Intact immunoglobulin heavy/light chain paired assays

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1 Intact immunoglobulin heavy/light chain paired assays Stephen Harding 1, Philip Young 1, Marina Di Fazio 2, Richard Hughes 1 1Research and Development, The Binding Site Group, Birmingham, UK 2Scientific Division, The Binding Site Italia, Grassobbio (BG) ABSTRACT International guidelines recommend serum protein electrophoresis (SPEP) and immunofixation as laboratory tests to improve monoclonal gammopathy patient management. In patients with multiple myeloma (MM) these measurements are required to assess patient response to therapy, which help guide treatment and may influence clinical decisions. Here we discuss the potential role of new immunoassays that quantify the intact immunoglobulin Ig κ and Ig λ serum concentrations for IgG, IgA, IgM and IgD isotypes. The Freelite assay revolutionised patient management, particularly in the case of AL amyloidosis, and, due to the polyclonal nature of the antisera used, had a broad recognition for the highly heterogeneous free light chains (FLC). Similarly, the Ig κ and Ig λ assays utilise polyclonal antisera, identifying all MM patient sera tested to date, including those not readily identifiable by SPEP. Furthermore, as with the assessment of FLC κ/λ ratios by Freelite, Ig κ/ig λ ratios have prognostic value at presentation and maximum response and may represent a hitherto unidentified MM cell biology that selectively suppresses isotype matched immunoglobulin production. Finally, we propose an algorithm utilising Ig κ and Ig λ and FLC measurements that may improve the detection of clonal evolution in MM patients. INTRODUCTION Monoclonal immunoglobulins (M-Ig) are associated with many different conditions, ranging from asymptomatic and common monoclonal gammopathies of undetermined significance (MGUS), reported in 3.2% of general population aged 50 years, to the pathological and rare multiple myeloma (MM) and AL-amyloidosis, showing an overall incidence of 4.5 per 100,000 per year and an estimated minimum incidence of 0.3 per 100,000 in British population, respectively (1-3). In all instances, the goals of laboratory are to identify [commonly by serum protein electrophoresis (SPEP)], characterise [commonly by immunofixation (IFE)] and quantify (commonly by SPEP densitometry - discussed later) M-Ig. The use of electrophoretic techniques is limited for samples in which M-Ig co-migrates with other proteins, commonly seen in IgA, IgM and, to a lesser extent, IgG M-Ig (4, 5). In such instances, intact immunoglobulin measurements can be used, as recommended by the International Myeloma Working Group (IMWG) (6). However, these are unable to distinguish between M-Ig and the physiologic polyclonal immunoglobulins, which can limit their utility in patients with subtle M-Ig production, in whom without IFE it would be impossible to determine whether the immunoglobulin is monoclonal or polyclonal. Furthermore, in patients with gross (>50 g/l) or subtle (<10 g/l) M-Ig production, SPEP densitometric analysis can be limited due to the non-linearity of M-Ig or poor analytical performance, respectively (7, 8). In the case of IgG M-Ig, the accuracy of SPEP becomes even more unreliable due to the variable catabolism of the molecule, which is dependent upon serum IgG concentrations and reflects the role of the neonatal Fc recycling receptor (9). Serum free light chains (FLC) are also poorly quantifiable by SPEP, either due to aggregation or poor analytical performance (10). However, the introduction of the Freelite assay, which utilises polyclonal sheep antisera, has overcome these limitations. Briefly, paired tests of anti-κflc and antiλflc are used to generate a κ/λ FLC ratio, which sensitively identifies monoclonal FLC production. In a recent audit we carried out on 651 published light chain multiple myeloma (LCMM) patients, the assay identified 651/651 patients (data not published). However, in a serie of 115 consecutive patients with AL amyloidosis, sensitivity of Freelite assay in detecting monoclonal amyloidogenic FLC was 97% for κ clones and 69% for λ (overall sensitivity, 76%), while high resolution IFE, Correspondence to: Stephen Harding, The Binding Site Group Ltd, 8 Calthorpe Road, Edgbaston, Birmingham, B15 1QT, UK. Tel. 44 (121) , Fax 44 (121) , stephen.harding@bindingsite.co.uk Received: Revised: Accepted: biochimica clinica, 2013, vol. 37, n

