Bone Marrow-Free Sequencing of M Protein Genes: A Liquid Biopsy Approach in Monoclonal Gammopathies
Authors: Nevone Alice , Sadeghi Arian , Mazzini Giulia , Milani Paolo , Mangiacavalli Silvia , Caminito Serena , Lattarulo Francesca , Russo Monica , Panno Giada , Basset Marco , Cartia Claudio Salvatore , Benvenuti Pietro , Nanci Martina , Bellofiore Claudia , Foli Andrea , Merlini Giampaolo , Arcaini Luca , Palladini Giovanni O. , Nuvolone Mario 
Published: December 2024

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 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.
Methods
Mononuclear cells from peripheral blood were subjected to single-molecule real-time sequencing of the M protein (SMaRT M-Seq) to obtain the expressed circulating repertoire of the affected light and heavy chain isotypes in a cohort of patients with monoclonal gammopathy of undetermined significance (MGUS, n=9), multiple myeloma (MM, n=40) and AL amyloidosis (n=39). 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. SMaRT M-Seq was performed on matched bone marrow samples to identify the bona fide clonal light +/- heavy chain sequences for confirmatory purposes.
Results
Reads with 100% identity to the bona fide clonal light chain sequences were identified in the peripheral blood of 87 patients (99%), representing a dominant clonal sequence in 73 cases (83%). In all cases where the most abundant, discrete clonal sequence represented at least 15% of all reads detected within the peripheral blood (n=57, 65%), such sequence invariably coincided with the bona fide clonal light chain sequence as defined by bone marrow sequencing studies.
In a subset of cases (n=50), we also analyzed the urinary proteome and mapped tryptic digestion peptides against each patient's peripheral blood antibody repertoire. In 43/50 (86%) 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 analysis of the urinary proteome to identify or confirm the clonal light chain sequence from the sequenced circulating repertoire, the full-length variable sequence of the bona fide clonal light chain was correctly identified in 84 out of 88 patients (95%). Noteworthy, digestion peptides identified through mass spectrometry to confirm or identify the M protein sequence include candidate clonotypic peptides that could be employed for highly sensitive and specific clonal tracking and minimal residual disease (MRD) detection.
Of the 53 patients with a conserved expression of a clonal heavy chain and an intact M protein, 31 patients underwent also IGHV sequencing in matched peripheral blood/bone marrow using SMaRT M-Seq. In 28 cases (90%), a bona fide clonal heavy chain sequence was identified in the bone marrow, representing 33% to 94% (median value: 85%) of a given repertoire. In 26 cases, this bona fide heavy chain sequence was identified in the peripheral blood, with a molecular clonal size ranging from 0.05% to &gt;99% (median value: 8,5%), being the first clone in the peripheral blood in 92% of cases.
Conclusion
Sequencing of the circulating expressed immunoglobulin repertoire through SMaRT M-Seq, eventually coupled with mass spectrometry-based analyses, enables the identification of the full-length clonal immunoglobulin sequence in most patients with a monoclonal gammopathy, including patients with low clonal burden and undetectable Bence Jones proteins at routine immunofixation, forming the bases for a liquid biopsy approach in monoclonal gammopathies.
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 MRD after anti-clonal therapy.