Clonotypic Peptide Mass Spectrometry to Define Functional Cure in Patients with Multiple Myeloma Undergoing Maintenance Cessation

Introduction: MRD2STOP is a prospective trial evaluating maintenance therapy cessation guided by measurable residual disease (MRD) negativity by next generation sequencing (NGS, clonoSEQ) in multiple myeloma (MM). We have previously reported that the estimated 3-year MRD-free survival (defined as death, progression, or MRD 10-6 resurgence) among patients with MRD < 10-6 at the time of discontinuation was 68% and was further improved to 78% when using a novel MRD < 10-7 cutoff (Derman et al. ASCO 2024). These results suggest that disease re-emergence can occur in patients achieving the deepest levels of MRD negativity. Clonotypic peptide mass spectrometry (MS), which has a limit of detection 1000x more sensitive than serum immunofixation and involves monoclonal protein (M-protein) de novo sequencing and peptide quantification to accomplish highly sensitive blood-based monitoring of a patient's M-protein over time. In this analysis, we assessed clonotypic peptide MS status among patients in the MRD2STOP cohort. Method: Clonotypic peptide MS was performed on peripheral blood in a subset of patients enrolled into the MRD2STOP trial, who had high-disease burden samples available (M-protein > 2 g/L) for patient. Eligibility for maintenance cessation required patients to be in a stringent complete response, with PET negativity and MRD < 10-6 as assessed by clonoSEQ (Adaptive Biotechnologies). Patient high-disease burden serum samples were digested with several combinations of proteolytic enzymes to permit sequencing of the M-protein heavy and light chain. Clonotypic peptides specific to the patient's complementarity-determining regions (CDR) were selected for quantification by MS using a bioinformatics algorithm and compared to RNA sequencing data. Clonotypic peptide quantification by MS was performed using M-InSight® (Sebia/Corgenix) on available tracking peripheral blood samples, typically at 6-month intervals. Results: To date, 11 patients had high-tumor burden samples enabling identification of clonotypic peptides; in 10 of the 11 cases, at least one of the clonotypic peptides predicted by clonoSEQ matched a clonotypic peptide identified by MS. An M-protein was detectable by clonotypic peptide MS at time zero in all 11 patients despite no detectable disease by serum immunofixation in 11/11 (100%), MRD < 10-6 in 11/11 (100%), and MRD <10-7 in 8/11 (73%). The median M-protein concentration at time zero was 0.0029 g/L (range 0.0000001 g/L - 0.03 g/L). At a median follow-up of 31.6 months from maintenance discontinuation, there were 4 MRD 10-6 resurgence events, including 2 progression events. Of the 4 patients with disease re-emergence, 3 occurred at 12 months and one occurred at 24 months. The 12-month rate of disease re-emergence from maintenance cessation was 33% and increased to 44% at 24 months. The median time zero MS M-protein concentration among those with disease re-emergence was 0.0035 g/L vs 0.00002 g/L for those without re-emergence. In follow-up from maintenance cessation, the MS M-protein concentration increased from baseline in 6 patients; of these 6 cases, MRD 10-6 resurgence at the 12-month mark occurred in 3 patients including 2 patients who had disease progression. There was a logfold increase in M-protein concentration at least 6 months prior to disease progression in both cases. No patient with stable or decreasing M-protein concentration in follow-up experienced disease progression though one had MRD 10-6 resurgence. Conclusion: Despite undetectable disease in the bone marrow at the 10-6 or even 10-7 threshold, trace M-proteins were still detectable in the blood by clonotypic peptide MS indicating the potential presence of MRD. The significance of MS M-protein kinetics over time requires additional evaluation to confirm this observation. In the search to identify cures in MM, clonotypic peptide MS may serve as a key marker for better defining the absence of disease in MM.