Mass Spectrometry (MS) Testing in Monoclonal Gammopathy (MG)

Language quoted from Centers for Medicare and Medicaid Services (CMS), National Coverage Determinations (NCDs) and coverage provisions in interpretive manuals is italicized throughout the policy. NCDs and coverage provisions in interpretive manuals are not subject to the Local Coverage Determination (LCD) Review Process (42 CFR 405.860[b] and 42 CFR 426 [Subpart D]). In addition, an administrative law judge may not review an NCD. See Section 1869(f)(1)(A)(i) of the Social Security Act.

Unless otherwise specified, italicized text represents quotation from one or more of the following CMS sources:

Title XVIII of the Social Security Act (SSA):
Section 1862(a)(1)(A) excludes expenses incurred for items or services which are not reasonable and necessary for the diagnosis or treatment of illness or injury or to improve the functioning of a malformed body member.

Section 1833(e) prohibits Medicare payment for any claim which lacks the necessary information to process the claim.

Section 1862(a)(7) excludes routine physical examinations, unless otherwise covered by statute.

CMS Publications:
CMS Publication 100-02, Medicare Benefit Policy Manual, Chapter 15, Section 80.1 – Laboratory services must meet applicable requirements of CLIA

CMS Publication 100-04, Medicare Claims Processing Manual, Chapter 16, Section 40.7 Billing for Noncovered Clinical Laboratory Tests Section and 120.1 Clarification of the Use of the Term “Screening” or “Screen”

CMS Publication 100-04, Medicare Claims Processing Manual, Chapter 30, Section 50 Advance Beneficiary Notice of Noncoverage (ABN)

CMS Publication 100-08, Medicare Program Integrity Manual, Chapter 13, Local Coverage Determinations

CMS National Correct Coding Initiative (NCCI) Policy Manual for Medicare Services, Chapter 10, Pathology/Laboratory Services, (A) Introduction

CMS Publication 100-02, Medicare Benefit Policy Manual, Chapter 15, Section 80.6.5 which describes the Surgical/Cytopathology Exception.

CMS National Correct Coding Initiative (NCCI) Policy Manual for Medicare Services, Chapter 10 Pathology/Laboratory Services which addresses reflex testing.

Code of Federal Regulations:
42 CFR, Section 410.32, indicates that diagnostic tests may only be ordered by the treating physician (or other treating practitioner acting within the scope of his or her license and Medicare requirements) who furnishes a consultation or treats a beneficiary for a specific medical problem and who uses the results in the management of the beneficiary's specific medical problem. Tests not ordered by the physician (or other qualified non-physician provider) who is treating the beneficiary are not reasonable and necessary (see Sec. 411.15(k)(1) of this chapter).

Indications of Coverage

The use of serum and urine mass spectrometry (MS) in monoclonal gammopathies (MGs), as a potential alternative to the use of serum or urine immunofixation electrophoresis (sIFE), is considered medically necessary for:

1. Diagnosis: Initial detection of M-protein in patients with suspected monoclonal gammopathy (MG) to confirm a serum or urine protein electrophoresis (SPEP) or serum free light-chain (sFLC) abnormality (1), or
2. Monitoring:
   1. Discrimination between therapeutic monoclonal antibodies and endogenous M-proteins (2), or
   2. Treatment response assessment per guidelines (3-5)

Limitations of Coverage (not covered)

1. Screening
2. Concurrent sIFE testing

Monoclonal gammopathy (MG) is characterized by the proliferation of a single clone of plasma cells which produces monoclonal immunoglobulin protein (M-protein). The M-protein can be an intact immunoglobulin (i.e., containing both heavy and light chains), only light chains (e.g., AL [light chain] amyloidosis), or rarely of heavy chains only (6). The prevalence of M-protein is relatively high, being detected in approximately 3% of the general adult population over 50 years old, and in up to 7% of those seeking medical evaluation (7). MGs represent a spectrum of at least 18 distinct entities, ranging from an asymptomatic limited clonal expansion of plasma cells (e.g., monoclonal gammopathy of undetermined significance (MGUS)), to the potentially life-threatening, such as multiple myeloma (MM) and AL amyloidosis. Therefore, timely diagnosis and treatment to prevent irreversible organ damage is critical. A search for M-protein should be considered in any patient with an elevated total serum protein or otherwise unexplained signs and symptoms suggestive of a plasma cell disorder (6).

