A 57-year-old man presented to his primary physician complaining of three weeks of sudden-onset “profound fatigue” and dyspnea on exertion. His medical history is insignificant. Initial laboratory studies (Figure 1a) are remarkable for a markedly elevated erythrocyte sedimentation rate and rouleaux formation in the CBC manual differential (Figure 1b), and serum hyperviscosity (Figure 2). Serum protein electrophoresis exhibited an M-spike of 3.4 g/dL and isotyping via MALDI-TOF mass spectrometry identified a monoclonal IgM kappa protein (Figure 1c/d).
The correct answer is ...
Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) is the method utilized at Mayo Clinic for M-protein isotyping, termed Mass-Fix (available since 2018 for clinical testing). It has replaced the traditional immunofixation electrophoresis (IFE) method for isotyping the M-protein identified by serum protein electrophoresis (SPE). Serum samples are immunopurified using five separate immunoglobulin (Ig) enrichment beads specific for IgG, IgA, IgM, and the two light chains, kappa and lambda. Following elution and a reduction step of each Ig-enrichment into heavy and light chains, the samples are spotted onto steel plates with matrix, and a laser ionizes each sample. The overlay of the resulting spectra (as seen in Figure 1d) is in a similar format to that of IFE and allows for isotyping of any monoclonal proteins in the specimen. Normal patients have near-gaussian mass-to-charge (m/z) distributions of light chains (lambda has one while kappa has two, corresponding to the two variant germ line kappa genes with one several amino acids heavier than the other). A patient with a monoclonal protein (M-protein) exhibits a spike in the light chain m/z distribution, and by inspecting all five spectra overlaid, one can determine the isotype of the M-protein.
Lymphoplasmacytic lymphoma (LPL)
A mature B cell lymphoma that is uncommon (only ~1% of hematologic malignancies in US), LPL is further characterized by the isotype of the clonal immunoglobulin secreted. When an IgM monoclonal gammopathy is present, is termed Waldenström macroglobulinemia (WM). Genomics studies show that LPL is phenotypically more comparable to chronic lymphocytic leukemia (CLL) than multiple myeloma (MM). The most common mutation seen in LPL is an activating point mutation (L256P) of MYD88, contributing to pathogenesis. Unfortunately, patients present with diverse symptoms, including most commonly weakness and fatigue (secondary to anemia), fever, diaphoresis, and weight loss (though around one-third of patients are asymptomatic at diagnosis). At diagnosis, over 70% of patients are in stage IV disease (as defined by involvement of the bone marrow). In the laboratory, the majority of patients exhibit a monoclonal gammopathy on serum protein electrophoresis or immunofixation. The monoclonal immunoglobulin class helps determine subtype of LPL and prognosis. For example, patients with a monoclonal IgM in their serum are diagnosed as having the clinical syndrome WM (more rare, with around 1,400 new cases per year in the U.S.). The original tumor can also produce different immunoglobulins, a mixed population of immunoglobulins (IgG and IgM, for example), mixed cryoglobulins and gamma heavy chains. Patients with an IgG monoclonal protein have a less aggressive clinical course compared to other classes.
This patient’s monoclonal gammopathy was isotyped as an IgM-kappa, with an M-spike of 3.4 g/dL. The initial concerning laboratory result was his elevated SED rate and manual differential, showing rouleaux formation, a linear stack of red blood cells that forms in the presence of high plasma protein concentrations. Further investigation yielded the laboratory results shown in Figures 1 and 2. He also exhibited bilateral retinal hemorrhages. Hyperviscosity is common with a diagnosis of WM, as IgM molecules form pentamers. The target of the IgM clone suggests further symptoms or associated diagnoses, such as anti-nerve components resulting in neuropathy or anti-red blood cells leading to autoimmune cold hemolytic anemia. His initial chief complaints are consistent with WM. Tumor infiltration of the tissue can lead to lymphadenopathy, hepatomegaly, and splenomegaly. A bone marrow aspirate was microscopically analyzed (low grade B-cell lymphoma with plasmacytic differentiation, 90%-95% cellular involvement of the marrow with 6% plasma cells), then genotyped and found positive for an activating mutation in MYD88. Due to the patient’s symptomatic presentation and initial IgM of 6,380 mg/dL, two sessions of plasma exchange were performed which reduced his IgM first to 3,890 mg/dL and finally to 1,850 mg/dL. His IgM concentrations rebounded quickly after plasma exchange and each early dose of drug therapy.
The 57-year-old man, who initially presented with profound fatigue, headache, weight loss, and tinnitus, received chemotherapy and careful monitoring of serum IgM concentration.
