Using Single Protein Biomarkers to Predict Health and Disease in Diverse Patient Populations: A New Role for Assessment of Immunoglobulin Free Light Chains

In the current issue of Mayo Clinic Proceedings, Dispenzieri and colleagues¹ describe the association between serum concentrations of immunoglobulin free light chain (FLC) and all-cause mortality in a normal population. The possibility that a single biomarker could have descriptive and predictive value for death from apparently diverse disease processes in an entire patient population is intriguing and is an extension of a long history of biomarker use to predict specific diseases in more highly selected populations.

Bence Jones Proteinuria as a Biomarker for Myeloma in the 19^th and 20^th Centuries

In the journal Lancet in 1847, Henry Bence Jones² described the characteristics of a protein in urine from a patient (of Drs Watson and Macintyre) who suffered with mollities and fragilitas ossium. Between 1897 and 1903, Drs T. R. Bradshaw and F. Parkes Weber increased and summarized the number of internationally published cases of “multiple myeloma (myelomatosis) with Bence-Jones proteid in the urine” [sic] from 6 to 34 cases.³ The development of serum protein electrophoresis in the 1950s enabled the identification of a band of protein of homogeneous isoelectric point (M protein) in the serum of many patients with myeloma. In the 1960s, Bence Jones protein was identified as FLC in the urine. This urinary FLC and the light and heavy chains bound into whole immunoglobulin (M protein) in serum were also then shown to be the products of the myeloma clone of plasma cells.^4,5

Between 1980 and 2002, the United Kingdom Medical Research Council multiple myeloma trials enrolled 2230 patients with either an IgG or IgA M protein in serum, and 72% of these patients also had FLC in urine.⁶ A further 361 patients had FLC in urine with no serum M protein (FLC-only myeloma [FLCO], often called Bence Jones myeloma). In many myeloma patients, their FLC are nephrotoxic, and in these United Kingdom trials, the incidence of renal impairment increased with levels of FLC in the urine. Ninety percent of the FLCO patients had lytic bone disease, 65% had renal impairment, and 45% had anemia. At diagnosis, FLCO patients were younger, had worse performance status, and had more lytic lesions than those patients with a serum M protein.⁶ It was postulated that these differences reflected delayed diagnoses in younger and missed diagnoses in older FLCO patients because serum M proteins were identified more readily than Bence Jones proteinuria (particularly when urine was not sent to the laboratory).

The Nature of FLCs

Immunoglobulins are composed of 2 identical heavy chains and 2 identical light chains. The light chains are either κ encoded on chromosome 2 or λ encoded on chromosome 22. Heavy chains are encoded on chromosome 14 by a cluster of immunoglobulin heavy chain C-region genes for the production of the 5 classes and subclasses of immunoglobulin that are IgM, IgD, IgG1-4, IgA1-2, and IgE.⁷

During response to antigen, a naïve B lymphocyte can switch from its original production of IgM and IgD to any of the other heavy chain isotypes. In contrast, the selection of light chain (κ or λ) is retained for the life of that B cell, all of its progeny (clone), and terminally differentiated plasma cells. B cells and immunoglobulin-secreting plasma cells manufacture nearly twice as many light chains in their cytoplasm as heavy chains, which prevents toxicity to the cell from aggregation of free heavy chain.^8,9 Both normal and neoplastic plasma cells secrete both whole immunoglobulin and FLC.

Whole immunoglobulin and FLC are secreted from hundreds of millions of clones of plasma cells in response to hundreds of millions of different antigens and spontaneously (natural antibody). These plasma cells are found in the medullary cords of lymph nodes and the red pulp of the spleen (most secreting IgM), the bone marrow (IgG, IgA, IgD, and IgE), and the mucosa (IgA). Secretion of κ and λ FLC by the total body plasma cell pool is about 1 g/d.⁸ These FLCs are mostly cleared through the renal glomeruli, with a serum half-life of 2 to 4 hours. Free light chains are not detectable in the urine of healthy individuals because they are metabolized in the proximal tubules of the nephrons.

Laboratory Detection of FLCs in Urine and Serum

Free light chains are detected by electrophoresis of concentrated urine followed by immunofixation to confirm that detected protein bands are κ or λ FLC. Quantification of urinary total protein and FLC excretion can be performed by densitometer tracing on a 24-hour urine collection or calculated in relationship to the urine creatinine on a random urine sample. A neoplastic clone of plasma cells must secrete more than 20 g of FLC per day to saturate FLC absorption in the proximal renal tubules of healthy kidneys and thus become detectable in urine. Accordingly, it is preferable to assess FLC secretion by measurement of FLC in serum, not urine; however, clinical laboratory tests for this purpose did not become available until 2002. This is because it is difficult to quantitate serum FLC (SFLC) levels (mg/L) in the presence of a 1000-fold greater concentration of light chain bound in whole serum immunoglobulin. Antibodies to detect FLC must have great specificity for epitopes that are exposed on FLC, hidden on light chain bound into whole immunoglobulin, and present on FLC from all patients.

