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Dutch Researchers Develop Mass Spec Test for Multiple Myeloma

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NEW YORK (GenomeWeb) – Researchers from Erasmus MC University Medical Center and Radboud University Medical Center in The Netherlands have developed a mass spec-based test for measuring M protein levels in multiple myeloma patients.

The test, detailed in a study published last week in the Journal of Proteome Research, could prove more sensitive than existing electrophoresis assays while also being less susceptible to interferences from therapeutic antibodies used to treat the disease, said Martijn VanDuijn, a researcher at Erasmus MC and senior author on the paper.

M proteins are established markers for plasma cell disorders including multiple myeloma and are a key analyte for diagnosis and management of that disease. Electrophoresis-based assays are commonly used to measure levels of these proteins, with higher levels indicating a higher disease burden, but such techniques are fairly time consuming and not highly sensitive.

Additionally, VanDuijn noted, increasing use of therapeutic monoclonal antibodies for treating multiple myeloma patients creates interferences that can affect the accuracy of electrophoresis-based tests.

"Therapeutic antibodies are an increasing modality within the clinic, and if you treat a patient with monoclonal antibodies you introduce yet another monoclonal protein," he said. "You have the M protein produced by the myeloma cells, and now you have a second antibody that you just infused as a therapeutic, or sometimes even multiple monoclonal antibodies you have given as a therapy, and this confuses a lot the existing diagnostics."

Specifically, the authors noted, therapeutic antibodies can register as M protein, making expression levels of the protein appear higher than they actually are and causing clinicians to underestimate patient response rates.

To address this problem, VanDuijn and his colleagues turned to mass spec, devising a bottom-up workflow in which they measure peptides specific to a patient's M protein, which, he said, allows for highly specific and sensitive measurement of the protein.

"We have access to the amino acid sequence of the M protein, so we know exactly what we are looking for," he said. The assay also uses stable isotope-labeled peptide standards, which enables absolute quantitation and improves quantitation at lower protein levels.

"If your patients have been treated and the disease load has been reduced, then your signal will get lower and lower, and you will start to see background noise in your mass spectra," VanDuijn said. "Having the stable isotope labeled reference peptides provides a big landmark giving confidence that [M protein] is what you are actually looking at."

He added that the assay can also be expanded to quantify the levels of therapeutic antibodies used to treat patients, allowing for "monitoring of the therapeutic levels of these antibodies as a side benefit."

The researchers also plan to explore whether the assay might be useful for measuring minimal residual disease in patients whose M protein levels have dropped below the amount measurable with electrophoresis-based assays. Currently, this is done using flow cytometry or PCR analysis of bone marrow biopsies. However, noted Marina Zajec, an Erasmus graduate student and first author on the paper, such biopsies are highly invasive, which makes frequent monitoring difficult. Additionally, VanDuijn said, these biopsies don't necessarily capture disease heterogeneity, which might be better observed in a sample like serum.

The researchers developed the test using parallel-reaction monitoring (PRM) on a Thermo Fisher Scientific Orbitrap Fusion Lumos. While PRM is not as widespread a targeted proteomic approach as triple-quad selected-reaction monitoring mass spec, a common clinical technique, adoption of the method is growing.

Like SRM, PRM approaches use an upfront quadrupole to isolate a target precursor ion, but because PRM uses high-resolution mass specs like the Fusion Lumos, such assays are able to then monitor not just a few but all of the resulting product ions. The larger number of product ions monitored via PRM can improve the specificity of the analysis, since more transitions will be available to confirm a peptide ID. It can also reduce the effects of co-isolating background peptides.

That said, high-resolution instruments are still not commonly used in the clinic, meaning that to achieve widespread adoption, the researchers will likely have to convert the assay to a more conventional SRM workflow using a triple quad instrument.

VanDuijn said that in experiments measuring M protein on a triple quad, the researchers acquired data that "was almost equivalent to the Lumos."

"The PRM method has some advantages in selectivity, but the triple quad machine has not disappointed us," he said. "So, there is no reason to believe that this could not be implemented on a triple quad machine in the future."

The Erasmus team's assay follows the development by Mayo Clinic researchers of a MALDI mass spec assay for monitoring M proteins published in 2016.

Like the Erasmus test, the Mayo assay is intended to offer better sensitivity than electrophoresis tests while also avoiding the problem of interferences from therapeutic antibodies. The Mayo researchers also focused heavily on maximizing the throughput of their assay, which led them to adopt MALDI measurements of intact M proteins, as opposed to a conventional bottom-up workflow like that used by the Erasmus researchers, which involves protein digestion and upfront sample separation by liquid chromatography.

VanDuijn said that avoiding protein digestion is a potential advantage of the Mayo Clinic's MALDI-based assay, but he added that the Erasmus approach using digestion and stable isotope-labeled standards makes for a more sensitive assay.

Indeed, Mayo researcher David Murray and his colleagues have also developed a more sensitive high-resolution mass spec assay for M protein to measure minimal residual disease. Like the group's MALDI assay, the test looks at intact M protein, but it has sensitivity comparable to the Erasmus team's peptide-based approach.

VanDuijn said he and his colleagues are now working to acquire patient samples for testing the assay in larger cohorts.

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