Traditionally, clinical laboratories have detected, measured, and quantitated monoclonal proteins (M-proteins) using either gel or capillary zone electrophoresis. However, despite being the centerpiece of screening panels and the most commonly performed biochemical tests for diagnosing monoclonal gammopathies, these methods are fraught with limitations. Recently, several mass spectrometry (MS) applications have shown the potential for delivering greater analytical sensitivity and deeper insight into this disease process. The road to translating these findings into routine clinical practice demonstrates the unique challenges in adopting MS for new applications.

Limits of Current Methods

The defining characteristic of monoclonal gammopathies is an expansion of clonal plasma cells that results in overproduction of a monoclonal immunoglobulin (Ig), referred to as M-protein. The M-protein is a serological biomarker of the disease-related plasma cell (PC) clone. The gold standard for detecting M-protein, electrophoresis, suffers from several well-established limitations. These include low analytical sensitivity, the inability to detect and accurately quantitate ß-migrating M-proteins, significant positive bias when quantitating M-proteins <0.2 g/dL, and variability in interpreting results across institutions. In addition to their analytical limitations, electrophoretic methods can be laborious, time-consuming, and difficult to automate. Yet they remain deeply rooted in the International Myeloma Working Group’s recommendations for diagnosing and monitoring patients, as well as in assessing treatment responses (1, 2). Despite this, given the analytical limitations of electrophoretic methods as well as the heterogeneous nature of M-proteins, laboratories are seeking new assays to ensure that patients with monoclonal gammopathies are managed properly.

Benefits of Mass Spectrometry

Why might MS be a practical solution to electrophoresis’s limitations in measuring M-proteins? One advantage is the depth of insight MS provides, enabling more personalized disease monitoring. Each clonal PC is defined by a unique random somatic recombination event that occurs early in differentiation at both the heavy chain (HC) and light chain (LC) gene loci. Consequently, each clonal PC contains the instructions to secrete a single Ig with a unique amino acid sequence that will have a specific molecular mass.

In a healthy adult, the repertoire of Igs in circulation represents the diversity of unique PCs in the bone marrow, each defined by a particular recombination event and thus a singular molecular mass. Since these events are largely random, Ig masses normally are distributed based on the central limit theorem (3). In the instance of a monoclonal gammopathy a particular clonal PC will be overrepresented, which should create an overabundance of Igs molecules with a specific mass. Therefore, a laboratory could use MS to separate Igs by mass (or m/z) to generate a composite of the Ig mass repertoire. The presence of a “spike” in the polyclonal Ig mass spectra at a specific mass would indicate the presence of an M-protein.

While clinical laboratories have begun to use MS-based platforms for other protein biomarkers (4, 5), MS analysis of M-proteins has lagged behind. However, progress over the last 10 years has brought forth compelling reasons to consider the application of MS for measuring M-proteins. For one, mass analyzers have better capability to measure complex mixtures of large proteins, such as Igs. Demand for technologies that can detect lower amounts of residual disease also has increased.

Two Approaches to Using Mass Spectrometry

Recently, two different applications of MS for measuring M-proteins have been reported (5, 7). In the first, MS serves as a potential replacement of current electrophoretic methods (gel or capillary zone electrophoresis and/or immunofixation/immunosubraction) such that a single MS-based assay would substitute for M-protein identification, isotyping, and quantitation. In this case, the researchers sought to generate a robust, high throughput method that could overcome the analytical limitations of electrophoretic methods—as well as generate a cost-effective and automatable technology as compared to the current battery of tests used to screen, diagnose, and monitor patients. They found that matrix assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS improved sensitivity and the ability to detect and quantitate ß-migrating­ M-proteins while also reducing the number of tests necessary for patient management and bettering analytical times. However, MALDI-TOF MS still requires subjective interpretation of complex mass spectra, and therefore many of the challenges in interpreting electrophoretic patterns remain.

In the second application, investigators used MS to detect residual disease in patients with a history of multiple myeloma who had received treatment and were subsequently found by electrophoretic methods to have undetectable disease. The priority was maximizing sensitivity as well as providing accurate molecular mass measurements of M-proteins. In patients receiving monoclonal antibody drugs, this information could help distinguish between residual disease and exogenous drugs. With high-resolution analyzers, this approach increases the sensitivity of detecting serum and urine M-proteins by >100-fold compared to gel-based immunofixation, with accurate molecular mass measurements within +/- 1 Da for Ig LCs. However, these initial methods have long analytical times of about 20 minutes per sample and the instruments necessary to achieve these performance characteristics cost in excess of $700,000.

