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Getting Specific: Mass Spectrometry Provides Detail for Diagnosing and Monitoring Plasma Cell Disorders

December 22, 2022

January 2023

Ruth Jessen Hickman, MD

Ruth Jessen Hickman, MD, is a freelance medical and science writer based in Bloomington, Indiana.

Research scientists and some medical institutions have begun applying mass spectrometry (MS)–based techniques in the diagnosis and monitoring of plasma cell disorders – for example, using them to analyze measurable residual disease (MRD) in multiple myeloma (MM). Although MS-based techniques offer certain advantages, they are still relatively new, and it isn’t yet clear how these assays will best fit into clinical practice and research.

Developed in the first half of the 20th century, MS, also called mass spectroscopy, can sort gaseous ions by their mass-to-charge ratio (m/z).1,2 The molecule under study is converted to gas phase ions so they can be manipulated and sorted as they travel through electric and magnetic fields in a vacuum flight tube. A detection system measures the separated ions in terms of their m/z ratio and relative abundance.

This information is presented as a mass spectrum, a plot that shows the amounts of different sample components in terms of their m/z ratio (see FIGURE). These measurements can be used to calculate the exact molecular mass of the sample being analyzed and identify and quantify compounds.

FIGURE. Example of MASS-FIX Revealing Residual Monoclonal Protein With Same m/z Post-Treatment, Even When CR Is Assessed Via Conventional Measures

CR = complete response; m/z = mass-to-charge ratio; MASS-FIX = matrix-assisted laser desorption ionization time-of-flight mass spectrometry Source: Dispenzieri A, et al. Blood. 2018;132(Suppl 1):4502. doi: 10.1182/blood-2018-99-119969

It took years for scientists to learn how to successfully ionize macromolecules such as proteins, and for many years the technique was limited to physics and the chemical sciences, such as identifying different isotopes as part of the Manhattan Project.1 Eventually, scientists developed more sophisticated and complex versions of MS, which allowed for successful ionization of proteins and opened the door for applications in biology and medicine.

For example, the matrix-assisted laser desorption ionization (MALDI) method was developed in the 1980s. It uses a laser striking a dried matrix of molecules to achieve ionization, typically combined with a “time-of-flight” (TOF) analyzer to help distinguish the components as they travel through the flight tube.2

Another approach to MS employs liquid chromatography (LC), another technique to separate a sample into its component parts, followed by a version of MS called electron spray ionization.2 LC reduces the number of different types of molecules present per scan, and thus this version of MS potentially provides a greater level of sensitivity.3

Ben Derman, MD, a hematologist and assistant professor of medicine at the University of Chicago Medical Center, noted, “[LC-MS] is more laborious and more expensive – it takes longer to do. MALDI-TOF can be done more cheaply, quickly, and it’s also currently being automated so that you may not need a human being interpreting every result.”

With these innovations, scientists have been interested in applying MS techniques clinically, partly inspired by its potential for highly sensitive and specific results with a quick turnaround time. The U.S. Food and Drug Administration has already approved multiple MS-based diagnostic approaches, such as identifying specific microbes and screening newborns for rare genetic diseases.4

Hematologists have been interested in applying this technology as well, particularly with respect to plasma cell disorders. These conditions, including MM, are characterized by the clonal expansion of plasma cells, which overproduce immunoglobulin components, M-proteins; their detection is central to both the diagnosis and monitoring of these diseases. However, M-proteins vary significantly among patients, contributing to the challenge of laboratory analysis in a single standardized test.5

Angela Dispenzieri, MD, a professor of medicine with joint appointments in hematology and clinical chemistry at Mayo Clinic in Rochester, Minnesota, pointed out that the monoclonal M-proteins produced by a patient with a plasma cell disorder have their own “molecular fingerprint” that MS can precisely detect.

ASH Clinical News spoke with Drs. Dispenzieri and Derman about the use of different MS techniques in plasma cell disorders, their potential benefits over other diagnostic and monitoring methods, how this technique is already being applied at some centers, and how it might be used more broadly in the future.

MS Methods in Plasma Cell Diagnosis

To help maximize sensitivity, serum protein electrophoresis, serum immunofixation electrophoresis (IFE), and serum free light chain assays are typically used in conjunction to screen and monitor patients with plasma cell disorders.6 However, the older electrophoretic techniques are cumbersome and labor intensive.

The new monoclonal therapeutic antibodies for MM, such as daratumumab, isatuximab, and elotuzumab, can complicate interpretation of electrophoretic assays.7 Dr. Derman explained, “If the patient’s plasma cell disorder produces the same immunoglobulin isotype as the monoclonal antibody, once the disease gets to low levels, it’s very hard to distinguish that protein from the monoclonal antibody.”

