides are synthesized. SIL peptides are heavy labeled, synthetic copies of the clonotypic peptide and currently the gold standard for MS-MRD quantification as these standards offer the best possible reference for clonotypic peptides. However, SIL peptides are patient specific and require the synthesis of new peptides for each new patient. To this end, an alternative, generic quantification method was explored using a heavy labeled monoclonal antibody (SILuMAB). Methods: In the new MS-MRD assay, both SILuMAB and clonotypic peptides were targeted. M-protein concentrations were determined by calibration on a sample with a known M-protein concentration quantified by SPE. The resulting M-protein concentration for each clonotypic peptide within a sample was averaged. Validation of MS-MRD using the off-the-shelf SILuMAB calibrator was performed using serum samples from patients participating in the IFM2009 trial. Results: Sensitivity and dynamic range of the SILuMAB-based MS-MRD assay was determined in two separate dilution series of two patient sera, serially diluted in control serum. We observed a linear signal until respectively 0.003 g/L (R2=0.994) and 0.001 g/L (R2= 0.995), indicating that the lower limit of quantification for SILuMAB-based MS-MRD is approximately between 0.003 g/L and 0.001 g/L. Overall, we observed a 1000-fold improved sensitivity for MS-MRD compared to SPE. Generating reproducible results is indispensable for quantitative clinical tests used for disease monitoring. To this end we have assessed the intra-assay variation, the inter-assay variation and the variation between laboratories. For both intra- and inter-assay variation, all observed CVs were below 20%, indicating a stable instrumental setup and sample preparation protocol. The variation between laboratories was tested by preparing and acquiring 16 longitudinal patient series (320 samples in total) by two different laboratories using different LC-MS/MS platforms. All M-protein concentrations were quantified using SILuMAB and results show an R² of 0.9087 and a slope of 1.03. Combined data from the reproducibility experiments indicate great robustness of the SILuMAB-based MS-MRD assay. Concordance between SILuMAB quantified data and SIL quantified data was assessed by preparing all available longitudinal samples from 3 patients with both SILuMAB and SIL. This experiment was performed by both the Radboudumc and Erasmus MC. Both sites reported R²s of >0.99 were, indicating great concordance between both SILuMAB and SIL quantified MS-MRD. Conclusion: MS-MRD was simplified by using an off-the-shelf calibrator. This reduces time and money spent on synthesis of personalized SIL and will allow robust M-protein quantification and leads to improved harmonization of MS-MRD analysis in the clinic.