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Minimal Residual Disease in Multiple Myeloma: Targeted Mass Spectrometry in Blood Vs Next Generation Sequencing in Bone Marrow

Background
 Multiple myeloma (MM) is a plasma cell malignancy arising in the bone marrow that is characterized by secretion of a monoclonal immunoglobulin (M-protein). M-protein detection is an integral part of MM diagnostics, performed using electrophoretic methods. This approach is reliable, fast and inexpensive, but not sensitive enough for detecting deeper responses, characterized by low M-protein concentration.
 Novel therapies have led to an increasing number of patients obtaining complete remission, however no curative therapy is available for MM. Even in remission, minimal residual disease (MRD) is present and can develop towards a relapse. MRD analysis plays an important role as primary endpoint in clinical trials, powerful prognostic marker, and MRD-status is increasingly used to guide therapy-decisions.
 Methods currently used for MRD detection are based on bone marrow aspirates (e.g. flow cytometry or next generation sequencing (NGS)). Not only are repetitive bone marrow aspirations uncomfortable for the patient, they also introduce a risk of non-representative sampling due to the possible patchy occurrence of the cancer cells in MM.
 Aim
 The aim is to measure MRD in a cohort of 41 multiple myeloma patients during follow-up using a targeted mass spectrometry assay. Unique peptides from the M-protein are selected based on RNA sequence information. Patient-specific peptides from M-proteins will be measured using mass spectrometry and MRD status will be compared to available NGS data.
 Methods
 Multiple myeloma patients were selected from the IFM 2009 Study (ClinicalTrials.gov number: NCT01191060) with available RNA sequence information and MRD data by NGS. We have selected 41 patients with three serum samples per patient; a baseline serum sample and two follow-up time points for MRD assessment.
 For each patient we have selected at least two peptides unique for the M-protein that could be observed in the baseline sample using mass spectrometry; one peptide from the light chain, and one from the heavy chain.(Zajec et al, J Proteome Res, 2018) In the case of light chain MM (3 patients in this study), only light chain peptides were selected.
 Serum samples were diluted 500 times, digested with trypsin and directly measured using targeted Orbitrap mass spectrometry technology.
 Results
 For all 41 patients we were successful in selecting and targeting unique peptides for M-protein quantification using mass spectrometry. MRD was assessed in follow-up serum samples (n=82, two time points for each patient) using NGS and mass spectrometry. In these follow-up patients, seven patients were negative by both NGS and mass spectrometry. Additionally, eight patients were negative by NGS but positive by mass spectrometry; and ten patients were negative by mass spectrometry and positive by NGS. The remainder of the follow-up samples (n=57) were positive by both mass spectrometry and NGS.
 Conclusion
 Unique peptides of the M-protein were found in all 41 multiple myeloma patients. Using these peptides, it was feasible to measure the M-protein in blood samples from multiple myeloma patients during follow-up. NGS and mass spectrometry MRD assessment have a concordance of 78% (64/82) in the follow-up samples. These are preliminary results without any immunoglobulin enrichment. We expect that further optimization can improve the sensitivity of mass spectrometry measurements.
 Future perspective
 Described experiments were performed on non-enriched serum without optimization of mass spectrometry parameters. Future experiments should be adapted accordingly and will increase the sensitivity of the mass spectrometry measurements. Internal standards for the peptides will be synthesized for absolute quantification (g/L value) of the M-protein during MRD.
 
 
 No relevant conflicts of interest to declare.
