Sharply Increased Serum Free Light-Chain Concentrations after Treatment for Multiple Myeloma

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1 Clinical Chemistry 56: (2010) Clinical Case Study Sharply Increased Serum Free Light-Chain Concentrations after Treatment for Multiple Myeloma Kazunori Murata, 1 Raynell J. Clark, 1 Karen S. Lockington, 1 Linda J. Tostrud, 1 Philip R. Greipp, 2 and Jerry A. Katzmann 1,2* CASE A 53-year-old woman presented to the orthopedic department with severe diffuse muscular and bone pain. An x-ray of her right upper extremity revealed a lytic destructive lesion in the right humerus. Computed tomography scans showed multiple lytic lesions in the spine and pelvis, and a biopsy confirmed the presence of 70% plasma cells, which were light chain restricted. The patient was referred to a hematologist. Although no monoclonal protein was detected in the serum by protein electrophoresis or by immunofixation electrophoresis (IFE), 3 the free light chain (FLC) was increased at 47.2 mg/l (reference interval, mg/l), with a / FLC ratio of 23 (reference interval, ). Serum concentrations of 2 - microglobulin and albumin were 248 nmol/l (reference interval, nmol/l) and 37 g/l (reference interval, g/l), respectively. The urine protein concentration was not increased, but protein electrophoresis revealed a small M (monoclonal) spike in the region (32 mg/24 h). In addition, IFE identified a monoclonal light chain (Bence Jones protein). On the basis of these findings, the patient was informed that she had stage I (International Staging System) oligosecretory/nonsecretory multiple myeloma (MM). The patient underwent surgical repair of her right humerus and was evaluated 1 month after her surgery. At that point, a second serum FLC measurement showed a FLC concentration of 68.2 mg/l. The patient s hematologist recommended close observation in lieu of initiating therapy because of her lack of symptoms. The patient s serum FLC concentration was monitored monthly and remained 50 mg/l for the next 3 months. Five months after diagnosis, the patient began to complain of mild fatigue, shortness of breath, and palpitations. A 10-fold increase in the urinary M QUESTIONS TO CONSIDER 1. Why was the patient s high serum FLC initially not detected by IFE? 2. What are the potential causes of increased FLC after treatment? 3. How can light-chain escape be differentiated from FLC antigen excess? protein to 333 mg/24 h was noted, along with a decrease in the blood hemoglobin concentration to 79 g/l (reference interval, g/l) and an increase in the calcium concentration to 2.80 mmol/l (reference interval, mmol/l). Her hematologist recommended initiation of treatment, and the patient was enrolled in a clinical trial protocol consisting of lenalidomide (25 mg/day) and dexamethasone (40 mg/ day) administered for 21 days of a 28-day cycle. At 1 month after initiation of treatment, the patient was noted to be tolerating the regimen well, with an increase in her hemoglobin to 91 g/l, a minimal decrease in serum IgG from a pretreatment value of 2.95 g/l to 2.71 g/l (reference interval, g/l), and a reduction in her serum creatinine concentration from 115 mol/l to 88 mol/l (reference interval, mol/l). The patient also stated that she felt less fatigued. Her serum FLC concentration, however, was inexplicably increased to 2180 mg/l (Fig. 1). By her next follow-up appointment a month later, the patient had completed 2 full cycles of the lenalidomide and dexamethasone regimen and showed evidence of continued response, as evidenced by a rise in the hemoglobin concentration to 105 g/l and a decrease in urinary protein excretion to 132 mg/24 h. The patient s serum FLC concentration remained increased at 1500 mg/l, however. DISCUSSION 1 Department of Laboratory Medicine and Pathology and 2 Division of Hematology, Mayo Clinic, Rochester, MN, MO. * Address correspondence to this author at: Division of Clinical Biochemistry and Immunology, Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st St. SW, Rochester, MN katzmann@mayo.edu. Received June 30, 2009; accepted October 1, DOI: /clinchem Nonstandard abbreviations: IFE, immunofixation electrophoresis; FLC, free light chain; MM, multiple myeloma; LEPP, light-chain escape from plateau phase. MM, a hematologic malignancy belonging to a class of diseases known as plasma cell proliferative disorders, is characterized by the neoplastic proliferation of a single clone of plasma cells producing a monoclonal immunoglobulin. The clone proliferates in the bone marrow and often produces osteolytic bone lesions, osteopenia, osteoporosis, and/or pathologic fractures. The mono- 16