However, the process for identifying patients with MGs is complex and depends on clinical and diagnostic testing information. Laboratories have developed disparate practices for M-protein detection and quantitative measurement, complicating result harmonization, likely resulting in suboptimal detection of treatable MGs (1). Currently, suspected MGs are initially detected using a combination of three serum-based diagnostic tests: serum protein electrophoresis (SPEP), serum immunofixation electrophoresis (sIFE), and serum measurement of free light-chain (sFLC) (1,5). SPEP testing for M-protein began in the 1930s and has since steadily improved in resolution. The M-protein usually presents as a single narrow peak; in contrast, a broad-based band usually suggests a polyclonal increase in immunoglobulins (usually an infectious, inflammatory, or reactive process). The initial SPEP should be performed in combination with sIFE (uses antibodies against heavy and light chain components) both to confirm monoclonality and determine isotype (the heavy and light chain class, e.g., IgG kappa). M-protein isotype has significance for prognosis and risk-stratification (e.g., progression of MGUS to MM) (8). The sFLC assay is an antibody-based system that can detect low concentrations of monoclonal free light chains (i.e., kappa or lambda) in the serum. FLC quantification is the most analytically sensitive blood-based method commercially available to diagnose and monitor patients with MGs, and is sometimes the only indication of a MG. Assessing changes in M-protein levels helps track disease progression and response to treatment (5).

Other emerging laboratory procedures to detect M-proteins include immunosubtraction (ISUB), mass spectrometry (MS), and heavy/light chain (HLC) isotype quantitative measurement. The MS method combines nanobody enrichment of immunoglobulins with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDITOF- MS) (also termed MASS-FIX, Mayo Medical Laboratories). The idea behind MS is that molecular mass can be used instead of electrophoretic patterns to identify and quantify the M-protein since each light and heavy chain has a unique amino acid sequence, and thus a unique molecular mass whose increased concentration could be distinguished from the normal polyclonal background (9). In addition to detection, MS might be able to isotype the M-protein because each immunoglobulin has a constant region with an amino acid sequence unique to each isotype. The multiple charged light and heavy chain ions are converted to their molecular masses, and reconstructed peak area calculations for light chains are used for quantification. Thus, in theory, the MASS-FIX assay uses the unique molecular mass signatures of the different Ig isotypes to generate mass spectra from which M-proteins could be identified, isotyped, and quantified.

A 2016 validation study found comparable analytic sensitivity of MASS-FIX with SPEP and IFE (10). MASS-FIX detected all M-proteins that were detectable by urine or serum protein electrophoresis. In serial dilution studies, MASS-FIX was more analytically sensitive than IFE (identified an M-protein in a higher percentage of samples at every dilution), and where they agreed provided the same primary isotype information for 98% of serum M-proteins (n = 152) and 95% of urine M-proteins (n = 55). A subsequent prospective study of paired serum and urine samples from 257 patients confirmed comparable sensitivity with serum/urine PEP/IFE and sFLC when serum and urine MASS-FIX results were combined (7). A more recent study of 226 patients diagnosed with MGUS or related gammopathy, considered negative for MGUS by protein electrophoresis and sFLC assay, found that M-protein could be detected at baseline in only 24 patients (10.6%) by IFE compared with 113 patients (50%) by MADLI-TOF mass spectrometry (11). IFE cannot distinguish if two bands of the same isotype represent biclonal proteins or M-proteins with some other feature. In a study of 81 serum samples with multiple IFE bands of the same isotype, MASS-FIX was able to characterize them as monoclonal or biclonal (12). In a study of 127 patient sera with abnormal FLC ratios, 43% of monoclonal proteins were confirmed by IFE, 57% by MALDI-TOF MS without FLC enrichment, and 87% with FLC enrichment MALDI-TOF MS (13). The authors conclude that FLC immunoenrichment coupled to MALDI-TOF MS enables direct detection of mFLCs, significantly increasing the confirmation of abnormal serum FLC ratios (a more indirect methodology), thus improving verification of disease in patients with light chain plasma cell disorders.

