Serum Free Light Chain (FLC) Analysis: A Guiding Light in Monoclonal Gammopathy Management

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1 Serum Free Light Chain (FLC) Analysis: A Guiding Light in Monoclonal Gammopathy Management Ellen L. Jenner, 1 Josie A.R. Evans, 1 and Stephen J. Harding 1 * Background: Monoclonal free light chains (FLCs) were first reported by Dr. Henry Bence Jones over 150 years ago in the urine of patients with multiple myeloma. Now established as important tumor markers, they aid not only in the diagnosis of monoclonal gammopathies but also in their clinical management by indicating the response to treatment and persistence of residual disease. Content: A particular focus over the past 15 years has been on the replacement of urine with serum analysis for monoclonal FLC measurement. Because of the limited sensitivity and practical constraints of urine assessment, a combination of serum electrophoresis and serum FLC analysis has been adopted by many laboratories as a first-line screen for patients with a suspected monoclonal gammopathy. Early myeloma diagnosis may translate into improved clinical outcomes, and a new study, istopmm, is underway to ascertain the benefit of populationwide screening protocols for early detection of the disease in its asymptomatic phase. Laboratory algorithms that include measurement of both monoclonal intact immunoglobulins and FLCs are important for assessing possible changes in myelomic clones in response to treatment, and recent data from Intergroupe Francophone du Myelome trials validate serum FLC as a clinically relevant disease biomarker. Whether sensitive serum techniques such as FLC analysis can be used to guide the use of more invasive procedures for detection of minimal residual disease is the subject of emerging studies. Summary: Here we review the current and evolving utility of serum FLC measurements for the management of patients with monoclonal gammopathies. IMPACT STATEMENT Serum free light chain (FLC) analysis represents a major advance in laboratory diagnostics. Greater sensitivity, compared to urine analysis, has heralded the widespread use and incorporation of serum FLC measurements into multiple guidelines for the management of myeloma, most recently as a myeloma defining event in asymptomatic patients. There has been much interest in recent years in the clonal heterogeneity of myeloma, and changes in serum FLCs can provide insight into plasma cell tumor biology. Moreover, new data have emerged which validates serum FLC as a clinically relevant biomarker for monitoring treatment response. 1 The Binding Site Group, Birmingham, UK. *Address correspondence to this author at: The Binding Site Group Ltd., 8 Calthorpe Rd., Edgbaston, Birmingham B15 1QT, UK. Fax ; stephen.harding@bindingsite.co.uk. DOI: /jalm American Association for Clinical Chemistry July : JALM 1 Copyright 2017 by American Association for Clinical Chemistry.

2 Serum FLCs in Monoclonal Gammopathy Management Monoclonal gammopathies are a diverse group of disorders generally characterized by the secretion of monoclonal immunoglobulins or their light-chain components. They range from malignant diseases, such as multiple myeloma (MM), 2 Waldenström macroglobulinemia, plasma cell leukemia, and amyloid light-chain (AL) amyloidosis, to precursor diseases such as smoldering MM and monoclonal gammopathy of undetermined significance (MGUS). The latter was once thought to be a benign entity that rarely transformed into malignancy; however, risk stratification models including serum free light chain (FLC) analysis now identify a subset of patients at high risk of malignant transformation. Laboratory methods to screen for monoclonal gammopathies historically comprise serum protein electrophoresis (SPE) for the detection of monoclonal intact immunoglobulins and urine protein electrophoresis (UPE) for the detection of monoclonal FLCs. However, the role of urine electrophoresis has come under increasing scrutiny since the introduction of highly sensitive assays that measure FLCs in the serum. Here we review the role of FLC testing in patients with monoclonal gammopathies and consider how this may evolve. POLYCLONAL SERUM FLC ASSAYS Immunoglobulins comprise 2 identical heavy chains and 2 identical light chains, and each of the 4 polypeptides are formed of 2 domains: one that is constant and one that is variable. Two serologically distinct light-chain types are recognized: κ and λ, which are encoded on chromosomes 2 and 22, respectively. At each κ or λ locus, a large number of V and J gene segments encode each protein; the combination of individual V and J segments contributes to the enormous genetic diversity in light-chain sequences in different B-cell clones. Further, allotypic, isotypic, and idiotypic variability adds to the amino acid diversity of these structures. Heavy-chain genetics