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1 Available online at Annals of Clinical & Laboratory Science, vol. 45, no. 2, The Utility of The Serum Heavy/Light Chain Assay as a Complementary Tool in the Monitoring of Patients with Plasma Cell Myeloma: a Report of Three Cases Heyjin Kim 1, Jin Kyung Lee 1,3, Young Jun Hong 1,3, Seok-Il Hong 1,3, Dong-Yeop Shin 2, Hye Jin Kang 2, and Yoon Hwan Chang 1,3 Departments of Laboratory Medicine 1 and Internal Medicine 2, Korea Cancer Center Hospital, Seoul, and Radiation Cancer Medicine 3, University of Science and Technology, Daejeon, Korea Abstract. We report three cases of plasma cell myeloma with obscure and discordant data for a monoclonal component. In this study, the results of serum heavy/light chain (shlc) were retrospectively compared with those of conventional methods during disease monitoring. All three patients achieved a complete response and experienced a relapse during follow-up, and the shlc ratio allowed early prediction of disease relapse and correlated well with other electrophoretic methods compared with the free light chain ratio. Therefore, we suggest that the shlc assay may be useful as a complementary tool; it has a good correlation with conventional methods and sensitivity in assessing disease status and treatment response in patients with plasma cell myeloma. Key words: Heavy/light chain, plasma cell myeloma, monitoring Introduction Plasma cell myeloma (PCM) is characterized by the production of monoclonal protein (M protein) in bone marrow and presents in serum and urine [1]. Conventional methods to detect monoclonal immunoglobulin (Ig), such as serum protein electrophoresis (spep), serum immunofixation electrophoresis (sife) and serum free light chain (sflc) assay, are mainly used for the diagnosis and serial monitoring of PCM according to international guidelines [2]. Because of the heterogeneous disease course of PCM and the introduction of novel agents and stem cell transplantation, both a small level of M protein overlaying normal serum levels of proteins on protein electrophoresis and a weak band or an abnormal protein band (APB) on sife are often obscured. Discordant results when using conventional methods make it difficult to assess the patient s disease status and treatment response. Although the sflc has excellent sensitivity, it cannot detect whole Igs or those with only a heavy chain protein or little free light chain [3-5]. A novel Address correspondence to Yoon Hwan Chang, MD, PhD; Department of Laboratory Medicine, Korea Cancer Center Hospital, 75 Nowon-ro, Nowon-gu, Seoul , Korea; phone: ; fax: ; e mail: cyhlabo@kcch.re.kr method for quantifying serum intact Ig heavy/light chain (shlc) pairs using antibodies specific for the junctional epitopes of each Ig was recently developed, and several studies have evaluated its performance in detecting M protein levels and determining disease progression and response to therapy. Those studies have correlated well with clinical responses to changes in shlc ratios [5-10]. Here, we report three patients with obscure and discordant data during monitoring of PCM. Materials and Methods Patients and serial samples. In this study, we retrospectively analyzed serial data from three patients with PCM who had discordant results among different M protein measurements and developed APBs such as oligoclonal bands, isotype-switching bands, or single protein bands during monitoring. In all three cases, M protein was serologically identified from diagnosis. Fifty-four frozen sera samples from the three patients were assayed for the shlc ratio. The results among those methods for detecting malignant plasma cell clones were compared, but only in the same periods. We evaluated only serological data for defining disease status in these patients. Each patient underwent a different treatment regimen with only chemotherapy or a combination of chemotherapy and autologous stem cell transplantation (ASCT) and/or /15/ by the Association of Clinical Scientists, Inc.