Other areas of potential utility include distinguishing endogenous M-proteins from therapeutic monoclonal antibodies; daratumumab, one therapeutic IgG kappa monoclonal antibody, can cause a false positive interference on both SPEP and sIFE, two assays routinely used to monitor a patient’s disease status and response to therapy (14). A study of 31 patients receiving daratumumab with a history of IgG kappa MG found that MASS-FIX could distinguish daratumumab from M-proteins in 26 out of 31 serum samples (84%) versus 14 out of 31 samples (45%) by sIFE (2). Another study of 311 AL amyloidosis patients showed that MASS-FIX can distinguish a subset with light chain glycosylation, providing a potential path to earlier AL amyloidosis diagnosis (15). Automation and integrated software may allow implementation of serum MASS-FIX in a high-throughput clinical laboratory (16).

Combined use of SPEP, sIFE, and sFLC is effective in the initial assessment of MG and is recommended in the International Myeloma Working Group (IMWG) guideline for the evaluation of MM and related disorders (17,18). The Myeloma Canada Research Network Consensus Guidelines Consortium and the European Myeloma Network make similar recommendations (19,20).

A just released collaborative guideline from the College of American Pathologists (CAP), the American Association for Clinical Chemistry (AACC), and the American Society for Clinical Pathology (ASCP) recommends that clinicians “should order both SPEP and sFLC for the initial detection of M-protein in all patients with suspected MG” (strong strength of recommendation, evidence strength moderate) (1). To confirm a SPEP abnormality, the guideline recommends a “sIFE or alternative method with similar sensitivity” (strong strength of recommendation, evidence strength moderate). To confirm a sFLC ratio abnormality, the guideline identically recommends a “sIFE or alternative method with similar sensitivity” (conditional recommendation, evidence strength low). That an “alternative method” might include MASS-FIX is supported by the statement that “a new technique involving immunoenrichment followed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MASS-FIX) has been shown to be a highly sensitive, specific, and cost-effective method comparable to sIFE to detect and identify M-proteins”. However, the guidelines specifically recommend against MS for screening.

UpToDate refers to MS for analyzing monoclonal proteins as “investigational” and “not a standard part of the evaluation of M-proteins in most centers” (6), but finds utility in the setting of AL amyloidosis, noting that to distinguish AL amyloidosis from other forms of amyloidosis “MS is the preferred method since immunohistochemistry and immunofluorescence have a greater risk of false positive and false negative results” (21). It is not mentioned for other MGs (22-24).

NCCN guidelines also recommend amyloid tissue subtyping with MS in the setting of AL amyloidosis “to confirm that the amyloid deposits are composed of light chains.“ NCCN recommends MS “only if clinically indicated such as in cases where two potential amyloid precursor proteins are present including patients with monoclonal gammopathies who are African-American or elderly men, or who have dominant peripheral or autonomic neuropathy, family histories of amyloidosis, or coexisting inflammatory disorders” (4). Something similar is noted in the NCCN guideline on Waldenstrom Macroglobulinemia (3). No mention is made of MS in the NCCN MM guideline (5).