Multiple myeloma (MM)
Over 10 times more prevalent than LPL, multiple myeloma (MM) makes up 10% of hematologic malignancies and it currently has no cure (though remission is possible). While LPL is cancer of white blood cells in the lymph system, MM is localized to the bone marrow (BM). IgM MM is very rare. Arising from an aberrant clone of BM plasma cells, MM is almost always preceded by a premalignant, asymptomatic condition called monoclonal gammopathy of undetermined significance (MGUS). Unlike MGUS, MM may be characterized by the CRAB criteria: hypercalcemia, renal dysfunction, anemia, and bone lesions. Another pre-malignant, intermediate stage of disease, termed smoldering MM (SMM), occurs when patients remain asymptomatic but meet the laboratory criteria for MM (i.e., an M-spike of >3 gdL, between 10%-60% bone marrow plasma cells, and no evidence of end-organ damage or amyloidosis). Diagnosis of MM requires >10% clonal plasma cells on bony or soft tissue plasmacytoma, plus one of the following additional characteristics: presence of any of the CRAB criteria or a biomarker associated with eventual end-organ damage (>60% bone marrow plasma cells, involved free light chain ratio >=100 or presence of one or more bone lesions on imaging).
In addition to the CRAB signs and symptoms, MM patients may rarely have neurological manifestations. MM patients are more susceptible to infections due to immune dysfunction. The detection of monoclonal proteins is paramount to diagnosing MM, and the laboratory methods include serum and urine protein electrophoresis and immunofixation. At Mayo Clinic, MALDI-TOF mass spectrometry is utilized for isotyping the monoclonal protein. Measuring free light chains in serum is not only part of the diagnostic criteria, but also used to monitor response to therapy; nephelometric or immunometric assays (ELISA) are available for quantitation. There are several phenotypes of MM: those characterized by a single immunoglobulin population (IgG-kappa is the most common), biclonal disease, light chain myeloma, and both oligo-secretory and nonsecretory forms of MM. All phenotypes can be differentiated by various laboratory tests, including serum and urine M-protein concentration, isotyping of the monoclonal protein, and bone marrow biopsy interpretation (including percent clonality and light chain restriction).
Immunoglobulin light chain amyloidosis (AL amyloidosis)
AL amyloidosis is a rare systemic disorder whose prevalence is unknown and can present as unexplained heart failure, heavy proteinuria, edema, and hepatosplenomegaly. Nonspecific symptoms, such as weight loss and fatigue, are common with AL amyloidosis. There are several forms of amyloidosis, as the term describes tissue deposition of protein fibrils (various subunits of normal serum proteins). When these fibrils are made of monoclonal immunoglobulin light chains, the diagnosis is AL amyloidosis, and it can present alone or in combination with other plasma cell proliferative disorders (such as MM or WM) or lymphomas. The degree of end-organ damage varies dramatically, from only one organ to widespread multi-system damage, with the kidneys being most commonly involved. Of note, AL amyloidosis can be a rare complication of an IgM-associated gammopathy, such as WM.
The diagnosis requires clinical or laboratory evidence of a plasmacytic proliferative disorder (e.g., serum or urine M-protein, elevated FLC ratio in serum, or clonal plasma cells in bone marrow) and positive Congo red staining in a tissue or bone marrow biopsy. Most patients with AL amyloidosis exhibit elevated monoclonal immunoglobulins, either IgG or free light chain isotypes. The same laboratory examinations used to detect MM are applied when AL amyloidosis is suspected. This patient does not have marked proteinuria or end-organ damage associated with AL amyloidosis.
Monoclonal gammopathy of undetermined significance (MGUS)
Approximately 3% of adults over age 50 have a MGUS and it is often discovered incidentally during evaluation for a number of other disorders, as it is asymptomatic. This patient had significant symptoms at presentation. Of the three subtypes of MGUS, non-IgM MGUS is the most prevalent followed by IgM MGUS, and finally light chain MGUS (LC-MGUS). While nearly all MM patients had a MGUS diagnosis prior, not all MGUS progresses to a more sinister plasma cell proliferative disorder. The diagnostic criteria for MGUS includes a serum M-protein (<3 g/dL), lack of end-organ damage, and if a bone marrow biopsy was performed, <10% plasma cell monoclonality. Many of the studies on MGUS progression were conducted at Mayo Clinic, starting in the 1960s, by Dr. Robert Kyle. The rate of progression of MGUS into advanced disease is 1% per year, yet 6.5 times more MGUS patients developed MM, WM, amyloidosis, or another lymphoproliferative disorder compared to the general population.
Ria Fyffe-Freil, Ph.D.
Fellow, Clinical Chemistry
Maria Alice Willrich, Ph.D.
Consultant, Clinical Biochemistry
Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science