2 194 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 Birmingham, England) was performed using the Roche/Hitachi Modular P800 (Roche Diagnostics, Naka, Japan). The heavy/light chain (HLC) pair assay was a nephelometric assay (SPAPLUS; Binding Site, Birmingham, England) using antibodies specific for the junctional epitopes of each Ig (i.e., IgGκ, IgGλ, IgAκ, and IgAλ) and allowed quantitative assessment of each Ig HLC combination (Hevylite ; Binding Site, Birmingham, England). Figure 1. Comparison of test results between the HLC ratio (IgAκ/IgAλ) and the FLC ratio (κ/λ) for case 1 (a patient with IgAκ) after the initiation of chemotherapy (one cycle of VAD). The horizontal dashed lines indicate the limit of normal ranges of the HLC ratio and the FLC ratio, respectively (red: IgAκ/IgAλ; blue: κ/λ). Earlier increases in the HLC ratio compared with the FLC ratio are marked with red arrows. Changes in the patient s drug regimen are marked in boxes with arrows at the top of the figure. Abbreviations: ASCT, autologous stem cell transplantation; CR, complete response; FLC, free light chain; HLC, heavy/light chain; Ig, immunoglobulin; M, melphalan; NR, normal range; PAD, bortezomib, doxorubicin, dexamethasone; VAD, vincristine, doxorubicin, dexamethasone; VD, bortezomib, dexamethasone. *Oligoclonal bands (IgG, IgM, λ) were shown on serum immunofixation electrophoresis (sife). **Oligoclonal bands (IgG, IgM, λ) and original monolclonal bands (IgAκ) were shown on sife. Association between shlc assay and sflc assay were tested using Pearson s correlation analysis. The statistical analyses were performed using SPSS (SPSS version 19, IBM, Armonk, NY, USA). Case Reports radiotherapy. All three patients achieved complete response (CR) after treatment and experienced a relapse at least once during monitoring. The diagnosis of PCM was established according to the 2008 World Health Organization classification [1], and the disease status of the patients was defined according to the European Blood and Bone Marrow criteria [2]. The study was approved by the Institutional Review Board of the Korea Cancer Center Hospital, and all patients gave written informed consent. Laboratory methods. Sequential analysis of M protein markers was performed using the following methods. Total IgG and IgA (Roche Diagnostics, Mannheim, Baden-Württemberg, Germany) were analyzed using the Cobas Integra 800 (Roche Diagnostics). Detection of the monoclonal components in spep and sife (Hydragel; Sebia, Evry, Essonne, France) was performed using the HYDRASYS 2 analyzer (Sebia). sflc using polyclonal sheep antisera (Freelite, Binding Site, Case 1. A 40-year-old woman was admitted to the hospital in August 2008 because of pain in the left leg that was unresponsive to treatment at local clinics. She presented with anemia (hemoglobin level of 10.8 g/dl) and an M protein level of 4.5 g/dl on spep. sife demonstrated monoclonal IgAκ type protein. The patient s IgA concentration was markedly increased to g/l (normal range: g/l), and osteolytic lesions of the proximal tibia and humerus were revealed on simple radiography and magnetic resonance imaging. Bone biopsy of the proximal tibia on the left side confirmed plasmacytoma. The patient achieved CR in February 2009 after undergoing chemotherapy (5 cycles of vincristine, doxorubicin, and dexamethasone as well as melphalan) and radiotherapy for the humerus bone lesions. After undergoing ASCT, no abnormal findings were shown on spep and sife. Two discrete free lambda bands appeared after four months and persisted for three months. A suspicious IgAκ band and M protein were detected, along with an abnormal sflc ratio. The sflc

3 Heavy/Light Chain Assay in Plasma Cell Myeloma 195 ratio became normal and remained so during followup, even though spep and sife showed an obvious monoclonal gammopathy pattern. After a recurrence of myeloma was confirmed, the patient restarted chemotherapy and achieved CR again. Oligoclonal bands were found after the second ASCT during monitoring. A second relapse was confirmed at the last follow-up in April 2011 based on abnormal serum measurements of M protein, although the sflc ratio was normal. Case 2. In December 2004, a 37-year-old man with a history of acute myocardial infarction was admitted to the hospital because of severe back and flank pain. IgAλ type PCM including anemia was diagnosed, with an M protein level of 5.5 g/ dl on spep. Bone marrow aspiration and