P10 an ultra-sensitive method for sequencing and monitoring of m-protein in peripheral blood (m-insight)

With the improvement of therapy (monoclonal antibody, CAR-T), detection of minimal residual disease (MRD) and early restart of therapy is of high importance to manage multiple myeloma disease (MM). Most sensitive MRD assays to date are based on quantification of clonal plasmocytes by next generation sequencing or flow cytometry in bone marrow aspirates. However, such methods have shown limitations such as being invasive with sample heterogeneity and lacking the possibility for frequent sampling. Frequent MM monitoring on blood at equivalent sensitivity than achieved with bone marrow could provide actionable information on disease activity and detect early signs of progression. M-protein is a well-established biomarker used for MM diagnostic and monitoring. Mass spectrometry (MS) has been introduced as a possibility to monitor M-protein. Intact protein measurement by MS has the drawback of lacking in sensitivity with high interference from the polyclonal background. Clonotypic peptides originating from the variable region of the M-protein are unique for each patient. Their detection by MS, which circumvent interferences from other immunoglobulins, has been demonstrated to quantify M-protein at MRD level. Several studies from our group have been published showing the use of targeted mass spectrometry-based MRD blood-test (M-InSight) that detects clonotypic peptides. In this study, M-InSight is used to sequence and select clonotypic peptides to allow highly specific and ultra-sensitive monitoring of the M-protein. Therapy response of 41 Multiple myeloma patients from the IFM-2009 clinical trial (NCT01191060) was used to evaluate the assay. M-InSight uses a novel de novo approach using Peaks Ab software to sequence the M-protein with mass spectrometry from serum that are further assembled into full length HC (Heavy Chain) and LC (Light chain) sequences. All 41 patients were sequenced by mass spectrometry, which was then compared to RNA sequencing data based on tala cDNA from all expressed genes. RNA assembly pipeline using Trust4 was used to construct clonotypes and to identify clonal molecular fingerprints and finally their clonotypic peptides based on transcriptomic datasets. Results showed a coverage of more than 90% of the entire LC and HC sequenced by mass spectrometry compared to the data obtain from RNA sequencing. Once the M-protein sequence was obtained, several clonotypic peptides were further chosen with the use of an in-house bioinformatics algorithm to select the best candidate. Each peptide is chosen to be specific to the patients (CDR region) from both chains. Clonotypic peptides are used to quantitate M-protein in patients’ serum after treatment. M-protein concentrations were determined by calibration on a sample with a known M-protein concentration quantified by an agarose gel electrophoresis system (Hydrasys 2, Sebia). Results showed a very high sensitivity with M-protein still detectable by M-InSight despite a MRD negativity determined by next generation sequencing data on bone marrow aspirate. The best sensitivity achieved by M-InSight, detecting 0.2 mg/L of M-protein, was 1000- and 100-fold more sensitive compared to SPE and intact protein MS method, respectively. In conclusion, the newly developed and validated M-InSight assay is presented as an ultra-sensitive fully blood based assay to sequence and monitor M-protein with the possibility for frequent non-invasive analysis.