2 REVIEWS Table 1 Mean value and 95% central range for intact immunoglobulin Ig κ/ig λ ratios obtained in blood donors n Mean value Reference interval IgGκ/IgGλ ratio Figure 1 The Hevylite immunoassays recognise conformational, junctional epitopes formed between the heavy chain (Hc) and light chain (Lc) of human immunoglobulins. IgAκ/IgAλ ratio IgMκ/IgMλ ratio A B C y= 0.90x r 2 = y= 0.92x r 2 = y= 1.007x r 2 = Intact IgGκ + IgGλ (g/l) Intact IgAκ + IgAλ (g/l) Intact IgMκ + IgMλ (g/l) Total IgG (g/l) Total IgA (g/l) Total IgM (g/l) Figure 2 Correlation between summated Ig κ+ig λ and total immunoglobulin nephelometric assays for IgG (n=130), IgA (n=138) and IgM (n=120). The coefficient of determination r 2 >0.9 gave confidence that the ratios produced (see Table 1) accurately reflected the Ig κ/ig λ value for each individual isotype. we present an overview of the published work utilising these assays and speculate on their role in the identification of heterogeneous MM clones. IgA lambda (g/l) IgA kappa (g/l) Figure 3 Analysis of presentation samples using Hevylite IgAκ/IgAλ ratio for 210 IgA multiple myeloma [145 IgAκ (blue); 65 IgAλ (red)] patients. Black squares and the parallel lines indicate the reference value for IgAκ/IgAλ ratio. carried out in agarose gel freshly prepared in laboratory for both serum and urine, showed an overall sensitivity of 100% (11). The Freelite assays are included in international guidelines and recommended as part of a screening algorithm alongside SPEP to identify monoclonal gammopathy patients (12). The success of the Freelite assays provided the impetus for the production of intact immunoglobulin Ig κ/ig λ assays (IgGκ/IgGλ, IgAκ/IgAλ, IgMκ/IgMλ, IgDκ/IgDλ), which recognise conformational epitopes spanning the junction between the heavy chain and light chain of the immunoglobulin molecule (Figure 1). Here ANALyTICAL ASpeCTS For intact immunoglobulin assays, polyclonal antibodies were raised against the conformational epitopes between the heavy and light chain of the immunoglobulin (13). These antibodies were raised using large pools of target M-Ig to minimise the impact of M-Ig variability (as with FLC recognition, monoclonal antibodies are liable to miss clones and thus making them poor candidates in these assays). Polyclonal reference materials were preferred to monoclonal as the latter may have unique structures. The summated Ig κ + Ig λ values were compared to nephelometric total immunoglobulin values and used to produce Ig κ/ig λ ratios (Figure 2). Table 1 shows reference intervals produced from >100 blood donor sera for each ratio. CLINICAL UTILITy OF Ig κ/ig λ RATIOS M-Ig identification Ig κ/ig λ ratios were compared to the respective reference intervals for assessing for their utility in the identification of M-Ig. 210 IgA MM patients (145 IgAκ and 65 IgAλ), where 40% of the M-Ig co-migrated with other proteins, were analysed and compared to the corresponding reference interval, showing that 100% of samples had abnormal IgAκ/IgAλ ratios (Figure 3). 366 biochimica clinica, 2013, vol. 37, n. 5

3 Relapse 25 mg Rev + 10 mg Dex Rev + Dex Rev only Rev + Dex Relapse Ratio IgAκ/IgAλ Hevylite ratio upper reference limit IgA normal upper reference limit IgA (g/l) Freelite ratio (κ/λ) upper reference limit Time (days) Figure 4 Biochemical monitoring of a patient with IgAκ multiple myeloma showing IgAκ/IgAλ ratio (red), free light chain (FLC) κ/λ ratio (blue) and total IgA nephelometric determination results (black). Grey empty circles denote negative immunofixation electrophoresis. Rev, revlimid (trade name for lenalidomide); Dex, dexametasone. Bradwell et al. reported similar sensitivities for 245 IgG MM patients (166 IgGκ and 79 IgGλ) (14). However, a retrospective analysis of 999 MGUS patients reported that whilst 97% of IgA and 90% of IgM MGUS patients were identified as having an abnormal Ig κ/ig λ ratio, only 56% of IgG MGUS patients were identified (15). This discordance between sensitivities for MM and MGUS is likely to be explained by the degree of uninvolved (or isotype matched) polyclonal suppression. This is supported by the fact that a subset analysis of data showed that in patients with <5 g/l M-Ig only 24% had an abnormal IgGκ/IgGλ ratio, whereas in patients with >21 g/l M-Ig 90% had an abnormal ratio. This can be explained mathematically: in patients with a physiological polyclonal IgG repertoire (up to 16 g/l total IgG), the presence of 1 g/l monoclonal protein will not result in an abnormal ratio; by contrast, as physiologic polyclonal IgA concentrations