biopsy confirmed 35.3% plasma cell involvement. The patient achieved CR after chemotherapy with thalidomidedexamethasone. After ASCT, oligoclonal bands including IgGκ, two κ, and λ bands appeared without M protein on spep, and the sflc ratio was within the normal range. After two months, the patient had no abnormal findings on sife and spep until January 2010, when suspicious monoclonal bands started to present and persisted on sife with increasing M protein on spep during monitoring. During that time, sflc ratio was normal until the M protein level reached 1.6 g/dl. Figure 2. Comparison of test results between the HLC ratio (IgAκ/IgAλ) and the FLC ratio (κ/λ) for case 2 (a patient with IgAλ) from diagnosis. The horizontal dashed lines indicate the limit of normal ranges of the HLC ratio and the FLC ratio, respectively (red: IgAκ/IgAλ, blue: κ/λ). An earlier decrease in the HLC ratio compared with the FLC ratio is marked with red arrows. Changes in the patient s drug regimen are marked in the boxes with arrows at the top of the figure. Abbreviations: ASCT, autologous stem cell transplantation; CR, complete response; FLC, free light chain; HLC, heavy/light chain; Ig, immunoglobulin; M, melphalan; NR, normal range; NA, not available; TCD, thalidomide, cyclophosphamide, dexamethasone; TD, thalidomide, dexamethasone. *Isotype-switching bands (IgGκ) were demonstrated on serum protein electrophoresis and serum immunofixation electrophoresis. developed, and abnormal ratios were found on sflc. The patient restarted chemotherapy with melphalanpamidronate after confirmation of relapse. The M protein level on spep decreased during treatment, but abnormal results continued for seven months. However, an abnormal sflc ratio was demonstrated for only four months. Two lambda bands appeared, but normal results were found for total IgG level, spep, and sflc in January Abnormal results (M protein level of 0.4 g/dl) for spep and sife were detected after two months, but the sflc ratio was normal. Case 3. A 69-year-old woman received a diagnosis of IgGλ type PCM in March The initial M protein level was 1.1 g/dl according to spep. Bone marrow aspiration and biopsy showed 13% plasma cells. The patient achieved CR after several cycles of chemotherapy (2 cycles of melphalan-pamidronate and 4 cycles of bortezomib-dexamethasone) in June During monitoring, weak bands indicating a suspicion of M protein on spep (0.3 g/dl) and an IgGλ band on sife were observed, but a normal sflc ratio was observed. After six months, prominent monoclonal bands on spep and sife Results and Discussion PCM is considered an incurable malignancy, but the disease usually progresses slowly, and survival is improving with the development of novel treatments. Serial measurement of serum M protein levels is useful for identifying the response to therapy for PCM and can assist in making therapeutic decisions. Early detection of M protein activity also allows early treatment and defends against further

4 196 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 Figure 3. Comparison of test results between the HLC ratio (IgGκ/IgGλ) and the FLC ratio (κ/λ) for case 3 (a patient with IgGλ) from a CR state. The horizontal dashed lines indicate the limit of normal ranges of the HLC ratio and the FLC ratio, respectively (red: IgGκ/IgGλ, blue: κ/λ). Earlier decreases in the HLC ratio compared with the FLC ratio is marked with red and blue arrows, respectively. Changes in the patient s drug regimen are marked in the boxes with arrow at the top of the figure. Abbreviations: CR, complete response; FLC, free light chain; HLC, heavy/light chain; Ig, immnunoglobulin; M, melphalan; NR, normal range; sife, serum immunofixation electrophoresis; spep, serum protein electrophoresis; TCD, thalidomide, cyclophosphamide, dexamethasone; VD, bortezomib, dexamethasone. *Two lambda bands were demonstrated on sife. disease progression. Therefore, monitoring patients with serological testing is essential for the detection of relapse. Recently, a new method for quantifying intact immunoglobulin (Ig) heavy/light chain (HLC) was developed and several studies showed that shlc ratios had good correlations with clinical responses and prognosis in PCM patients [5-10]. In this report, we retrospectively analyzed serial samples from three patients to determine the utility of HLC assay in the monitoring of PCM. The specific Ig HLC pairs showed equivocal and/or discordant results for