**Indications of Coverage**

The use of serum mass spectrometry (MS) in monoclonal gammopathies (MGs), as a
potential alternative to serum immunofixation electrophoresis (sIFE), may be
considered medically necessary for:

  1. Diagnosis: Initial detection of M-protein in patients with suspected monoclonal gammopathy (MG) to confirm a serum 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
  3. Urine MS 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.