2 Serum FLC (mg/l) chemotherapy initiated day 152 Despite its utility, the FLC assay is not without its limitations. Lot-to-lot variation in assay reagents can be an issue. In addition, both polyclonal and monoclonal serum FLCs have often been found to dilute in a nonlinear fashion, leading to underestimation in the absence of off-line dilution (3). False high results may occur due to polymerization of light chains (4, 5). Finally, falsely low serum FLC results may be obtained in cases of antigen excess (6) Days Fig. 1. Serum FLC concentrations reported for the patient over time. A noteworthy spike occurred immediately after initiation of treatment with lenalidomide/dexamethasone. clonal proteins may cause further organ damage, often leading to renal insufficiency and/or renal failure. The median age of diagnosis for MM is 66 years; only 2% of patients are younger than 40 years (1). MM patients often present with bone pain. Weakness and fatigue are also a common manifestation of MM and are often associated with anemia. Radiographic imaging of the skeleton reveals punched-out lytic lesions, diffuse osteopenia, osteoporosis, or fractures in nearly 80% of patients. Computed tomography and magnetic resonance imaging may be helpful for patients presenting with no abnormalities apparent on conventional x-rays. Examination of the bone marrow reveals bone marrow involvement by plasma cells, which constitute 10% of all nucleated cells in 96% of MM patients; this finding is confirmatory for MM. Laboratory findings at the time of diagnosis include anemia (73% of patients), increased serum creatinine (50%), hypercalcemia (28%), and leukopenia (20%). Serum 2 - microglobulin may also be increased (75%) and serves as a prognostic indicator, with higher values predicting poorer survival prospects. M protein is detected by IFE in the serum and/or urine in 97% of patients (1). QUANTIFICATION OF M PROTEINS IN THE DIAGNOSIS AND MANAGEMENT OF MM The serum FLC assay was developed in the early 2000s to detect light-chain epitopes that are exposed only when not bound to a heavy chain. The assay quantifies and FLCs and has come into routine use in the diagnosis and management of several plasma cell proliferative disorders, including monoclonal gammopathy of undetermined significance, light-chain amyloidosis, and MM. The clinical utility of the FLC assay has been reviewed elsewhere (2). WHAT CAUSED THE INCREASE IN SERUM FLC AFTER TREATMENT IN THIS PATIENT? For our patient, the hematologist sought the consultation of the laboratory director after consecutive monthly measurements of the serum free chain produced values much higher than before the initiation of treatment. The sudden increase in serum FLC after treatment was worrisome to the hematologist because it may have been indicative of a phenomenon referred to as light-chain escape from plateau phase (LEPP) (7). LEPP was documented in a series of 3 MM patients who were noted to undergo a shift in secretion from intact immunoglobulin to FLCs after treatment with either thalidomide or lenalidomide. LEPP is thought to be a novel manifestation of relapsed disease due to selective pressure brought on by thalidomide/lenalidomide treatment. It typically follows an aggressive clinical course, with subsequent therapies offering only marginal benefits. Fortunately, the sample from the patient s second pretreatment visit had been stored frozen. Retesting of this sample at a starting dilution of 1 part in 400 (i.e., 400-fold dilution) instead of 1 part in 100 (100-fold dilution) indicated the need for further sample dilutions and yielded a final FLC concentration of mg/l instead of the initially reported 68.2 mg/l. Nephelometric assays measure light scatter caused by the formation of immune complexes in solution and are subject to limitations inherent in antigen antibody reactions. This method requires that antigen concentrations fall within a certain range known as the antibody excess of the Heidelberger Kendall curve. Higher antigen concentrations produce falsely low readings. In this particular case, the patient had extremely high FLC concentrations that caused antigen excess and an artifactually low reading before treatment. Treatment with dexamethasone and lenalidomide caused a drop in this patient s FLC value to within the region of equivalence for the assay, causing what was perceived to be a spurious increase in concentration. WHAT IS THE PREVALENCE OF ANTIGEN EXCESS IN THE FLC ASSAY? After this case, our laboratory identified an additional 4 MM patients (2 cases of light-chain MM, 1 of nonse- Clinical Chemistry 56:1 (2010) 17

3 cretory MM, and 1 of IgA MM) who had artifactually low serum FLC concentrations during the course of disease monitoring. This experience prompted us to further investigate the incidence of serum FLC antigen excess. During a 4-month period (August 1, 2006, to November 30, 2006), all clinical FLC assays ordered in our laboratory were performed at the recommended 100-fold dilution as well as at a second (400-fold) dilution (8). FLC assays were performed on a Dade Behring BN II nephelometer with FLC reagent sets from The Binding Site (Birmingham, UK). Of the 7538 serum FLC studies that were clinically ordered and assayed in duplicate during this period, no samples exhibited FLC antigen excess, but 9 patients (0.12%) were identified to have FLC excess. These 9 