spep, sife, and sflc ratio during monitoring. The patient in Case 1 presented with an abnormal IgAκ/IgAλ ratio that correlated relatively well with total IgA level, M protein level of spep, and IgAκ band of sife, except the κ/λ ratio. During both the first and the second relapse, abnormal results were observed for spep, sife, total IgA level, and shlc ratio, but not sflc ratio. The sflc ratio was abnormal during the early period of the first relapse but returned to normal. However, abnormal results were found for the other monoclonal parameters before the second CR (Figure 1). In Case 2, an abnormal IgAκ/IgAλ ratio persisted for longer during the early treatment course before ASCT; however, the sflc ratio became normal. During the relapse, an abnormal result was detected earlier in the shlc ratio than in the sflc ratio (Figure 2). Lastly, in Case 3, an abnormal IgGκ/IgGλ ratio was observed in the sample that had previously showed weak bands. In addition, an abnormal transition of the shlc ratio was observed earlier than that of the sflc ratio during the first and second relapses (Figure 3). During monitoring, there was no relationship between the shlc ratios and the sflc ratios (r= , p=0.415, Table 1) in Case 1, although good correlation between them was shown in Cases 2 and 3 (r=0.903, p=0.000 and r=0.702, p=0.000, respectively). Because persistently normal FLC ratios were seen in these three patients who were suspected of experienced a relapse on the basis of other serum parameters, this did not appear to be a proper tool to monitor disease progression in our series. Although the sflc assay has been approved for diagnosis and monitoring with high sensitivity, and the relationship between the shlc ratios and the sflc ratios was highly comparable in Case 2 and 3, it could not

5 Heavy/Light Chain Assay in Plasma Cell Myeloma 197 Table 1. The comparison between shlc ratio and sflc ratio in Case 1 (IgAκ type) during monitoring. Time (years) shlc ratio (IgAκ/IgAλ, sflc ratio (κ/λ, Pearson s correlation after diagnosis normal range: ) normal range: ) r= Abbreviations: shlc, serum heavy/light chain; sflc, serum free light chain. Arrows: the time of diagnosis of relapse. detect the M protein found earlier in shlc assay during follow-up of the three patients, especially in determination of relapse. No correlation between the shlc ratios and the sflc ratios in Case 1 might indicate discordant amount of the production of heavy chains and light chains in malignant plasma cells during early relapse [11]. In addition, the sflc assay was essentially unable to detect whole Igs, and low levels of monoclonal FLC in serum can be masked by sufficient levels of polyclonal heavy chains [3-5]. It was much more difficult to judge the disease state with normal results using conventional methods and/or sflc assay around the time of relapse, even though other clinical findings or symptoms were abnormal. The use of serial shlc assays in patients treated with chemotherapy and/or ASCT correlated well with other parameters for the monitoring of disease progression or regression. In addition, all three cases involved APBs on sife during therapy. When APBs appeared, the shlc ratio was measured and demonstrated normal values, except when oligoclonal bands (IgG, IgM, λ) were shown and in both original monoclonal bands (IgAκ) in Case 1 (Figure 1). The appearance of APBs causes difficulties in decisions regarding patient management, although immune reconstruction after chemotherapy and transplantation has been proposed in many studies [12-17]. The use of the shlc assay may help to assess APBs. Unfortunately, this study was based on a small number of patients, and the samples in each method were not strictly sequential from the time of diagnosis of PCM. Each patient had different intervals of follow-up, and the method of measurement of M protein was not always identical to the previous methods. Also, serial HLC measurements were not allowed from the first diagnosis of PCM. Welldesigned further studies are needed to determine the beneficial effect of HLCs for the early detection of treatment response and relapse. In our study, serial serum levels of M protein in spep, sife, sflc, and shlc ratio were all useful for monitoring disease activity during therapy, and the HLC assay was not always superior to the other conventional methods. To date, the HLC assay may not be a substitute for sife but can serve as an adjunct in the diagnosis of early recurrence. In summary, we present serial data demonstrating that the