Published methods for measuring SFLC levels with polyclonal antisera have been available since 1975 and those using monoclonal antibodies since 1983. In 2001, immunoassays using latex particle–conjugated sheep polyclonal antisera to detect either κ or λ SFLC became available (Freelite; Binding Site, Birmingham, England).¹⁰ The central 95% reference intervals of normal SFLC levels are 0.33-1.94 mg/dL for κ and 0.57-2.63 mg/dL for λ light chains, with a 100% κ to λ ratio range of 2.6 to 1.65 mg/dL (to convert to mg/L, multiply by 10). Serum FLC levels (but not ratio) increase up to 20-fold with reduced glomerular filtration. In persons aged 50 years or older, SFLC levels (but not ratio) increase with age, but this is a function of reduced glomerular filtration. A neoplastic clone of plasma cells only has to secrete 1 g/d of monoclonal FLC (either κ or λ) to perturb the normal SFLC κ to λ ratio. The greater sensitivity of serum vs urine FLC assays for the detection of myeloma was first demonstrated by the detection of abnormal SFLC κ to λ ratios in patients classified as nonsecretory because neither M protein nor FLC could be detected in serum or urine by the criterion standard sensitive test, immunofixation.¹¹ A quarter of all patients with myeloma who have a serum M protein with no detectable FLC in urine are found to have abnormal SFLC κ to λ ratios. Serum FLC assays have been shown to be more sensitive than urine values for measuring FLC response and detecting FLC relapse.¹²

SFLC as a Biomarker in the 21^st Century

Serum FLC assays are now established worldwide for diagnosis, prognostication, and monitoring of all plasma cell dyscrasias and are particularly useful in oligosecretory and FLCO myeloma and in light chain amyloidosis.¹³ In their article in this issue of Mayo Clinic Proceedings¹ and in other seminal papers, Dispenzieri and colleagues summarize how the SFLC assay provides prognostic information in other clonal B-lymphoid diseases including monoclonal gammopathy of undetermined significance, chronic lymphocytic leukemia, and non-Hodgkin lymphoma.¹⁴

An abnormal SFLC κ to λ ratio is pivotal to detecting and monitoring neoplastic B-lymphoid and plasma cell expansions. An abnormal SFLC κ to λ ratio is also found in association with immune dysregulation in which oligoclonal B-cell expansions occur, including in patients with human immunodeficiency virus infection and patients who have received hematopoietic stem cell rescue.

A normal SFLC κ to λ ratio indicates that the FLCs are the product of the many millions of normal plasma cell clones with no important contribution from a neoplastic expansion. Reduced SFLC levels with normal κ to λ ratios are found in patients with antibody deficiency, both primary and secondary to causes other than B-cell neoplasia.

Elevated SFLC levels (2- to 20-fold) with normal κ to λ ratios are found in patients with increased immunoglobulin production as a result of bacterial and viral infection and autoimmune disease. Similar elevation of SFLC levels with normal κ to λ ratios are found in patients with normal immunoglobulin production but reduced FLC clearance through reduced glomerular filtration. In 1394 patients with chronic kidney disease and in some other patient groups, elevated levels of polyclonal FLC has been shown to be prognostic for change in glomerular filtration rate, all-cause mortality, and mortality from cardiovascular disease, infections, and cancer (Colin Hutchison, MD, PhD, written communication, January 2012). Elevated levels of polyclonal FLC are prognostic for developing non-Hodgkin lymhoma in patients with human immunodeficiency virus infection, for short event-free survival in patients with non-Hodgkin lymphoma, and for inferior survival in patients with myeloid neoplasms.¹

Excluding persons with plasma cell disorders, Dispenzieri and colleagues¹ measured SFLC levels in a cohort of 15,859 Olmsted County residents (median age, 63 years; range, 50-109 years). In subsequent follow-up (median, 12.7 years), 4348 subjects had died. Compared with the other subjects, those with the highest-decile SFLC levels had a risk ratio of 4.4 for death. Multivariate analysis showed this excess risk was still present after correction for age, gender, and renal insufficiency measured by serum creatinine with a relative risk for death of 2.1. The increased mortality risk existed for most causes including the commonest: circulatory, neoplasms, respiratory, mental, nervous system, and endocrine.

Why are elevated SFLC levels associated with a worse outcome for nearly all cause-of-death categories in the few years following diagnosis? In the inflammatory and innate immune systems, increased blood levels of C-reactive protein and white blood cells are both associated with increased cardiovascular and cancer deaths. Increased lymphocyte counts are associated with increased cardiovascular and all-cause mortality in Vietnam veterans,¹⁵ as are increased serum immunoglobulin levels (Douglas Carroll, PhD, and Anna C. Phillips, PhD, written communication, February 2010). It remains to be discovered which plasma cells are responsible for the increased FLC secretion that portends earlier death, whether that is associated with increased secretion of the different classes of whole immunoglobulin, and what the stimuli to increased secretion are. The excess mortality found in the 10% of people with the highest SFLC levels is comparable to that of many common diseases in which therapy can improve survival, but we do not know to what extent the high SFLC levels are causal or just associated with increased mortality. We do not know whether those elevated SFLC levels are derived from marrow, mucosa, or lymphoid plasma cells, which whole immunoglobulin types they are secreted with, or the stimulus to that secretion. Answers to these questions may provide insight into common disease processes and perhaps new treatment strategies.