Comparison of MALDI-TOF Mass Spectrometry Analysis of Peripheral Blood and Bone Marrow Based Flow Cytometry for Tracking Measurable Residual Disease (MRD) in Patients with Multiple Myeloma
Authors: Eveillard Marion , Rustad Even H , Roshal Mikhail , Zhang Yanming , Ciardiello Amanda Kathryn , Korde Neha , Hultcrantz Malin , Hassoun Hani , Smith Eric L , Lesokhin Alexander M. , Mailankody Sham , Landgren Ola , Thoren Katie 
Published: November 2019

Introduction
In multiple myeloma (MM), the absence of measurable residual disease (MRD) after completed therapy is associated with longer progression free survival. Different techniques are available to detect low levels of plasma cells in bone marrow (BM) either by flow cytometry or by next-generation sequencing as a gold standard of molecular methods. But these techniques are limited because they require a representative bone marrow sample obtained by an invasive procedure. Therefore, detecting low levels of disease in blood would be ideal, because serial sampling is much easier and fully representative, and it would allow for the detection of extramedullary disease. Mass spectrometry-based methods have been shown to be more sensitive for detecting monoclonal proteins (M-protein) in serum. In this study, we were motivated to evaluate MALDI-TOF mass spectrometry (MALDI-TOF MS) head-to-head with an established BM-based MRD assays.
Patients and Methods
This cohort included 71 patients treated at Memorial Sloan Kettering Cancer Center (MSKCC) who had serum samples available at 2 timepoints including during active disease and within 60 days of MRD results as determined by flow cytometry of bone marrow aspirates (Flow-BM-MRD). The cohort enrolled 26 females and 45 males with a median age of 61 years (range 37-78 years). Twenty-seven patients had high-risk cytogenetics at baseline. The median time between diagnosis and the MRD timepoint was 13.4 months (3.4-91 months). MALDI-TOF MS analysis was performed according to the method published by Mills et al. Immunoglobulins were purified from serum samples using CaptureSelect beads specific of each isotype and were then eluted from the beads. Light chains and heavy chains were separated by the addition of a reducing agent. Purified samples were mixed in matrix and spotted onto a stainless steel MALDI plate and were analyzed using a Microflex LT MALDI-TOF mass spectrometer (Bruker). Samples taken during active disease were used to identify the mass to charge ratio (m/z) of the M-protein and served as a surrogate marker in the analysis of subsequent samples. MALDI-TOF MS results were compared to the Flow-BM-MRD assay, performed using the MSKCC's ten-color, single-tube method.
Results
MALDI-TOF MS detected an M-protein in all 71 active disease samples and in 25 MRD samples. MALDI-TOF-MS results at the MRD timepoint were concordant with Flow-BM-MRD for 44/71 patients (p=0.342, chi-square test). Eight patients were positive and 36 negative by both techniques. Twenty-seven patients were discordant, including 10 patients detectable only by Flow-BM-MRD and 17 detectable only by MALDI-TOF MS. Among the 10 patients detectable by flow cytometry but not by MALDI, the median MRD level was 0.00092% (+<0.0001% - 0.011%). The M-protein could have been present but below the polyclonal background. Regarding the 17 patients positive only by MALDI-TOF-MS, the BM sample for flow analysis was not suitable for 3 patients due to hemodilution. The others 14 samples reached the target of sensitivity with a limit of detection of 0.0001%. Alternatively, the MALDI-TOF result could be a false positive in terms of disease detection. MS is likely not falsely detecting M-proteins and indeed, immunofixation was also positive in 11/17 of these samples. However, low levels of M-protein may not indicate the presence of active disease. Indeed, a confounding factor is that immunoglobulins have a long half-life in serum.
To determine the clinical utility of more sensitive M-protein detection, we evaluated the clinical outcome for the 48 newly diagnosed MM patients in CR at the MRD timepoint with a median follow-up of 11 months. Of these 48 patients, 2 of the 3 that were positive by both techniques relapsed during follow-up. One out of 27 patients that were negative by both techniques relapsed. None of the 10 patients who were positive only by MALDI-TOF relapsed and 1 of the 8 patients who were positive only by Flow-BM-MRD relapsed.
Conclusions
This study is important because it is a first step in understanding how to use a more sensitive blood test for the follow-up of MM patients. MALDI-TOF MS analysis may provide complementary results to Flow-BM-MRD especially for the follow-up of patients in CR and during maintenance therapy to detect poor responders that would be positive by both techniques. In summary, our results suggest that MALDI-TOF may be quite useful for early detection of relapse.

Disclosures
Roshal: Physicians' Education Resource: Other: Provision of services; Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services. Hassoun:Celgene: Research Funding; Janssen: Research Funding; Novartis: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy. Lesokhin:Takeda: Consultancy, Honoraria; Serametrix Inc.: Patents & Royalties; Genentech: Research Funding; GenMab: Consultancy, Honoraria; BMS: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Juno: Consultancy, Honoraria. Mailankody:Juno: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda Oncology: Research Funding; CME activity by Physician Education Resource: Honoraria. Landgren:Abbvie: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Theradex: Other: IDMC; Adaptive: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Other: IDMC; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.