is similarly complex, and a unique immunoglobulin is formed by the pairing of unique heavy- and light-chain molecules. The vast repertoire of B-cell clones, each expressing a unique immunoglobulin, has been estimated to equate to individual immunoglobulins (1). During immunoglobulin synthesis by plasma cells, an excess of FLC molecules are produced and these are released, along with the intact immunoglobulin molecule. These FLC molecules that remain unbound to heavy chains are the target of the Freelite immunoassays (The Binding Site Group). To recognize the considerable diversity in κ and λ light chains, Freelite immunoassays are based on polyclonal antisera, directed at epitopes found on the constant domain of κ or λ light chains, which are only exposed in unbound light-chain molecules. Latex enhancement increases the assay sensitivity to a few milligrams per liter. Polyclonal antisera reliably detect the full range of epitopes present in patients with monoclonal gammopathies, as demonstrated by their ability to correctly detect monoclonal FLCs in all light-chain MM patients (805/805) reported in more than 16 independent studies over the past 15 years. This result is in contrast to other commercial assays based on monoclonal antisera that reportedly miss a number of patients (2 5). The polyclonal FLC assays are available across a number of turbidimetric and nephelometric automated laboratory instruments. For each platform, assays undergo extensive validation according to CLSI guidelines, before they are judged suitable for routine laboratory use. There have been limited cross-analyzer comparison studies to date. In one study, Matters et al. (6) showed reasonable concordance across the platforms. Freelite assays were calibrated to the same primary standard, and 2 Nonstandard abbreviations: MM, multiple myeloma; AL, amyloid light-chain; MGUS, monoclonal gammopathy of undetermined significance; FLC, free light chain; SPE, serum protein electrophoresis; UPE, urine protein electrophoresis; IFE, immunofixation; NICE, National Institute for Health and Care Excellence; IMWG, International Myeloma Working Group; CR, complete remission; IFM, Intergroupe Francophone du Myelome; MRD, minimal residual disease; HLC, heavy-light chain. 2 JALM :01 July 2017

3 Serum FLCs in Monoclonal Gammopathy Management MINI-REVIEW as part of the routine validation, healthy samples were run to confirm the healthy range [published by Katzmann et al. in 2002 (7)]. However, it is advisable to rebaseline patients when moving from one platform to another. In addition, when interpreting patient results, it is important to consider the biological variability of serum FLC measurements, which was reportedly 28% in a 5-year follow-up study of patients with stable monoclonal disease (8). SCREENING FOR MONOCLONAL GAMMOPATHIES Serum electrophoretic techniques that are routinely used to identify monoclonal intact immunoglobulins have inadequate sensitivity for identifying monoclonal FLCs. As such, UPE and urine immunofixation (IFE) have historically been performed alongside SPE and serum IFE for identifying immunoglobulin light-chain disorders, but many patients with nonsecretory MM, AL amyloidosis, and other FLC-associated disorders are still missed. This finding should not be surprising considering that FLCs are rapidly cleared from the blood and metabolized by the kidneys. The reabsorption process in the proximal tubule is usually extremely efficient, and the amount of FLCs in urine is more dependent upon renal function than synthesis by the tumor, even for some myeloma patients for whom FLC production is considerably increased compared to healthy controls. In a hypothetical patient producing monoclonal FLCs, we can envisage that, from low initial starting concentrations, FLCs in the serum increase steadily with growing tumor mass, while concentrations in the urine show little change until the proximal tubular metabolism is exceeded and overflow proteinuria develops. Hence, early disease and oligosecretory disease may not be identified by urine tests. Before discussing the merits of replacing urine electrophoresis with the serum FLC assay further, it is worth commenting on the poor patient compliance documented for providing a urine sample, which has ranged from 5% to 52% across different studies (9 13). Overall, urine compliance rates were just 14% in a UK pathology review (9); without a serum FLC assessment, 86% of patients would have been evaluated by SPE alone, with the potential to miss monoclonal FLCs. The issue of urine availability was fundamental to the recent update in guidelines from the National Institute for Health and Care Excellence (NICE) (14). The revision notes that failure to obtain a urine sample could result in potential missed diagnoses if the serum FLC test was not performed. Thus, serum testing is far more convenient and