By A.R. Bradwell, FRCP
Serum immunoassays for immunoglobulin free light chains (FLCs) have important theoretical and practical advantages over urine electrophoresis tests. Published data indicates that 100% of patients with light chain multiple myeloma, 85% of patients with nonsecretory myeloma, and more than 95% of patients with primary amyloidosis have abnormal serum FLC concentrations. This improvement in disease detection rates and their superior utility for disease monitoring may obviate the need for urine FLC tests in most patients with monoclonal plasma cell diseases.
Background on Serum Free Light Chain Assays
Monoclonal FLCs, traditionally termed Bence Jones proteins, are homogeneous populations of kappa (k) or lambda (l) immunoglobulin light chain molecules produced by malignant clones of B-cells. They are important tumor markers for identifying and monitoring patients with light chain multiple myeloma (LCMM) and light chain amyloid disease (AL amyloidosis). (See Figure 1.)
Figure 1. Distribution of clinical diagnoses in 1,026 patients with a serum monoclonal protein detected at the Mayo Clinic in 1992.1 The classification of multiple myeloma patients by immunoglobulin type is also shown.
Identification of FLCs has traditionally been by protein electrophoresis or immunofixation electrophoresis (IFE) of urine. These tests can detect as little as 10 mg/L to 20 mg/L of FLCs; however, urine tests have a variety of problems. Samples need to be accurately collected over 24 hours, properly preserved, and then transported to the laboratory. Subsequent concentration is usually required before final analysis by electrophoresis assays.2 These procedures are time-consuming and relatively insensitive and can be inaccurate. Furthermore, the renal proximal tubules can metabolize up to 30 g per day of FLC so urine measurements are poorly related to tumor synthesis rates when production is low. These issues suggest that serum analysis should be preferable but the existing serum protein electrophoresis (SPE) tests and IFE are too insensitive to detect FLC in most patients. They are also unsatisfactory for quantitative purposes.
An alternative approach to serum analysis is to use particle enhanced immunoassays for FLC that can distinguish between free light chains and light chains bound to intact immunoglobulins. Recent studies show that these assays are sensitive, quantitative, and more precise than electrophoresis assays. Furthermore, FLC clonality, as judged by precipitation bands on gels can be replaced by the numerical FLC kappa/lambda (k/l) ratios.3,4 Results of many laboratory studies have shown that they are considerably more sensitive than serum electrophoresis tests (see Table 1). The assays are also automated and available on a variety of routine clinical laboratory turbidimeters and nephelometers.
Table 1. Representative sensitivity levels of serum free light chain assays.
Clinical Use of Serum FLC Immunoassays
In some situations serum FLC measurements are having considerable clinical impact and in others the tests are under evaluation. From a clinical standpoint, 85% of patients with nonsecretory multiple myeloma (NSMM), more than 95% of AL patients, and all LCMM patients were identified using serum samples. These considerable improvements in disease detection rates over current methods may obviate the need for most urine tests. Similar benefits, in terms of sensitivity and precision of results, were also observed when monitoring these patients. Diseases with excess production of monoclonal FLC are shown in table 2. The current state of these investigations is described below.
Multiple Myeloma
The concentrations of serum FLC in patients with different types of monoclonal gammopathies at the time of diagnosis is shown in table 2. Most patients with nonsecretory multiple myeloma (NSMM) can be identified when all other tests for monoclonal proteins are normal.5 By definition, these patients do not have detectable monoclonal proteins in serum or urine by electrophoresis tests. In multiple myeloma patients producing only monoclonal light chains (LCMM), all had elevated serum FLC. The implication of this observation is that urine tests in LCMM are no longer necessary.6
The remaining 80% to 85% of multiple myeloma patients have intact monoclonal immunoglobulins in the serum. However, by immunoassays, over 95% also have elevated serum FLC (see Table 2).
Table 2. Incidence of diseases commonly producing monoclonal proteins in the United States.
IIMM: intact immunoglobulin multiple myeloma. LCMM: light chain multiple myeloma. NSMM: nonsecretory multiple myeloma. MGUS: monoclonal gammopathy of undetermined significance. B-CLL: B cell chronic lymphocytic leukemia. *Monoclonal proteins by traditional electrophoresis methods. B-CLL#: Other B cell lymphoid malignancies occasionally produce monoclonal immunoglobulins.
While serum FLC measurements are particularly useful in the diagnosis of light chain only multiple myeloma patients, serial measurements may be useful in nearly all patients.1 The half-life of serum FLC (2 to 4 hours) is much shorter than intact IgG (3 weeks) so responses to treatment can be identified in hours or days rather than weeks. This is shown in figure 3 in a myeloma patient producing both monoclonal IgGk and free k who was studied retrospectively using stored serum samples. During the initial therapy, k concentrations fell rapidly while IgGk, quantified from protein electrophoresis strips, only gradually returned to normal. The half-life of IgGk was between 100 and 200 days. A similar pattern of response was seen during a subsequent relapse and treatment period. Plasma cell counts from bone marrow biopsy specimens and serum B2 microglobulin concentrations followed the changes in serum FLC and not the monoclonal IgGk. It may be that selection of effective chemotherapeutic agents could be made more quickly from serial FLC measurements rather than using IgG (see Figure 3).
Figure 3. Monitoring of a myeloma patient using IgGk and free k. Electrophoresis gels are shown for each sample. CVAMP: cyclophosphamide, vincristine sulfate, Adriamycin, melphalan, prednisolone. HDM: high dose melphalan and stem cell transplant.
An additional study has shown abnormal serum FLC concentrations in patients in complete remission. Some have subsequently relapsed, suggesting that serum FLC may be a prognostic marker for patients who are in complete remission by other criteria. Further studies are awaited.