In summary, the use of MALDI-TOF MS to detect and characterize MGs is a novel application of well-established technology for detection of proteins, now applied to a well-established clinical indication (e.g., detection of M-proteins to characterize MGs). The MASS-FIX assay is a well validated, characterized, and published application of this technology in this context. Therefore, NGS considers serum MS use in MG as a possible alternative to sIFE in confirming a SPEP or sFLC abnormality, largely in concordance with the CAP/AACC/ASCP collaborative guideline. Serum MS may also provide advantages in distinguishing between therapeutic and endogenous M-proteins. While published studies and guidelines on MS primarily focus on initial diagnosis, since sIFE also figures prominently in MG monitoring guidelines, it seems logical to also cover serum MS as a sIFE alternative in that setting. MS for screening for MGs is not covered.

Reconsideration Request- January 2024

The Reconsideration Request recommended that NGS add urine Mass Spectrometry to this LCD. In the article by Danelle H. Moonen et al titled, “Utilizing Mass Spectrometry to Detect and Isotype Monoclonal Proteins in Urine: Comparison to Electrophoretic Methods” concluded that u-Mass-Fix is a reliable alternative to u-IFE. It was demonstrated that u-IFE and u-MASS-FIX methods are substantially equivalent, with 91% result agreement. These methods revealed no statistical difference in confirming UPEP M-spikes (25). Another article by Paola Milani et al, was titled “The Utility of MASS-FIX to detect and monitor monoclonal proteins in the clinic.” The article compared MASS-FIX to protein electrophoresis, IFE and quantitative serum free light chains for the identification of M-proteins in different clinical diagnoses. Paired urine and serum samples from 257 patients were tested. It was found that MASS-FIX has a comparable sensitivity with protein electrophoresis, IFE and quantitative free light chains. It was felt that the MASS-FIX works across the spectrum of plasma cell disorders and patients with renal failure (7). A final article was submitted titled “Comprehensive Assessment of M-Proteins Using Nanobody Enrichment Coupled to Maldi-TOF Mass Spectrometry by John R. Mills, et al demonstrated that MASS-FIX detected all M-proteins that were detectable by urine and serum protein electrophoresis (10).

In conclusion, NGS will allow the use of serum and urine mass spectrometry (MS) in monoclonal gammopathies (MGs).

