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Editorials |
Department of Immunity and Infection, The Medical School, University of Birmingham, Birmingham B15 2TT, United Kingdom, and, The Binding Site Ltd., Birmingham B14 4ZB, United Kingdom, E-mail A.R.BRADWELL{at}bham.ac.uk
For more than 150 years, the presence of Bence Jones protein [immunoglobulin free light chains (FLCs)] in the urine has been an important diagnostic marker for multiple myeloma. Indeed, it was the first cancer test, and 100 years before any others (1). Over the last few years, however, interest in FLCs has undergone a renaissance. Development of serum tests for free 
and free 
has opened the door to new applications and increased their clinical importance (2). By way of comparison, the management of diabetes mellitus was hugely improved when blood replaced urine for glucose analysis.
The report by Katzmann et al. (3) in this issue of Clinical Chemistry adds valuable confirmatory data on serum FLC testing. It is the first report of the assays being used in routine clinics with analysis of results on 1020 samples. As the authors point out, the performance of the tests has matched up to the retrospective studies that have been published previously.
From a physiologic viewpoint, blood tests for small proteins have clear advantages over urine tests. Serum FLCs are cleared rapidly through the renal glomeruli with a serum half-life of 2–6 h and are then metabolized in the proximal tubules of the nephrons. Under ordinary circumstances, little protein escapes to the urine (4), and serum FLC concentrations have to increase manyfold before the absorption mechanisms are overwhelmed. This makes urinalysis a fickle witness to changing FLC production. Conversion to a serum test provides clarity in assessing disease processes that were previously hidden from view.
Serum concentrations of FLCs are dependent on the balance between production (by plasma cells and their progenitors) and renal clearance. When there is increased polyclonal immunoglobulin production and/or renal impairment, both and FLC concentrations can increase 10- to 20-fold. However, the relative concentration of to , i.e., the / ratio, remains unchanged. In contrast, tumors produce a monoclonal excess of only one of the light chains, often with bone marrow suppression of the other light chain, so that / ratios become highly abnormal. Accurate measurement of / ratios underpins the utility of the serum FLC immunoassays and provides a numerical indicator of clonality (5). Urine / ratios are not as dependable because the nontumor light chain production is too low to pass consistently through the nephrons. Electrophoretic tests are used only to quantify the monoclonal light chain peak because they are not sensitive enough to identify the nontumor FLC concentrations.
Early clinical studies with serum FLC tests were in patients with Bence Jones (light chain) multiple myeloma. In two studies, on 270 sera taken at the time of clinical presentation, highly abnormal serum FLC concentrations were found in every case (6)(7). Furthermore, during chemotherapy, urine tests frequently normalized, whereas serum tests remained abnormal, indicating their increased sensitivity for residual disease. In this patient group, urinalysis can now be replaced by serum FLC tests. This is particularly helpful for frail, elderly patients because 24-h urine samples are difficult to collect and results may be unreliable (8).
Of patients with multiple myeloma, 3–4% have so-called nonsecretory disease. By definition, these patients have no monoclonal proteins by serum and urine electrophoretic tests. Nevertheless, in a study by Drayson et al. (9), serum FLC tests identified monoclonal proteins in 70% of 28 patients. The current study by Katzmann et al. (3) found that all five patients with nonsecretory myeloma had abnormal FLC concentrations. It is apparent that these patients’ tumor cells produce small amounts of monoclonal protein. Their serum FLC concentrations are below the detection limits of serum electrophoretic tests and below the threshold for clearance into the urine. Importantly, these patients can now be closely monitored by serum FLC tests rather than repeated bone marrow biopsies or whole-body scans.
Approximately 20% of all patients with myeloma have light chain or nonsecretory disease. Among the remaining patients who produce intact monoclonal immunoglobulins, FLCs are abnormal in 96% at disease presentation (10). Interestingly, the serum concentrations of FLCs and intact monoclonal immunoglobulins are not correlated (R <0.02). Monoclonal serum FLCs are, therefore, independent markers of the disease process. This is of potential clinical use when the tumor produces large amounts of FLCs and small amounts of intact monoclonal immunoglobulins. Patients who are in apparent remission, as judged by study of their intact monoclonal immunoglobulins, may still have monoclonal FLCs, indicating residual disease. Using a similar argument, when these patients relapse, FLC concentrations may increase first. FLC "breakthrough" is thought to occur in 2–5% of patients who relapse after modern, intensive treatment.