Figure 2. Composite figure of serum free light chain concentrations in various diseases, including 31 patients with renal impairment (courtesy H. Lachmann). LCMM: light chain multiple myeloma. IIMM: intact immunoglobulin multiple myeloma. High pIgG: polyclonal hypergammaglobulinemia. NSMM: nonsecretory multiple myeloma.
AL Amyloidosis
Monoclonal free light chains occasionally form polymers that deposit causing AL amyloidosis or more rarely light chain deposition disease (LCDD). While less common than multiple myeloma, patients have a median life expectancy of little more than 12 months and die from heart, renal, or liver failure. The origin of the FLC is usually a slowly growing clone of plasma cells in the bone marrow. However, the resulting monoclonal gammopathies can be subtle and are undetectable by conventional electrophoresis techniques in 10% to 30% of patients. In a recent study, serum FLC were quantified in 98% of 262 patients attending the National Amyloidosis Centre in London (see Figure 2).7 Although 76% of the patients had detectable monoclonal proteins by conventional tests, serum FLC could only be quantified in 3% by serum protein electrophoresis. Since serum FLC are the depositing proteins, it is logical that they should be accurately quantified for disease assessment. In a recently published retrospective study,7 reductions in serum FLC concentrations during chemotherapy appeared to be the best indicator of long-term survival (see Figure 4). These important observations are being used to study disease progress in a variety of clinical situations.8
Figure 4. Kaplan-Meier estimate of survival in 137 patients with AL showing that a reduction of free light chain concentrations by greater than 50% following chemotherapy was associated with increased survival. FLC = free light chain. (Courtesy PN Hawkins).
Monoclonal Gammopathies of Undetermined Significance (MGUS)
The majority of observed monoclonal immunoglobulins (see Table 1) are chance findings on electrophoresis gels and are of undetermined clinical significance. In these patients, the risk of progression to multiple myeloma, Waldenström’s macroglobulinemia or other B cell diseases is approximately 1% per year, 30 times that of the normal population. Once identified, these MGUS individuals are monitored on an annual basis and early signs of disease progression are treated appropriately. One risk factor for progression is the amount of monoclonal protein, such that individuals with 30 g/L are three times more likely to progress than those with low concentrations. In a preliminary study of 97 patients at the Mayo Clinic, increased concentrations of monoclonal serum FLC indicated an additional risk factor for progression of 2.5-fold (P < 0.002). This needs to be confirmed in a larger study but the observations suggest that it may be useful to measure serum FLC on a regular basis in MGUS patients.1
MGUS proteins nearly always comprise intact immunoglobulin molecules and these indicate a premalignant condition for the development of intact immunoglobulin multiple myeloma. However, the light chain MGUS counterpart for light chain multiple myeloma has not been identified. The likely explanation is that serum concentrations of light chains in FLC MGUS are insufficient to exceed the absorption capacity of the renal tubules so they do not enter the urine. Recent studies from the Mayo Clinic have shown that approximately 0.7% of elderly, healthy individuals have serum monoclonal FLC using immunoassays.1 These are probably the “missing,” premalignant, LCMM patients. Again, larger studies are under way.
| Advantages of Serum FLC Immunoassays
• Up to 500 times more sensitive than SPE or UPE |
Other Diseases Associated With the Production of Monoclonal Immunoglobulins
There are several other B cell dyscrasias that are associated with the production of monoclonal immunoglobulins. These include Waldenström’s macroglobulinemia, solitary plasmacytomas, light chain deposition disease, and some leukemias and lymphomas. Monoclonal free light chains have been identified in some of these patients but the clinical relevance is yet to be determined.1
Serum FLC assays are likely to become part of the routine clinical assessment of patients with monoclonal gammopathies. They are particularly useful when existing tests cannot identify monoclonal proteins but their short serum half-life suggests they will be the markers of choice for assessing responses to therapy in a variety of clinical situations.
A.R. Bradwell, FRCP, Division of Immunity and Infection, University of Birmingham Medical School, can be reached at 0121-414-2025 or [email protected].
References
1. Bradwell AR. Serum Free Light Chain Assays. Birmingham, England: The Binding Site Ltd; 2003.
2. Keren DF, Alexanian R, Goeken JA, Gorevic PD, Kyle RA, Tomar RH. Guidelines for clinical and laboratory evaluation of patients with monoclonal gammopathies. Arch Pathol Lab Med. 1999;123(2): 106-107.
3. Bradwell AR, Carr-Smith HD, Mead GP, et al. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem. 2001;47(4): 673-680.
4. Katzmann JA, Clark RJ, Abraham RS, et al. Serum reference intervals and diagnostic ranges for free kappa and free lambda immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem. 2002;48(9): 1437-1444.
5. Drayson M, Tang LX, Drew R, Mead GP, Carr-Smith HD, Bradwell AR. Serum free light-chain measurements for identifying and monitoring patients with nonsecretory multiple myeloma. Blood. 2001;97(9): 2900-2902.
6. Bradwell AR, Carr-Smith HD, Mead GP, Harvey TC, Drayson MT. Serum test for assessment of patients with Bence Jones myeloma. Lancet. 2003;361:489-491.
7. Lachmann HJ, Gallimore R, Gillmore JD, et al. Outcome in systemic AL amyloidosis in relation to changes in concentration of circulating immunoglobulin light chains following chemotherapy. Br J Haematol. 2003;122(1):78-84.
8. Abraham RS, Katzmann JA, Clark RJ, Bradwell AR, Kyle RA, Gertz MA. Quantitative analysis of serum free light chains: A new marker for the diagnostic evaluation of primary systemic amyloidosis. Am J Clin Pathol. 2003; 119(2):274-278.