In order to optimize analytical sensitivity and simplify downstream analysis, both applications use the light chain component of the M-protein as a surrogate of the intact M-protein. Larger intact proteins are more challenging to analyze due to reduced ionization efficiencies and the difficulty of achieving mass accuracy for large molecules. An alternative approach involves measuring clonotypic peptides from the ­complementarity-determining region of Igs as surrogate markers­ of the M-protein. However, this requires a priori knowledge about the M-protein amino acid sequence and adds considerable time and variability to the analysis.

Next Steps for Wider Adoption

While preliminary work has highlighted the potential benefits of MS-based approaches to analyze M-proteins, the journey to implementing this technology into clinical practice is just beginning. Researchers’ success in showing that MALDI-TOF MS could potentially replace a decades old electrophoretic technique for detection of M-proteins echoes the role MALDI-TOF MS played in revolutionizing routine bacterial and fungal identification, the adoption of which has been ongoing since the 1990s (8, 9). The high cost, need for bioinformatics, and lack of reagents initially delayed MALDI-TOF adoption. Now with lower cost and more powerful instruments, proven cost savings, and literature reporting superior performance, MALDI-TOF is poised to become a standard practice in microbiology in the near future.

It is hard to overstate the challenge of converting a disruptive technology into standard practice. In one regard use of MALDI-TOF MS for measuring M-proteins has a head start: Many clinical laboratories now have expertise in and understanding of MS with access to MALDI-TOF MS instruments. 

However, before laboratories start replacing electrophoresis with MS, follow-up studies need to replicate the initial reports, and reagents, software, and instrumentation for automation need to be developed further. Clinical laboratories also will need significant training and expertise in MS-based testing before they will be comfortable interpreting the raw data these methods generate. Many smaller hospital laboratories are not prepared for such a transition. Academic laboratories may be interested in MS-based assays to measure minimal residual disease, personalize disease monitoring, or gain insight into the biology of monoclonal gammopathies. Similarly, large reference laboratories with resources such as bioinformatics support, technical MS expertise, and access to instrumentation may be enticed by the ability to automate and streamline workflows. 

At the same time, even with MS’s clear cost savings and undeniable analytical superiority, clinicians and laboratorians need practice guidelines on how to incorporate MS-based measurements of M-proteins into diagnostic criteria, monitoring recommendations, and response criteria. Reimbursement also is uncertain, so the financial incentives to convert to MS-based testing remains hazy. In the case of MALDI-TOF MS-based bacterial identification, companies such as Bruker and bio­Mérieux were instrumental in driving the application of MALDI-TOF MS to the clinical market. Similarly, for MS-based M-protein measurement to advance quickly into clinical practice, commercial producers of reagents and instrument manufacturers will have to see this as a worthwhile endeavor. 

John R. Mills, PhD, is a fellow in the department of laboratory medicine and pathology at Mayo Clinic in Rochester, Minnesota.+Email: mills.john2@mayo.edu 

References

1. Rajkumar SV, Dimopoulos MA, Palumbo A, et al. International myeloma working group updated criteria for the diagnosis of multiple myeloma. The Lancet Oncology 2014;15:e538–48.

2. Kumar S, Paiva B, Anderson KC, et al. International myeloma working group consensus criteria for response and minimal residual disease assessment in multiple myeloma. The Lancet Oncology 2016;17:e328–46.

3. Wada Y. Mass spectrometry of transferrin and apolipoprotein c-iii for diagnosis and screening of congenital disorder of glycosylation. Glycoconj J 2016;33:297–307.

4. Das R, Mitra G, Mathew B, et al. Mass spectrometry-based diagnosis of hemoglobinopathies: A potential tool for the screening of genetic disorder. Biochem Genet 2016;54:816–25.

5. Barnidge DR, Dasari S, Botz CM, et al. Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy. J Proteome Res 2014;13:1419–27.

6. Mills JR, Kohlhagen MC, Dasari S, et al. Comprehensive assessment of m-proteins using nanobody enrichment coupled to maldi-tof mass spectrometry. Clin Chem 2016;62:1334–44.

7. Kohlhagen MC, Barnidge DR, Mills JR, et al. Screening method for m-proteins in serum using nanobody enrichment coupled to maldi-tof mass spectrometry. Clin Chem 2016;62:1345–52.

8. Claydon MA, Davey SN, Edwards-Jones V, et al. The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 1996;14:1584–6.

9. Holland RD, Wilkes JG, Rafii F, et al. Rapid identification of intact whole bacteria based on spectral patterns using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 1996;10:1227–32.


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