patients all had increased FLC concentrations and abnormal / FLC ratios when tested at the initial 100-fold dilution, but the instrument did not indicate a need for further dilutions. When retested at a 400-fold dilution, however, all 9 samples gave substantially higher results or indicated that additional dilutions were needed. Four of the 9 patients had 2 samples submitted for testing, and each pair of samples yielded similar results. The sample with the largest change in the FLC result was from 77 mg/l to mg/l. On average, the concentrations of these samples increased approximately 200-fold when they were diluted. Diagnostically, the assay identified all 9 samples as abnormal. From a monitoring standpoint, however, the initial FLC results were misleadingly lower than the results obtained at a higher starting dilution. Some nephelometers, including the BN II, have automated detection algorithms designed to detect antigen excess. Such algorithms may include monitoring the initial rate of the precipitin reaction as well as a separate prereaction step. The manufacturer s protocol for the Freelite assay does not include a prereaction step and relies on whether the result lies beyond the linear portion of the calibration curve, as well as on the initial rate of precipitin formation, to determine if further dilutions are necessary. This protocol may have led to the analyzer missing certain cases of antigen excess. Because of these findings, we have modified the laboratory operating procedure. All sera with a FLC / ratio 2 and a FLC concentration that has not been obtained at test dilutions 100-fold are retested at a serum dilution of 400-fold. In samples that are not in antigen excess, the 400-fold dilutions give concentrations that are 50% higher on average than those obtained with the 100-fold dilutions. For consistency, the 100-fold dilutions are reported. Our choice of the 100- fold dilution for reporting purposes is based on the fact that most samples have the 100-fold dilution within the linear portion of the calibration curve. POINTS TO REMEMBER 1. Serum FLC is a sensitive test for the diagnosis of monoclonal FLC disease. 2. Increases in the FLC concentration after treatment for MM may be due to a light-chain escape phenomenon, referred to as light-chain escape from plateau phase (LEPP). 3. Our practice has a high percentage (55%) of abnormal FLC ratios in submitted samples, and approximately 0.1% of the sera in our practice are in FLC excess. No cases of FLC excess have been observed. 4. Because of the potential for FLC antigen excess, newly identified cases with abnormally high ratios should be retested with a further dilution. declared any potential conflicts of interest. Acknowledgments: FLC reagent sets for the dilution studies were provided at no charge by The Binding Site Ltd. 1. Kyle RA, Gertz MA, Witzig TE, Lust JA, Lacy MQ, Dispenzieri A, et al. Review of 1027 patients with newly diagnosed multiple myeloma. Mayo Clin Proc 2003;78: Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, et al. International Myeloma Working Group guidelines for serum-free light chain analysis in multiple myeloma and related disorders. Leukemia 2009;23: Tate JR, Mollee P, Dimeski G, Carter AC, Gill D. Analytical performance of serum free light-chain assay during monitoring of patients with monoclonal light-chain diseases. Clin Chim Acta 2007;376: Abraham RS, Charlesworth MC, Owen BA, Benson LM, Katzmann JA, Reeder CB, Kyle RA. Trimolecular complexes of lambda light chain dimers in serum of a patient with multiple myeloma. Clin Chem 2002;48: Emond JP, Harding S, Lemieux B. Aggregation of serum free light chains (FLC) causes overestimation of FLC nephelometric results as compared to serum protein electrophoresis (SPE) while preserving clinical usefulness [Abstract]. Blood 2007;110:265. Abstract Daval S, Tridon A, Mazeron N, Ristori JM, Evrard B. Risk of antigen excess in serum free light chain measurements. Clin Chem 2007;53: Dawson MA, Patil S, Spencer A. Extramedullary relapse of multiple myeloma associated with a shift in secretion from intact immunoglobulin to light chains. Haematologica 2007;92: Clark RJ, Lockington KS, Tostrud LJ, Katzmann JA. Incidence of antigen excess in serum free light chain assays [Abstract]. Clin Chem 2007;53(S6):A Clinical Chemistry 56:1 (2010)