urine testing is not included in the NICE recommendations for monoclonal protein screening. The largest study to date that has compared the relative diagnostic contributions of serum and urine assays in the detection of monoclonal gammopathies was reported by Katzmann et al. (15). Samples from 1877 untreated patients diagnosed with various plasma proliferative diseases underwent a full panel of 5 screening tests (SPE, serum IFE, UPE, urine IFE, and serum FLCs). This step allowed different screening protocols to be compared and addressed the question of whether serum FLC analyses could replace urine studies. The highest diagnostic sensitivity was achieved by use of all available tests: this approach identified 98.6% of all gammopathies, but still a small number of AL amyloidosis, plasmacytoma, and almost all extramedullary plasmacytoma patients were not detected. A simplified algorithm of SPE and serum FLC testing yielded comparable sensitivity overall at 94.3%, and proved an extremely efficient initial diagnostic screen for monoclonal gammopathies with a high tumor burden such as MM (100% sensitivity), Waldenström macroglobulinemia (100% sensitivity), and smoldering MM (99.5% sensitivity). AL amyloidosis presents a particular diagnostic challenge, since tumor burden is low and renal damage is a common occurrence. When renal reabsorption is compromised, small quantities July : JALM 3

4 Serum FLCs in Monoclonal Gammopathy Management Table 1. Definition of myeloma-defining events (17). a MDEs b Evidence of end-organ damage that can be attributed to the underlying plasma cell proliferative disorder Any 1 or more of the following biomarkers of malignancy Details Hypercalcemia: serum calcium >0.25 mmol/l (>1 mg/dl) higher than the upper limit of the reference interval or >2.75 mmol/l (>11 mg/dl) Renal insufficiency: creatinine clearance <40 ml/min or serum creatinine >177 μmol/l (>2 mg/dl) Anemia: hemoglobin value of >2 g/dl below the lower limit of the reference interval, or a hemoglobin value <100 g/l Bone lesions: 1 or more osteolytic lesions on skeletal radiography, computerized tomography, or positron emission tomography/computerized tomography Clonal bone marrow plasma cell percentage 60% Involved/uninvolved serum FLC ratio 100 c >1 focal lesion on MRI studies a A diagnosis of MM requires at least 10% clonal plasma cells in the bone marrow or biopsy-proven bony or extramedullary plasmacytoma and 1 or more MDE. b MDE, myeloma-defining event. c The iflc must be 100 mg/l. These values are based on the serum Freelite assays. of monoclonal light chains may pass into the urine, and serum testing may no longer be the most sensitive approach. In cases of suspected AL amyloidosis, the International Myeloma Working Group (IMWG) (16) recommend that a combination of serum electrophoresis and serum FLC testing should be used alongside urine IFE. Until recently, the diagnosis of MM was solely based on the presence of excess monoclonal plasma cells in the bone marrow alongside related organ or tissue impairment (i.e., hypercalcemia, renal insufficiency, anemia, or bone lesions, collectively known as the CRAB criteria). However, guidelines (17) now define additional biomarkers of malignancy (also known as SLiM criteria) that include serum FLCs far outside the reference interval (defined as an involved/uninvolved Freelite serum FLC ratio 100) as part of the diagnostic criteria (Table 1). This revision has significant implications, since asymptomatic patients with active disease may now be considered for treatment, before the onset of potentially irreversible end-organ damage. Work by Mateos et al. (18) suggests that the treatment of high-risk asymptomatic patients leads to a decreased rate of malignant transformation and an improvement in overall survival. Furthermore, early diagnosis results in fewer complications at presentation of symptomatic disease (19). Can serum FLC testing ever become part of a routine diagnostic screen to detect early MM in asymptomatic individuals? Almost 50 years ago, Wilson and Jungner (20) published criteria to guide the selection of suitable conditions for populationwide screening, which are still considered the gold standard today. Factors included the following: importance of the health problem, effectiveness and accessibility of diagnostic tests and treatments, and economic implications. Ultimately, the overall benefits should outweigh the harm. In favor of this approach for monoclonal gammopathies, an effective diagnostic screen was developed, and asymptomatic patients are likely to benefit from earlier intervention. However, these benefits need to be weighed against potential concern for false-positive results associated with overdiagnosis of MGUS and with the costs of a population-wide screening approach. Will such an approach for monoclonal gammopathies fulfill Wilson and Jungner's criteria? A new study, istopmm, headed by Prof. Sigurdur 4 JALM :01 July 2017