6 198 Annals of Clinical & Laboratory Science, vol. 45, no. 2, 2015 shlc ratio may be a useful monitoring tool for the interpretation of obscure or discordant findings for spep, sife, and sflc ratio that may subsequently indicate relapse. Therefore, monitoring of the shlc ratio may allow more knowledgeable patient management. Acknowledgment We thank the DOW biomedica, Inc. for providing the HevyliteTM kits. The specimens used in this study were distributed by the Korea Institute of Radiological and Medical Sciences (KIRAMS) Radiation Biobank (KRB). References 1. Mckenna RW, Kyle RA, Kuehl WM, Grogan TM, Harris NL, Coupland RW. Plasma cell neoplasms. In: WHO classification of tumours of haematopoietic and lymphoid tissues, 4th ed (Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW, Eds), IARC, Lyon, 2008; pp Bladé J, Samson D, Reece D, Apperley J, Björkstrand B, Gahrton G, Gertz M, Giralt S, Jagannath S, Vesole D. Criteria for evaluating disease response and progression in patients with multiple myeloma treated by high-dose therapy and haemopoietic stem cell transplantation. Myeloma Subcommittee of the EBMT. European Group for Blood and Marrow Transplant. Br J Haematol 1998;102: Levinson SS. Polyclonal free light chain of Ig may interfere with interpretation of monoclonal free light chain κ/λ ratio. Ann Clin Lab Sci 2010;40: Uljon SN, Richardson PG, Schur PH, Anderson KC, Tanasijevic MJ, Lindeman LI. Serial serum free light chain measurements do not detect changes in disease status earlier than electrophoretic M-spike measurements in patients with intact immunoglobulin myeloma. Clinica Chemica Acta 2011;412: Bradwell AR, Harding SJ, Fourrier NJ, Wallis GL, Drayson MT, Carr-Smith HD, Mead GP. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios. Clin Chem 2009;55: Keren DF. Heavy/light chain analysis of monoclonal gammopathies. Clin Chem 2009;55: Ludwig H, Milosavljevic D, Zojer N, Faint JM, Bradwell AR, Hubl W, Harding SJ. Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients. Leukemia 2012;27: Bradwell AR, Harding SJ, Fourrier NJ, Mathoiot C, Attal M, Moreau P, Harousseau JL, Avet-Loiseau H. Prognostic utility of intact immunoglobulin Ig'κ/Ig'λ ratio in multiple myeloma. Leukemia 2013;27: Tovar N, Fernández de Larrea C, Elena M, Cibeira MT, Aróstegui JI, Rosiñol L, Filella X, Yagüe J, Bladé J. Prognostic impact of serum immunoglobulin heavy/light chains ratio in patients with multiple myeloma in complete remission after autologous stem cell transplantation. Biol Blood Marrow Transplant 2012;18: Koulieris E, Panayiotidis P, Harding SJ, Kafasi N, Maltezas D, Bartzis V, Tzenou T, Dimou M, Georgiou G, Mirbahai L, Bradwell AR, Kyrtsonis MC. Ratio of involved/uninvolved immunoglobulin quantification by HevyliteTM assay: clinical and prognostic impact in multiple myeloma. Exp Hematol Oncol 2012;1:9 doi: / Mead GP, Carr-Smith HD, Drayson MT, Morgan GJ, Child JA, Bradwell AR. Serum free light chains for monitoring multiple myeloma. Br J Haematol 2004;126: Zent CS, Wilson CS, Tricot G. Jagannath S, Siegel D, Desikan KR, Munshi N, Bracy D, Barlogie B, Butch AW. Oligoclonal protein bands and Ig isotype switching in multiple myeloma teated with high-dose therapy and hematopoietic cell transplantation. Blood 1998;91: Hovenga S, de Wolf JThM, Guikema JEJ, Klip H, Smit JW, Sibinga CThS et al. Autologous stem cell transplantation in multiple myeloma after VAD and EDAP courses; a high incidence of oligolconal serum Igs post transplantation. Bone Marrow Transplant 2000;25: Hall SL, Tate J, Gill D, Mollee P. Significance of abnormal protein bands in patients with multiple myeloma following autologous stem cell transplantation. Clin Biochem Rev 2009;30: Yoon CE, Hong YJ, Lee JK, Hong SI, Kang HJ, Chang YH. Clinical significance of oligoclonal bands in patients with multiple myeloma after autologous stem cell transplantation. Lab Med Online 2012;2: Maisnar V, Tichý M, Smolej L, Zák P, Radocha J, Palicka V, Malý J, Bláha V. Isotype class switching after transplantation in multiple myeloma. Neoplasma 2007;54: Alejandre ME, Madalena LB, Pavlovsky MA, Facio ML, Corrado C, Milone G, Bresciani PD, Fraind SA, Pavlovsky S, Pizzolato MA. Oligoclonal bands and immunoglobulin isotype switch during monitoring of patients with multiple myeloma and autologous hematopoietic cell transplantation: 16- year experience. Clin Chem Lab Med 2010;48: