Mass-Fix for M-protein measurement in multiple myeloma

Traditionally, immunofixation electrophoresis (IFE) has been used to measure immunoglobulins (Igs) from patients with multiple myeloma (MM), using the differential velocity of the Igs through a gel to split them into distinct bands. However, mass spectrometry is rising as a novel technique with great promise and potentially greater sensitivity for the analysis of Ig clones.

During the 2nd European Myeloma Network Meeting, David Murray discussed this technique and explained why mass measurement is more valuable than velocity for monoclonal (M)-protein detection and analysis.¹

Background to using mass spectrometry in MM

The basic paradigm for mass spectrometry in MM is that plasma cell clones generate specific Igs with unique heavy and light chains. As the heavy chain may be subject to glycosylation, it often gives multiple peaks on mass spectrometry compared with the light chain, which gives a single peak. Interpretation of the light chain read-out is simpler, and therefore, this is used as a marker for the specific clone. The intensity of the reading indicates the amount of clone present.

High-resolution machines show two peaks for the light chain, which represent the kappa and lambda light chains. In patients with MM, a large spike indicates that the M-protein is present. Post-translational modifications may also be identified using this technique as smaller peaks at different masses may be present.

Method

A novel method, which is called Mass-Fix as it combines the elements of the traditional immune fixation assays with the measurement using mass spectrometry, has been used by David Murray and colleagues to measure M-proteins.

In this Mass-Fix assay, samples from serum are immunopurified into the five Ig classes: G, A, M, K, and L. The heavy and light chains are dissociated before adding the samples to a matrix-assisted laser desorption ionization (MALDI) plate. A rapidly analyzing mass spectrometer assesses the samples, and custom software is used to identify and quantify the M-protein present.

Advantages of Mass-Fix over IFE

Increased analytical sensitivity was found using Mass-Fix compared with IFE when samples were diluted. Serial dilution comparisons showed that at 1:200 sample dilutions, Mass-Fix was able to identify ~60% of M-proteins compared with ~30% for IFE.

Having baseline samples from patients allows the identification of M-protein changes and aids interpretation of results with multiple clones.

Using monoclonal antibodies as treatment has caused interpretation interference historically using IFE. Standard gates can be set up where the normal mass of rituximab, isatuximab, and daratumumab should be, which helps minimize interference. Unfortunately, this does not completely solve the issue, as certain M-proteins may have a mass close to these monoclonal antibody agents.

Other advantages of Mass-Fix over IFE include the following:

• Results are obtained more rapidly with a less intensive workflow.

• Patient histories can be integrated with results, as an electronic format is used.
• The patient sample and identification are kept together throughout the process, which reduces sample mix-up.

Patient outcomes

Mass-Fix can be more sensitive to the presence of disease compared with next-generation flow (NGF). Patients who are negative on both Mass-Fix and NGF show improved progression-free survival compared with those who are NGF negative but Mass-Fix positive.

While heavy chain glycosylation is frequently detected in healthy individuals, light chain glycosylation is not observed. However, light chain glycosylation was detected unexpectedly with Mass-Fix in some patients with amyloid light-chain (AL) amyloidosis. Using data collected over 25 years, an association was identified between patients with light chain glycosylation and AL amyloidosis (p < 0.001). A positive association was found between light chain glycosylation even when this was expanded to progression to any plasma cell disease, with these patients progressing faster than those without the post-translational modification (p < 0.001).

International Myeloma Working Group recommendations²

The International Myeloma Working Group (IMWG) recommendations agree with this presentation that liquid chromatography MALDI-time of flight mass spectrometry can be used in lieu of IFE in both clinical assessment of patients and in clinical trials and that mass spectrometry can be used to distinguish therapeutic antibodies from M-proteins.

Additional points:

• As mass spectrometry methods may lead to lower rates of complete response compared with IFE, the IMWG does not recommend comparing complete response rates between trials that use these different methods.
• Further data collection to assess if mass spectrometry can be used to measure measurable residual disease negativity status and as a guide for the timing of bone marrow sampling for next-generation sequencing and NGF is required.
• The IMWG also acknowledge that the link between N-linked glycosylation and progress from monoclonal gammopathy of unknown significance to MM and AL amyloidosis is an interesting one that requires further investigation.

Conclusion

Mass-Fix is a sensitive assay that not only reduces lab workflow but also demonstrates increased sensitivity of M-protein detection compared with IFE. Mass-Fix has the potential to identify patients who may be at a greater risk of relapse. Even if they have a negative NGF, patients who are positive for Mass-Fix do fare worse than double-negative patients. The association identified between plasma cell disease progression and light chain glycosylation is very promising and requires further investigation to validate the use of this post-translational modification as a marker of risk of disease progression.