1. Keren DF, Bocsi G, Billman BL, et al. Laboratory Detection and Initial Diagnosis of Monoclonal Gammopathies: Guideline From the College of American Pathologists in Collaboration With the American Association for Clinical Chemistry and the American Society for Clinical Pathology. Arch Pathol Lab Med. 2021.
2. Moore LM, Cho S, Thoren KL. MALDI-TOF mass spectrometry distinguishes daratumumab from M-proteins. Clin Chim Acta. 2019;492:91-94.
3. NCCN Waldenstrom Macroglobulinemia/Lymphoplasmacytic Lymphoma V1.2022. https://www.nccn.org/professionals/physician_gls/pdf/waldenstroms.pdf. Accessed 8/24/21.
4. NCCN Systemic Light Chain Amyloidosis V1.2022. https://www.nccn.org/professionals/physician_gls/pdf/amyloidosis.pdf. Accessed 8/24/21.
5. NCCN Multiple Myeloma V1.2022. https://www.nccn.org/professionals/physician_gls/pdf/myeloma.pdf. Accessed 8/24/21.
6. UpToDate: Laboratory methods for analyzing monoclonal proteins. https://www.uptodate.com/contents/laboratory-methods-for-analyzing-monoclonal-proteins?search=mass%20spectrometry&source=search_result&selectedTitle=1~150&usage_type=default&display_rank=1. Accessed 8/24/21.
7. 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.
8. Kyle RA, Larson DR, Therneau TM, et al. Long-Term Follow-up of Monoclonal Gammopathy of Undetermined Significance. N Engl J Med. 2018;378(3):241-249.
9. 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(3):1419-1427.
10. 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(10):1334-1344.
11. Murray D, Kumar SK, Kyle RA, et al. Detection and prevalence of monoclonal gammopathy of undetermined significance: a study utilizing mass spectrometry-based monoclonal immunoglobulin rapid accurate mass measurement. Blood Cancer J. 2019;9(12):102.
12. Fatica EM, Martinez M, Ladwig PM, et al. MALDI-TOF mass spectrometry can distinguish immunofixation bands of the same isotype as monoclonal or biclonal proteins. Clin Biochem. 2021.
13. Sepiashvili L, Kohlhagen MC, Snyder MR, et al. Direct Detection of Monoclonal Free Light Chains in Serum by Use of Immunoenrichment-Coupled MALDI-TOF Mass Spectrometry. Clin Chem. 2019;65(8):1015-1022.
14. McCudden CR, Jacobs JFM, Keren D, Caillon H, Dejoie T, Andersen K. Recognition and management of common, rare, and novel serum protein electrophoresis and immunofixation interferences. Clin Biochem. 2018;51:72-79.
15. 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.
16. Kohlhagen M, Dasari S, Willrich M, et al. Automation and validation of a MALDI-TOF MS (Mass-Fix) replacement of immunofixation electrophoresis in the clinical lab. Clin Chem Lab Med. 2020;59(1):155-163.
17. Dispenzieri A, Kyle R, Merlini G, et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia. 2009;23(2):215-224.
18. Rajkumar SV, Dimopoulos MA, Palumbo A, et al. International Myeloma Working Group updated criteria for the diagnosis of multiple myeloma. Lancet Oncol. 2014;15(12):e538-548.
19. Bergstrom DJ, Kotb R, Louzada ML, et al. Consensus Guidelines on the Diagnosis of Multiple Myeloma and Related Disorders: Recommendations of the Myeloma Canada Research Network Consensus Guideline Consortium. Clin Lymphoma Myeloma Leuk. 2020;20(7):e352-e367.
20. van de Donk NW, Palumbo A, Johnsen HE, et al. The clinical relevance and management of monoclonal gammopathy of undetermined significance and related disorders: recommendations from the European Myeloma Network. Haematologica. 2014;99(6):984-996.
21. UpToDate: Clinical presentation, laboratory manifestations, and diagnosis of immunoglobulin light chain (AL) amyloidosis. https://www.uptodate.com/contents/clinical-presentation-laboratory-manifestations-and-diagnosis-of-immunoglobulin-light-chain-al-amyloidosis?search=mass%20spectrometry&topicRef=6665&source=see_link. Accessed 8/24/21.
22. UpToDate: Clinical manifestations, pathologic features, and diagnosis of lymphoplasmacytic lymphoma. https://www.uptodate.com/contents/clinical-manifestations-pathologic-features-and-diagnosis-of-lymphoplasmacytic-lymphoma?search=mass%20spectrometry&topicRef=6655&source=see_link. Accessed 8/24/21.
23. UpToDate: Epidemiology, pathogenesis, clinical manifestations, and diagnosis of Waldenström macroglobulinemia. https://www.uptodate.com/contents/epidemiology-pathogenesis-clinical-manifestations-and-diagnosis-of-waldenstrom-macroglobulinemia?search=mass%20spectrometry&topicRef=6655&source=see_link. Accessed 8/24/21.
24. UpToDate: Diagnosis of monoclonal gammopathy of undetermined significance. https://www.uptodate.com/contents/diagnosis-of-monoclonal-gammopathy-of-undetermined-significance?search=mass%20spectrometry&topicRef=6655&source=see_link. Accessed 8/24/21.
25. Moonen DH, Kohlhagen M, Dasari S, et al. Utilizing Mass Spectrometry to Detect and Isotype Monoclonal Proteins in Urine: Comparison to Electrophoretic Methods. Clin Chem. Jul 5 2023;69(7):746-753. doi:10.1093/clinchem/hvad053
26. Gillmore JD, Maurer MS, Falk RH, et al. Nonbiopsy Diagnosis of Cardiac Transthyretin Amyloidosis. Circulation. Jun 14 2016;133(24):2404-12. doi:10.1161/CIRCULATIONAHA.116.021612

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