5 Serum FLCs in Monoclonal Gammopathy Management MINI-REVIEW Fig. 1. Changes in monoclonal protein type at relapse or progression of disease (POD) in patients who achieved a complete response. Figure was generated using published data (23). Kristinsson, will address this question by screening the Icelandic population over 40 years old; more information on this effort can be found at (accessed May 8, 2017). Unfortunately, we will have to wait approximately 5 years for the answer. AN INDEPENDENT MARKER OF RESPONSE Over 40 years ago, the phenomenon of lightchain escape was first characterized by J.R. Hobbs. He described 15 myeloma patients at disease relapse in which Bence Jones proteinuria dramatically increased without a rise in the serum intact immunoglobulin concentration (21). In a larger, recent study of 520 myeloma patients at relapse, Brioli et al. (22) reported that 183 individuals (35%) had a significant increase in intact immunoglobulin and serum FLC concentrations, 258 (50%) had an increase in intact immunoglobulin only, and 54 (10%) had light-chain escape (25 patients had relapse detected clinically). Similar patterns of serum protein changes were reported elsewhere (23) (Fig. 1). Over the past decade, the cellular basis underlying the changes in monoclonal protein production has become better understood. First, Ayliffe et al. (24, 25) challenged the perception that a patient's disease is the result of a single tumor clone. Using double immunofluorescence staining to study immunoglobulin heavy-chain and light-chain expression by bone marrow tumor cells, they found dual clonal plasma cell populations in 18% of patients, which expressed FLCs with or without immunoglobulin heavy chains. These dual populations were the first indicators that, within a single patient, multiple clones may be present that express different types of monoclonal protein. Subsequently, using array comparative genomic hybridization and fluorescence in situ hybridization, Keats et al. (26) elegantly demonstrated that a large number of genetically distinct clones are present in an individual at diagnosis. Furthermore, the clonal composition may change during the course of the disease, giving rise to tides of myelomic clones that compete for dominance in a landscape that is continually changed by therapy. The presence of FLCs at relapse confers a poor outcome. Brioli et al. (22) found that myeloma patients who relapsed with an FLC alone or an FLC in association with an intact immunoglobulin had a significantly shorter overall survival after relapse (28 and 24 months, respectively) compared to those without a rising FLC at relapse (37 months). It has been suggested that this may be because monoclonal FLC production acts as a marker of a tumor clone with a more aggressive behavior. However, it is also worth exploring compelling data highlighting potential toxicity of FLCs (27). Urine electrophoresis is included in international guidelines to define response in light-chain MM patients (28). However, an increasing weight of evidence supports the replacement of urine with serum FLC analysis. The physiological reasons are well described; FLCs are small molecules that are readily cleared by the renal glomerulus and reabsorbed in the proximal tubule. Therefore, urine remains insensitive for detecting low concentrations of monoclonal FLCs. Also, urine is seldom favored July : JALM 5

6 Serum FLCs in Monoclonal Gammopathy Management by myeloma patients, because 24-h collections are cumbersome and particularly difficult to collect for frail patients. A number of publications have shown the relative insensitivity of urine electrophoresis compared to serum FLC measurements for monitoring disease. In an initial study of 82 light-chain MM patients by Bradwell et al. (29), 32% achieved complete remission (CR) according to urine results, while only 11% achieved normalization of their serum FLC ratios. Similar results have now been reported in 2 studies by the French Intergroupe Francophone du Myelome (IFM) (30, 31). In their most recent publication, Dejoie et al. (31) reported that the serum FLC ratio was outside the reference interval, and monoclonal FLCs were at a concentration deemed measurable for monitoring ( 100 mg/l), in all of their 111 light-chain MM patients at diagnosis. By contrast, urine BJP (Bence- Jones protein) concentrations were measurable in only 64% of cases. Both IFM studies showed that the serum FLC assay continued to be a more sensitive indicator of disease than urine analysis at clinically relevant time points during follow-up (Fig. 2). The most likely explanation is that urine assays underestimated the amount of FLC production (and overestimated the response to treatment) due to the renal metabolism of FLCs. The greater sensitivity afforded by serum FLC