Dr. Derman noted that additional tests are available to help resolve this interference, but MS alone can easily distinguish between the monoclonal M-protein and any therapeutic monoclonal antibodies that the patient is receiving, whether for myeloma or for another clinical application, such as a rheumatologic disease. Dr. Dispenzieri also noted that particularly when a patient’s immune system is reconstituting itself after intensive chemotherapy treatment, one can see findings on IFE that are more difficult to interpret compared to results from MS.

At Mayo Clinic, researchers have developed an MS technique termed MASS-FIX. It couples nanobody technology to first separate immune light chains and heavy chains of immunoglobulins (e.g., IgA, IgM, kappa light chains), followed by MALDI-TOF. Since 2018, they have employed the assay in screening and diagnosis for all patients with potential plasma cell disorders who would have previously received IFE, using it in conjunction with serum free light chains and other tests as appropriate.8

“The turnaround times are much faster than for IFE; it’s very efficient to do in the laboratory once you have it set up,” Dr. Dispenzieri said.

In multiple papers, her group has demonstrated the utility of the assay in thousands of patients with monoclonal gammopathies of clinical significance (MGCS), MM, amyloid light-chain (AL) amyloidosis, and other plasma cell disorders, with equivalent or higher sensitivity and specificity compared to IFE.6,8

The researchers at Mayo discovered another potential benefit of the assay over IFE as well. They found some complex spikes on the mass spectrum of some patients, which they eventually identified as N-glycosylation of light chains. This provided extra information not available via IFE.8

“We don’t know why, but we found that it was more prevalent in patients that have AL amyloidosis or in cold agglutinin disease than in other plasma cell disorders like MGUS [monoclonal gammopathies of unknown significance] or myeloma,” Dr. Dispenzieri explained. Thus, it might raise the suspicion for these diseases in the right clinical context.9,10

She added, “We also found that patients with MGUS who have N-glycosylation have a higher rate of progression to myeloma or amyloid.”11 Because of this higher risk of progression, she said, clinicians might want to keep a closer eye on such patients.

The International Myeloma Mass Spectrometry Committee endorses the use of the MALDI-TOF method of MS as a potential alternative to IFE for detecting M-proteins and distinguishing them from therapeutic monoclonal antibodies in both clinical practice and clinical trials.7

“When you go to meetings, people are pretty excited about [MS], because compared to immunofixation, what it is replacing, it is more specific and more sensitive, and it has higher accuracy,” Dr. Dispenzieri said.

MS Methods in Monitoring and MRD

Disease monitoring is another key area in which scientists have been applying MS techniques, particularly in evaluating MRD in myeloma. The dramatic improvements in the available treatments for MM have made it more important for clinicians to detect very low levels of disease, which electrophoretic methods may miss.

To better detect MRD, which has important prognostic implications, many institutions have relied on bone marrow sampling, detecting residual malignant plasma cells with high-sensitivity flow cytometry and next-generation sequencing (NGS): the approach is considered by some to be the gold-standard of MRD testing.12 However, bone marrow sampling is invasive, and analysis may require a high level of expertise.7 Although it has very high sensitivity, it may be prone to false negatives because of extramedullary disease or inadequate sampling.13

Like bone marrow NGS testing, serum MS methods detecting M-proteins are also more sensitive and specific than IFE. The blood test is less painful compared to bone marrow sequencing with NGS, and it is more suited to serial sampling.

“This is a very promising peripheral blood monitoring tool for MRD,” Dr. Derman said, “one that could be performed frequently.”

Both Dr. Dispenzieri’s and Dr. Derman’s groups have studied MRD sensitivity and specificity compared to the bone marrow NGS approach. Speaking of her work with the STAMINA trial, Dr. Dispenzieri said, “We found that it was additive to the bone marrow test. It performs much better than immunofixation; it’s much more predictive for progression-free survival and overall survival post-transplant.”14

In a phase II study of patients with newly diagnosed MM who were receiving carfilzomib, lenalidomide, and dexamethasone (KRd), autologous hematopoietic cell transplant, and KRd consolidation, Dr. Derman and colleagues found that patients who did not show detectable M-protein via MS had better health outcomes than those who had detectable disease. Moreover, patients who had undetectable disease via bone marrow NGS testing but detectable disease via serum MS did worse than those who had undetectable disease by both methods.13

“What that tells us is that the blood may be actually a very important compartment to analyze,” Dr. Derman said. He sees these techniques being employed with bone marrow NGS testing in a complementary, synergistic way in the future. He added that although we don’t yet know the best way to employ MS in this context, a patient who was found to have detectable disease in the blood might be able to forgo a bone marrow biopsy.

Challenges and Limitations

It will take time, but automated diagnostic MS tests may become more widespread in coming years. Like all tests, these techniques have their limitations and should be ordered and interpreted in the specific clinical context.