An additional feature of serum FLCs is that, in contrast to intact immunoglobulin molecules, they are potentially nephrotoxic. In many patients with intact monoclonal immunoglobulins, the serum FLC concentrations are >1000 mg/L (50–100 times the upper limit of the reference interval). This is characteristic of patients with IgD multiple myeloma, but is also apparent in 5–10% of IgG- and IgA-producing patients. The FLC assays now allow assessment of the prerenal load of monoclonal light chains. There is early evidence that in some patients, treatment should be aimed at normalizing serum FLC concentrations to prevent renal damage (Nowrousian MR, et al. Using serum free light chain assays in the myeloma clinic, submitted for publication).
One particularly interesting aspect of serum FLCs involves their short half-lives in the blood: 2–3 h for , and 5–6 h for . This is 
150 times shorter than the 21-day half-life of IgG molecules. Hence, responses to treatment are seen in "real time". This is apparent from the good correlation between bone marrow assessment of disease status and FLC concentrations but poor correlation with IgG concentrations (11). Thus, FLC concentrations allow more rapid assessment of the effects of chemotherapy than does monoclonal IgG. The impact of this is likely to be considerable. For example, the resistance of patients to particular drugs or drug combinations can be observed quickly and alternative treatments chosen. The short half-lives of FLCs may also allow distinction between partial and complete tumor responses before stem cell transplantation. This is typically performed several months after the start of induction chemotherapy. The 21-day half-life of IgG hides complete responses, whereas FLC analysis should allow more accurate assessments.
Serum FLC tests are also having considerable impact in AL (primary) amyloidosis. Characteristically, light chain fibrils are deposited in various organs and tissues and lead directly to disease. The origin of the fibrils is monoclonal FLCs produced by a slowly growing clone of plasma cells. Concentrations are usually insufficient for measurement by serum electrophoretic tests. However, serum FLC assays provide quantification of the circulating fibril precursors in 90–95% of patients (12)(13). Furthermore, the tests allow assessment of treatment responses and disease relapses that, in turn, correlate with survival. As recently stated by Dispenzieri et al., "The introduction of the serum immunoglobulin free light chain assay has revolutionized our ability to assess hematological responses in patients with low tumor burden" (14).
Additional support for a role of serum FLCs in AL amyloidosis is given by Katzmann et al. (3). The combination of serum FLC and serum immunofixation electrophoretic tests identified 109 of 110 patients at diagnosis. The FLC analysis alone identified 91% of the patients, whereas immunofixation electrophoresis identified only 69% and urinalysis failed to identify the sole patient who was normal by both serum tests. A similar high sensitivity for the FLC assays has been found in light chain deposition disease (3)(5).
The new international consensus for the management of AL amyloidosis includes use of serum FLC measurements (15). Reduction of the / ratio to normal, alongside the intact monoclonal immunoglobulins, will become the benchmark for complete serologic responses to therapy in AL amyloidosis and multiple myeloma.
An emerging role of serum FLC analysis is for assessing the risk of progression in individuals with monoclonal gammopathies of undetermined significance (MGUS). These are precancer markers, and patients progress to multiple myeloma, AL amyloidosis, or other plasma cell dyscrasias at a rate of 1% per year. Rajkumar et al. (16) recently showed that the presence of an abnormal serum FLC / ratio is a major independent risk factor for progression. In particular, the 50% of MGUS patients with low concentrations of M-spike (<15 g/L) and normal / ratios had a sevenfold lower risk of progression than patients with an M-spike of >30 g/L and abnormal / ratios. It seems that the low-risk patients can be reassured about their disease and may not need to be monitored on a long-term basis.
The high sensitivity of serum FLC immunoassays for tumor detection suggests that they have a role in screening for plasma cell dyscrasias. Currently, symptomatic patients are assessed by use of serum and urine protein electrophoretic tests. Because urine is frequently unavailable, it is logical to add serum FLC analysis to existing test protocols. In a study of 1003 consecutive samples by Bakshi et al. (17), serum FLC analysis identified an additional 16 patients with monoclonal proteins in addition to the 39 detected by serum capillary zone electrophoresis. B-Cell/plasma cell tumors were present in 9 of the 16, including 3 with light chain myeloma. These first results indicate that the combination of serum protein electrophoresis and FLC analysis is a clinically sensitive strategy for identifying patients with monoclonal gammopathies. Adding serum immunofixation electrophoresis is of modest extra clinical consequence. Katzmann et al. (3) showed that there was an additional AL amyloidosis detection rate of 8%. However, this is a rare disease. If the choice is between serum FLCs and serum or urine immunofixation electrophoresis, then FLC tests are more useful.
