MSACL 2023 Abstract

Objective: To demonstrate the roles of MALDI-TOF and ESI-TOF mass spectrometry in monitoring patients with monoclonal gammopathies.

Introduction: Over the past few years, our lab has transitioned the majority of clinical testing for serum M-proteins from gel-based immunofixation to immunoaffinity purification with MALDI-TOF MS analysis (Mass-Fix). Mass-Fix performs comparably to immunofixation, but with several important advantages. Mass spectrometry allows for improved M-protein tracking, detection of clinically relevant post-translational modifications of M-protein light chains, and distinguishes therapeutic monoclonal antibodies (tmAbs) from endogenous M-proteins with increased analytical sensitivity and specificity. MALDI-TOF MS has rapid high throughput testing which has increased the productivity of our lab. Chromatographic separation and higher-resolution ESI-TOF mass spectrometry has been advantageous as the increased resolution affords the ability to track M-proteins in serum to levels comparable to bone marrow based minimal residual disease detection. These advantages result in better care of patients with monoclonal gammopathies.

Methods:
Immunoaffinity purification with MALDI-TOF MS: 10 mcL of serum was added to 50 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After a 15 minute sample incubation, 3 50 mcL PBS washes and 3 50 mcL water washes were performed. Isolated immunoglobulins were eluted with 30 mcL of 20 mM TCEP in 0.1% TFA with a 15 minute incubation for reduction. The isolates were spotted with 10 mg/mL CHCA matrix in 50% ACN with 0.1% TFA and acquired on a MALDI TOF mass spectrometer (microflex smart LS, Bruker) in positive ion mode, with spectra collection from 6000-32000 m/z. Spectra were reviewed using in-house developed software.

Immunoaffinity purification with high resolution ESI-TOF MS: 30 mcL of serum was added to 200 mcL of a 10% v/v slurry of CaptureSelect resin targeting each IgG, IgA, IgM, kappa or lambda. After sample incubation for 15 minutes, resin was washed 3 times with 500 mcL water. Isolated immunoglobulins were eluted with 100 mcL of 5% acetic acid, followed by reduction with 50 mcL DTT in 1M ammonium bicarbonate and incubated at 55°C for 30 minutes. Isolates were analyzed via an Eksigent Ekspert 200 microLC (Dublin, CA) for separation; mobile phase A was water + 1% FA, and mobile phase B was 80% acetonitrile + 10% 2-propanol + 0.1% FA. A 7 μL injection was made onto a 1.0 × 75 mm Poroshell 300SB C3, 5 μm column flowing at 25 μL/min. A 15 min gradient from 25%B to 50% B was used for immunoglobulin elution. Spectra were collected on an Sciex TripleTOF 5600 quadrupole time-of-flight mass spectrometer (Sciex, Vaughan, ON, Canada) in ESI positive mode with a Turbo V dual-ion source with an automated calibrant delivery system (CDS). Source conditions were IS, 5500; temp, 500; CUR, 45; GS1, 35; GS2, 30; and CE, 50 ± 5. TOF MS scans were acquired from m/z 600−2500 with an acquisition time of 200 ms. The instrument was calibrated every ten injections through the CDS using calibration solution supplied by the manufacturer. Data analysis was performed using Analyst TF v1.6 and PeakView version 2.2. The mass spectra of the multiply charged light-chain ions were deconvoluted to accurate molecular mass using Bio Tool Kit version 2.2 plug-in software. Deconvoluted mass spectra were reviewed manually.

Results:
Analytical comparison between mass spectrometry methods: For an IgG kappa monoclonal protein spiked at decreasing concentrations into normal human serum, the limit of detection measured by MALDI-TOF MS was 50 mcg/mL, and by ESI-TOS MS was 3.13 mcg/mL. Current gel-based limits of detection range from 20-200 mcg/mL. In a study spiking tmAbs into samples with endogenous IgG kappa clones, MALDI-TOF MS was able to resolve 87% of endogenous M-proteins from tmAbs, while ESI-TOF MS was able to resolve 100% of clones (Kohlhagen. Clin BioChem 2021 Jun: 92:61-66).

Light chain N-glycosylation: In a study from 2020, among 414 MGUS patients, 25 (6%) displayed N-glycosylated light chains and were found to have a higher likelihood of progression to AL amyloidosis over time (hazard ratio =10.1, 95% CI 2.9,34.7) (Dispenzieri. Leukemia 2020 Oct; 34 (10): 2749-53). N-glycosylated light chains were also observed in a high number of patients with cold agglutinin disease (N=9/14, 64%) compared to other IgM related gammopathies (N=31/438, 7%) (Sidana. Am J Hematol 2020 Sep: 95(9):E222-5). In an additional cross-sectional study of 6315 patients, N-glycosylation was observed at higher rates in patients with LC amyloidosis and cold agglutinin disease than in other disease groups (Mellors. Blood Cancer J 2021 Mar; 11(3):50).

Minimal residual disease: For 251/431 patients enrolled in the STAMINA trial having MRD bone marrow testing by high sensitivity flow cytometry at 1 year post induction, serum Mass-Fix negativity and MRD negativity predicted better progression free survival and overall survival (median follow-up time was 6 years with overall survival of 76%), while serum immunofixation negativity and complete response did not (Dispenzieri. Blood Cancer J 2022 Feb; 12(2): 27).

Conclusion: Mass spectrometry has advanced the way we detect and monitor M-proteins in the clinical laboratory. Observation of post translational modifications such as N-glycosylation of light chains and more sensitive detection of M-proteins already has had a positive impact on the care of patients with monoclonal gammopathies.