Some have noted that the greater sensitivity of MS techniques compared to electrophoretic techniques may be of less benefit in terms of routine screening diagnostics, because M-protein levels are high then, unlike in treated patients.15 In contrast, in screening for MRD, the deepest level of sensitivity possible may be desired.

Both Drs. Derman and Dispenzieri agreed that because the MS technique is so sensitive, some patients may be overdiagnosed, particularly if the test isn’t performed in the right clinical context. In other words, MS may be so sensitive that it detects a small M-protein that may not be clinically significant, one which would have been undetectable by standard electrophoretic techniques.

“You are hopefully ordering that test because you think the patient may have myeloma, amyloid, or an MGCS, etc.,” Dr. Dispenzieri said. “You want to do it because you are actually looking for something.”

Another thing for clinicians to keep in mind when considering MS to assess treatment response: the lab needs blood samples from both pre-treatment and post-treatment to get the best sensitivity and specificity. Otherwise, it may be more challenging for the lab to distinguish background findings from low levels of disease M-proteins.

“If you had sent us a sample from before treatment to give us a fingerprint, then we can compare the second sample to the first,” Dr. Dispenzieri explained.

Dr. Derman also pointed out that when using MS to evaluate MRD, clinicians should consider timing carefully. Because of immunoglobulin recycling, myeloma proteins can persist for weeks to months in the blood, even after the patient has received treatment and no evidence of disease is detectable in the bone marrow.

“You need to pick later time points to use [MS] as MRD – if you do it too early, everyone is going to be positive, and it’s not really that helpful,” Dr. Derman said.

But the promise of MS in plasma cell disorders is evident.

“This is still a very nascent field,” Dr. Derman said. “Further studies are needed to validate this approach, some of which are ongoing. But it’s clear that [MS] is the future of paraprotein monitoring – how we can best use it remains to be seen.”

References

  1. Griffiths J. A brief history of mass spectrometry. Anal Chem. 2008;80(15):​5678-5683.
  2. Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass spectrometry: an emerging technology for microbial identification and diagnosis. Front Microbiol. 2015;6:791.
  3. Heaney LM, Jones DJ, Suzuki T. Mass spectrometry in medicine: a technology for the future?. Future Sci OA. 2017;3(3):FSO213.
  4. Banerjee S. Empowering clinical diagnostics with mass spectrometry. ACS Omega. 2020;5(5):2041-2048.
  5. Mills JR, Barnidge DR, Murray DL. Detecting monoclonal immunoglobulins in human serum using mass spectrometry. Methods. 2015;81:56-65.
  6. Milani P, Murray DL, Barnidge DR, et al. The utility of MASS-FIX to detect and monitor monoclonal proteins in the clinic. Am J Hematol. 2017;92(8):772-779.
  7. Murray DL, Puig N, Kristinsson S, et al. Mass spectrometry for the evaluation of monoclonal proteins in multiple myeloma and related disorders: an International Myeloma Working Group Mass Spectrometry Committee report. Blood Cancer J. 2021;11:24.
  8. Mellors PW, Dasari S, Kohlhagen MC, et al. MASS-FIX for the detection of monoclonal proteins and light chain N-glycosylation in routine clinical practice: a cross-sectional study of 6315 patients. Blood Cancer J. 2021;11:50.
  9. Sidana S, Murray DL, Dasari S, et al. Glycosylation of immunoglobulin light chains is highly prevalent in cold agglutinin disease. Am J Hematol. 2020;95(9):E222-E225.
  10. Kumar S, Murray D, Dasari S, et al. Assay to rapidly screen for immunoglobulin light chain glycosylation: a potential path to earlier AL diagnosis for a subset of patients. Leukemia. 2019;33(1):254-257.
  11. Dispenzieri A, Larson DR, Rajkumar SV, et al. N-glycosylation of monoclonal light chains on routine MASS-FIX testing is a risk factor for MGUS progression. Leukemia. 2020;34(10):2749-2753.
  12. Burgos L, Puig N, Cedena MT, et al. Measurable residual disease in multiple myeloma: ready for clinical practice?. J Hematol Oncol. 2020;13(1):82.
  13. Derman BA, Stefka AT, Jiang K, et al. Measurable residual disease assessed by mass spectrometry in peripheral blood in multiple myeloma in a phase II trial of carfilzomib, lenalidomide, dexamethasone and autologous stem cell transplantation. Blood Cancer J. 2021;11(2):19.
  14. Dispenzieri A, Krishnan A, Arendt B, et al. Mass-Fix better predicts for PFS and OS than standard methods among multiple myeloma patients participating on the STAMINA trial (BMT CTN 0702 /07LT). Blood Cancer J. 2022;12(2):27.
  15. Thoren KL. Will mass spectrometry replace current techniques for both routine monitoring and MRD detection in multiple myeloma? Hemato. 2021;2(4):764-768.

 

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