analysis also provides valuable prognostic information. After 3 cycles of treatment, light-chain MM patients whose serum FLC results remained outside the reference interval had a significantly worse outcome than patients whose results normalized (31). By contrast, a positive UPE or urine IFE did not provide prognostic information. The authors concluded that the improved sensitivity and prognostic value of serum over urine measurements provide a strong basis for recommending the former for monitoring light-chain MM patients. Moustafa et al. (32) extended these findings in patients with intact immunoglobulin MM; normalization of the serum FLC ratio conferred a more favorable outcome in intact immunoglobulin MM Fig. 2. The sensitivity of urine and serum FLC measurements at presentation and during response assessment. (A), Percentage of patients with increased serum involved FLC (iflc) and positive UPE. (B), Percentage of patients with measurable disease according to UPE ( 200 mg/24 h) and serum FLC (sflc) values (iflc 100 mg/l). Chart was generated using published data (31). patients, including those individuals who did not achieve a CR after therapy. These findings support the inclusion of serum FLC analysis in all levels of response criteria. GATEWAY TO MINIMAL RESIDUAL DISEASE ASSESSMENT Normalization of the serum FLC ratio has been included in the definition of a stringent CR for over a decade. The recommendation to include serum FLC analysis was initially based on the theoretical ability of the assays to provide a sensitive indicator 6 JALM :01 July 2017

7 Serum FLCs in Monoclonal Gammopathy Management MINI-REVIEW of clonality and provide a stricter indication of CR. In recent years, this has proven true, with strong evidence that supports a survival advantage for those patients who achieve a healthy serum FLC ratio (33 37). Novel therapies for MM have dramatically improved patient outcomes in recent years. With higher rates of CR comes the requirement for new methodologies to identify patients with better responses. Minimal residual disease (MRD) negativity is the ultimate aim of therapy, and the IMWG recently updated their consensus guidelines (38) to include new response categories for MRD assessment based on advanced imaging techniques, next-generation flow cytometry, and gene sequencing. There is a logical argument for using simple biochemical tests to guide when MRD assessment is required. A residual disease marker such as a serum FLC ratio outside the reference interval and/or heavy-light chain (HLC; Hevylite) ratio in patients achieving a CR may circumvent the requirement for invasive procedures, such as bone marrow biopsies, and advanced imaging such as MRI or positron emission tomography/computerized tomography scan. An initial prospective study by Campbell et al. (39) concluded that normalization of both HLC and FLC ratios serves as a useful surrogate marker for MRD negativity by flow cytometry and correlates with improved MM progression free survival. Moreover, Dejoie et al. (31) showed that all patients whose serum FLC ratio normalized after 1 and 3 cycles of therapy went on to achieve MRD negativity (by flow cytometry). The authors quoted that this further underlines the clinical relevance of this measurement and suggests that routine monitoring could be a useful aid in guiding treatment (31). In conclusion, since the introduction of serum FLC assays over 15 years ago, there has been mounting evidence to support their use in the management of a variety of monoclonal gammopathies. As a result, they are now in routine clinical use and are recommended in numerous clinical guidelines. In addition to the practical advantages of using serum samples rather than urine, the principle advantages of monitoring patients with serum FLC tests derives from their greater clinical sensitivity and prognostic utility, as demonstrated most recently by the IFM group. Considering the strength of data supporting the relationship between serum FLC assessment and clinical outcome, it seems reasonable to propose that, as experience and confidence in the serum FLC assays grows, they will increasingly replace urine assays for monitoring MM patients. Furthermore, new and interesting data evaluating the use of these simple, sensitive serum tests as a gateway to more complex and often invasive MRD assessment are also emerging; larger studies are required to further investigate this utility. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Employment or Leadership: E.L. Jenner, J.A.R. Evans, S.J. Harding, The Binding Site Group. Consultant or Advisory Role: None declared. Stock Ownership: None declared. Honoraria: None declared. Research Funding: None declared. Expert Testimony: None declared. Patents: None declared. Role of Sponsor: No sponsor was declared. July : JALM 7

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