P37 multiple myeloma monitoring from archived serum protein electrophoresis gels by mass spectrometry and de novo sequencing

In multiple myeloma (MM), the plasma cells overproduce monoclonal immunoglobulins, called M-proteins. Serum protein electrophoresis (SPEP) is a blood-based analysis commonly used to detect and measure the M-protein, but it has a relative limited sensitivity of 0.5 g/L. More sensitive techniques exist to monitor minimal residual disease (MRD) in MM, such as flow cytometry or next-generation sequencing, but they require samples from invasive bone marrow procedures. Mass spectrometry (MS) is arising as a sensitive approach for longitudinal M-protein monitoring that relies on blood samples rather than bone marrow procedures. By using RNA data obtained from the initial bone marrow sample, clonotypic peptides of the M-protein can be used as a surrogate to monitor MM. In this study, de novo sequencing of the M-protein was performed using routine SPEP gels as starting material for MS analysis. With this approach an SPEP band is sufficient to detect M-protein without use of bone marrow. Sensitivity of MS to longitudinally monitor M-proteins was compared with routine diagnostics. Nine MM patients were retrospectively selected based on the data in the Erasmus MC hospital information system, without available RNA data. Each patient had SPEP detectable M-protein at diagnosis (>1 g/L) and at the end of the disease course, with at least one period in between when the M-protein was absent by routine diagnostics. To validate the de novo sequencing approach and quantitative performance of our method, one sample of a MM patient with available RNA data was used as a reference to prepare a dilution series. The M-protein bands were digested with trypsin, extracted from gel, and measured with MS. Patient-specific M-protein peptides were selected based on unique presence in the patient and homology of the de novo sequence to the immunoglobulin germline sequences. The selected peptides were used as surrogates to monitor the M-protein in all patient’s samples with MS. With de novo sequencing, at least one patient-specific peptide of the M-protein was identified for all of the MM patients, showing 100% feasibility. For the reference patient, 2 patient-specific M-protein peptides from the heavy chain were identified. When compared to the translated DNA sequence, one patient-specific peptide differed in only one amino acid while the other had all the correct amino acids but some were misplaced in the sequence. Nevertheless, neither of the inaccuracies interfere with monitoring the associated signals in the MS. The dilution series of the reference patient showed that MS can detect M-protein concentrations as low as 5 mg/L, while with SPEP no visible M-protein band was detected when the M-protein concentration was less than 664 mg/L. One patient is displayed in the Figure as an example where the M-protein was detected in all time points (blue), compared to routine diagnostics (orange) that did not detect the M-protein between 265-485 days and 938-1259 days. Additionally, relapse was detected sooner by MS than by SPEP. MS-analysis on M-protein from archived SPEP gels is feasible by using de novo sequencing. This approach enables retrospective analysis of patients in the clinic or from completed clinical trials. Additionally, MS-analysis on patient blood can be used to measure MRD instead of or complimentary to bone marrow MRD detection, lowering the burden of sequential MRD measurements using invasive bone marrow-based techniques.