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

Introduction

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 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.

Methods

Mononuclear cells from peripheral blood were subjected to single-molecule real-time sequencing of the M protein (SMaRT M-Seq)¹ to obtain the circulating repertoire of the affected light chain isotype in a cohort of patients with MGUS (n=3), MM (n=8) and AL amyloidosis (n=36) at diagnosis. Tryptic digestion peptides from urinary proteins were subjected to mass spectrometry analysis to identify or validate the clonal light chain sequence from the obtained circulating repertoire². 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 44 patients (94%) and were the dominant clonal sequence in 35 cases (75%). In all cases where the most abundant, discrete clonal sequence represented at least 15% of all reads detected within the peripheral blood (n=26, 55%), such sequence invariably coincided with the bona fide clonal light chain sequence as defined by bone marrow sequencing studies (Figure 1A).

In a subset of cases (n=21), we also analyzed the urinary proteome and mapped tryptic digestion peptides against each patient's peripheral blood antibody repertoire. In all 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 (Figure 1B).

The percentage of peripheral blood sequences corresponding to the bona fide clonal light chain sequence significantly, but modestly correlated with bone marrow plasma cell infiltration (R² 0.30).

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 most 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 far². It could also facilitate approaches to personalized medicine, including the implementation of sequence-based predictive models for the identification of potentially pathogenic M proteins^3,4, and the sensitive detection of patients' specific M proteins at diagnosis and MRD after anti-clonal therapy⁵.

References

1. Cascino P, Nevone A, Scopelliti C, Girelli M, Mazzini G, Caminito S, et al. Single-molecule real-time sequencing of the M Protein: toward personalized medicine in monoclonal gammopathies. 63rd Annual Meeting of the American Society of Hematology; 2021; 2021. p. Abstract 2673.

2. Nevone A, Girelli M, Mangiacavalli S, Paiva B, Milani P, Cascino P, et al. An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis. Leukemia 2022; Online ahead of print

3. Garofalo M, Piccoli L, Romeo M, Barzago MM, Ravasio S, Foglierini M, et al. Machine learning analyses of antibody somatic mutations predict immunoglobulin light chain toxicity. Nat Commun. 2021;12(1):3532.

4. Rawat P, Prabakaran R, Kumar S, Gromiha MM. Exploring the sequence features determining amyloidosis in human antibody light chains. Sci Rep. 2021;11(1):13785.

5. Langerhorst P, Brinkman AB, VanDuijn MM, Wessels HJCT, Groenen PJTA, Joosten I, et al. Clonotypic Features of Rearranged Immunoglobulin Genes Yield Personalized Biomarkers for Minimal Residual Disease Monitoring in Multiple Myeloma. Clin Chem. 2021;67:867-875.

Milani:Pfizer: Honoraria, Research Funding; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mangiacavalli:Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings; Sanofi: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Participation in meetings. Basset:Janssen-Cilag: Honoraria. Foli:Janssen-Cilag: Honoraria. Merlini:Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees. Arcaini:Janssen-Cilag: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; EUSA Pharma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Celgene/Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Research Funding. Palladini:Argobio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Prothena: Honoraria; Protego: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; The Binding Site: Honoraria, Research Funding; Pfizer: Honoraria; Gate Bioscience: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sebia: Honoraria; Siemens: Honoraria. Nuvolone:Janssen-Cilag: Honoraria, Other: Travel grant; Oncopeptides, Inc.: Research Funding; Gate Bioscience: Research Funding; Pfizer: Research Funding.