FLC concentrations have been assessed in cerebrospinal fluid (CSF). In a study by Fischer et al. (18), concentrations provided information comparable to oligoclonal band measurements. They concluded that CSF FLC measurements may be a useful diagnostic procedure for detecting, and potentially monitoring, intrathecal immunoglobulin synthesis.
Is there a remaining role for urine FLC analysis? The answer is a qualified yes. When both serum and urine tests are available, it is always clinically reassuring when different tests provide similar results. In addition, samples do occasionally get incorrectly analyzed, mislabeled, or misplaced; therefore, additional evidence for making a diagnosis or changing treatment is always helpful. Moreover, there are rare patients who have normal serum FLCs but low concentrations of monoclonal proteins in the urine, although the clinical relevance of the urinary finding is doubtful.
In summary, serum FLC tests are assuming an increasing role in the detection and monitoring of monoclonal gammopathies. This new approach is bringing benefits to many patients with plasma cell dyscrasias.
Acknowledgments
The author is a director and major shareholder in The Binding Site Ltd., which manufactures and distributes serum FLC assays.
References
and free light chain assays in clinical practice. Clin Chem 2005;51:878-881. and free immunoglobulin light chains: relative sensitivity for detection of monoclonal light chains. Clin Chem 2002;48:1437-1444.The following articles in journals at HighWire Press have cited this article:
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  M. M. Giarin, L. Giaccone, R. Sorasio, C. Sfiligoi, B. Amoroso, F. Cavallo, A. Cipriani, A. Palumbo, and M. Boccadoro Serum Free Light Chain Ratio, Total {kappa}/{lambda} Ratio, and Immunofixation Results Are Not Prognostic Factors after Stem Cell Transplantation for Newly Diagnosed Multiple Myeloma Clin. Chem., August 1, 2009; 55(8): 1510 - 1516. [Abstract] [Full Text] [PDF]
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 P. Zhou, J. Teruya-Feldstein, P. Lu, M. Fleisher, A. Olshen, and R. L Comenzo Calreticulin expression in the clonal plasma cells of patients with systemic light-chain (AL-) amyloidosis is associated with response to high-dose melphalan Blood, January 15, 2008; 111(2): 549 - 557. [Abstract] [Full Text] [PDF]
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 M. A. Gertz, M. Q. Lacy, A. Dispenzieri, S. R. Hayman, S. K. Kumar, N. Leung, and D. A. Gastineau Effect of hematologic response on outcome of patients undergoing transplantation for primary amyloidosis: importance of achieving a complete response Haematologica, October 1, 2007; 92(10): 1415 - 1418. [Abstract] [Full Text] [PDF]
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 J. A. Katzmann, A. Dispenzieri, R. A. Kyle, M. R. Snyder, M. F. Plevak, D. R. Larson, R. S. Abraham, J. A. Lust, L. J. Melton III, and S. V. Rajkumar Elimination of the Need for Urine Studies in the Screening Algorithm for Monoclonal Gammopathies by Using Serum Immunofixation and Free Light Chain Assays Mayo Clin. Proc., December 1, 2006; 81(12): 1575 - 1578. [Abstract] [Full Text] [PDF]
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J. A. Katzmann Serum Free Light Chain Specificity and Sensitivity: A Reality Check Clin. Chem., September 1, 2006; 52(9): 1638 - 1639. [Full Text] [PDF]
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P. G. Hill, J. M. Forsyth, B. Rai, and S. Mayne Serum Free Light Chains: An Alternative to the Urine Bence Jones Proteins Screening Test for Monoclonal Gammopathies Clin. Chem., September 1, 2006; 52(9): 1743 - 1748. [Abstract] [Full Text] [PDF]
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 A. M. S. Muller, A. Geibel, H. P. H. Neumann, A. Kuhnemund, A. Schmitt-Graff, J. Bohm, and M. Engelhardt Primary (AL) Amyloidosis in Plasma Cell Disorders Oncologist, July 1, 2006; 11(7): 824 - 830. [Abstract] [Full Text] [PDF]
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