Bone Marrow-Free Sequencing of M Protein Genes in Monoclonal Gammopathies

Topic: 13. Myeloma and other monoclonal gammopathies - Biology & Translational Research Background: In patients with monoclonal gammopathies, M proteins are patient-unique, can cause potentially fatal organ damage and can be used to track the B cell/plasma cell tumor after therapy. The presence of circulating tumor cells and the possibility to analyze the antibody repertoire through next generation sequencing (NGS) and proteomics approaches may provide a window of opportunity to identify patients’ specific M protein genes in the peripheral blood, without invasive bone marrow investigations. Aims: To establish and validate an experimental workflow to for bone marrow-free sequencing of M protein genes in monoclonal gammopathies. Methods: Mononuclear cells from peripheral blood were subjected to single-molecule real-time sequencing of the M protein (SMaRT M-Seq) to obtain the circulating repertoire of the affected light chain isotype in a cohort of patients with monoclonal gammopathy of undetermined significance (MGUS, n=3), multiple myeloma (MM, n=40) and AL amyloidosis (n=37). Tryptic digestion peptides from urinary proteins were subjected to mass spectrometry analysis to identify or confirm the clonal light chain sequence from the obtained circulating repertoire (Figure 1A). SMaRT M-Seq on matched bone marrow samples to identify the bona fide clonal light chain sequence was performed for confirmatory purposes. Results: Reads with 100% identity to the bona fide clonal light chain sequences were identified in the peripheral blood of 77 patients (96%) and was the dominant clonal sequence in 65 cases (81%). In all cases where the most abundant, discrete clonal sequence represented at least 15% of all reads detected within the peripheral blood (n=52, 65%), such sequence invariably coincided with the bona fide clonal light chain sequence as defined by bone marrow sequencing studies (Figure 1B). In a subset of cases (n=36), we also analyzed the urinary proteome and mapped tryptic digestion peptides against each patient’s peripheral blood antibody repertoire. In 34/36 (94%) cases, peptide mapping correctly identified the bona fide clonal light chain sequence as the light chain sequence with the highest relative abundance, sequence coverage, and number of unique peptides among all light chain sequences identified in the patient’s peripheral blood. Overall, when combining NGS sequencing results in peripheral blood with mass spectrometry-based analysis of the urinary proteome to identify or confirm the clonal light chain sequence from the obtained circulating repertoire, the full-length variable sequence of the bona fide clonal light chain was correctly identified in 77 out of 80 patients (96%). Summary/Conclusion: Sequencing of the circulating light chain repertoire through SMaRT M-Seq, eventually coupled with mass spectrometry-based analysis of the urinary proteome, enables the identification of the full-length clonal light chain sequence in the majority of patients with a monoclonal gammopathy. The possibility of reliably determining disease-related immunoglobulin gene sequences even from the peripheral blood from large cohorts of patients has the potential to uncover molecular mechanisms of M protein-related clinical manifestations which have remained largely unexplored so far. It could also facilitate approaches of personalized medicine, including the implementation of sequence-based predictive models for the identification of potentially pathogenic M proteins, and the sensitive detection of patients’ specific M proteins at diagnosis and for minimal residual disease (MRD) after anti-clonal therapy.Keywords: Monoclonal gammopathy, AL amyloidosis, Multiple myeloma, MGUS