4 Commentary Jim D. Faix Quantitative measurement of serum free light chain is an important part of the diagnosis and management of multiple myeloma and related disorders. Recent consensus guidelines (1) focus on screening for the presence of monoclonal immunoglobulin and monitoring treatment in patients with amyloidosis or oligosecretory disease. As we expand our use of this assay to monitoring patients with intact monoclonal immunoglobulin, it is clear that some technical limitations still need to be resolved. This clinical case study reminds us that one of the original drawbacks of nephelometric determinations antigen excess may be important when measuring serum free light chain. Antigen excess is due to inhibition of immune complex formation in the presence of very high antigen concentrations (2). Although manufacturers have largely eliminated this problem in noncompetitive immunoassays by either the use of sequential incubations or increasing the amount of solid-phase antibody, these approaches are not feasible for nephelometric assays. Consequently, unless the instrument s software is able to detect possible antigen excess, the laboratory must rule it out by repeating the analysis with a higher dilution. If antigen excess is present, the final result will be higher, paradoxically, because the inhibition of immune complex formation has been removed. Although this problem has been described before with the serum free light chain assay, the authors of the case study provide important context by documenting its relatively low prevalence. This fact might indicate that it is not necessary to routinely test serum samples for free light chain at more than the initial dilution. As in all areas of laboratory medicine, however, no individual result should be interpreted without consideration of other results, as well as the clinical picture. decalred any potential conflicts of interest. Stanford University School of Medicine, Department of Pathology, Stanford, CA. Address correspondence to the author at: Stanford University, Department of Pathology, 300 Pasteur Dr., Rm. H1507, Stanford, CA jim.faix@stanford.edu. Received September 8, 2009; accepted September 25, Previously published online at DOI: /clinchem Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, Hajek R, et al. International Myeloma Working Group guidelines for serum free light chain analysis in multiple myeloma and related disorders. Leukemia 2009;23: Hortin GL, Remaley AT. Introduction to protein analysis. In: Detrick B, Hamilton RG, Folds JD, eds. Manual of molecular and clinical laboratory immunology. 7th ed. Washington, DC: ASM Press; Commentary Morey A. Blinder Departments of Internal Medicine and Pathology & Immunology, Washington University School of Medicine, St. Louis, MO. Address correspondence to the author at: Departments of Internal Medicine and Pathology & Immunology, Washington University School of Medicine, 660 S. Euclid Ave. Box 8125, St. Louis, MO Fax ; mblinder@ dom.wustl.edu. Received November 6, 2009; accepted November 6, DOI: /clinchem In the mid-19th century, Henry Bence Jones described the presence of free light chains (FLCs) in urine (1).A century later, work by Korngold and Lipari (2) characterized Bence Jones proteins and showed that they reacted with those found in myeloma. Although rarely appreciated, the designation of and for the 2 forms of Bence Jones proteins has remained standard nomenclature in tribute to those investigators. Nearly another half-century passed before immunoassays were developed to identify serum FLCs and begin to define their role (3). Coincident with the development of serum FLC assays have been major improvements in the treatment of myeloma. Patients now have therapeutic options that include combinations of corticosteroids, thalidomide, lenalidomide, bortezomib, and stem cell transplant, along with newer investigational agents; thus, Clinical Chemistry 56:1 (2010) 19

5 the prognosis for patients with myeloma continues to improve. The consequence of heightened awareness, a cavalcade of therapy, and prolonged survival have led to increased laboratory monitoring, reflected in the explosion of investigations using serum FLCs. Yet, as with many such advances, cautionary tales arise. In the case presented here, a patient with myeloma was followed for 4 months without symptoms, and apparently comforted by the presence of a low concentration of serum FLCs. As her disease burden worsened, traditional laboratory test results, including hemoglobin, serum calcium, and urinary M-protein, indicated advancing myeloma, and treatment was begun. Although the patient improved, the serum FLCs markedly increased. This contradiction was resolved when previously stored samples were retested at a higher dilution, indicating that large antigen excess led to an artificially low result. The authors noted that this occurred in approximately 0.1% of samples, although it would be difficult to predict who might be at risk for this finding and whether earlier identification would have ultimately changed the outcome. As highlighted in this case, discordant results need to be further investigated, and there is no substitution for careful clinical observation. declared any potential conflicts of interest. 1. Jones HB. Papers on chem pathology. Lecture III. Lancet 1847;50: Korngold L, Lipari R. Multiple-myeloma proteins. III. The antigenic relationship of Bence Jones proteins to normal gamma-globulin and multiple-myeloma serum proteins. Cancer 1956;9: Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson MT, Drew R. Highly sensitive automated immunoassay for immunoglobulin free light chains in serum and urine. Clin Chem 2001;47: Clinical Chemistry 56:1 (2010)