are lower (up to 4 g/l total IgA) a 1 g/l of IgA M-Ig is likely to result in an abnormal ratio. Monitoring of MM patients One key advantage that the use of IgAκ/IgAλ ratios may have over SPE/IFE is the ability to monitor the patient irrespective of the migration of the M-Ig by SPEP. Briefly, if a patient has an IgA M-Ig that co-migrates with other serum proteins, three tests will be required to monitor the patient, i.e., SPEP, IgA determination and IFE to confirm clonality, which is a lengthy and costly process. By contrast, utilising paired IgAκ/IgAλ antisera may be a more sensitive and cost-effective solution (Figure 4) (5, 14, 16). Donato et al. highlighted the difficulties in monitoring IgA MM patients (16). They described the disease course of a 71-year old woman with IgA MM achieving a complete remission (CR) following stem cell transplantation and immunotherapy. However, at CR the patient s serum IgA concentrations remained high and the physician was uncertain as to whether this elevation was due to polyclonal expansion of IgA in response to a lung infection or an indication of relapse of the monoclonal IgA. When the patient subsequently relapsed, analysis of Igκ/Igλ ratio confirmed re-emergence of clonal disease and, importantly, analysis of retrospective samples demonstrated that the IgAκ/IgAλ ratio had remained abnormal throughout monitoring, including when the patient had achieved a CR using traditional M-Ig measurement techniques. The authors concluded that IgAκ/IgAλ pair analysis provided a measure of clonal synthesis that is difficult to detect due to the migration of the M-Ig on SPEP (16). Similarly, Bradwell et al. (14) identified abnormal IgGκ/IgGλ ratios present when IFE remained negative which also indicated early relapse. The patient was an IgGκ MM patient who achieved a CR at day 250 following cyclophosphamide therapy. Between days , SPE and IFE remained negative, while an increasingly abnormal intact immunoglobulin heavy/light chain ratio provided an earlier indication of relapse (14). To date, published studies show that Ig κ/ig λ ratios have a greater sensitivity than electrophoretic techniques in the identification of relapse; in the majority of cases this appears associated with suppression of the uninvolved isotype matched immunoglobulin; it is, therefore, tempting to hypothesise that the malignant clone can have an impact upon its surrounding environment before immunoglobulin production. However, further work is required to support these hypotheses and to date no relapse criteria using Ig κ/ig λ ratios have been established. biochimica clinica, 2013, vol. 37, n

4 REVIEWS Light chain & Intact IgA Intact IgA only Light chain & Intact IgA IgAκ/IgAλ FLC kappa/lambda ratio Time (days) Figure 5 Detection of an intact monoclonal immunoglobulin escape during monitoring of an IgAκ multiple myeloma patient with Hevylite and Freelite. The IgAκ/IgAλ ratio (red), free light chain (FLC) κ/λ ratio (green) and serum protein electrophoresis gels are shown. Yellow boxes highlight clonal disease characterised by monoclonal IgAκ and monoclonal FLCκ expression; the blue box indicates clonal disease characterised by monoclonal IgAκ expression only. Dashed lines indicate upper reference limits of ratios Changes in the dominant monoclonal protein produced during the course of disease was first documented by Hobbs in 1969, who identified light chain escape in MM patients (17). More recently, clonal switches have been elegantly described in some studies (18-20). Zamarin et al. (19) described changes in M-Ig expression post maximum response after autologous stem cell transplantation. Evolution of clones expressing only FLC was identified in MM patients with intact immunoglobulins or intact immunoglobulins with FLC. Interestingly, in patients with LCMM the ability to produce intact immunoglobulins did not return (19). Magrangeas et al. (20) reported in a study of 24 MM patients that at relapse there are three sub-types of clonal disease: 1) re-emergence of the original clone, 2) emergence of a secondary clone (termed clonal tiding) and 3) reemergence of the primary clonal after a genetic modification (termed clonal evolution). Each of these events was identified in one third of the studied population (20). These studies support a multiparametric approach to MM patient monitoring and it is important that monitoring algorithms are introduced that allow the detection and characterisation of these changes. Freelite and intact immunoglobulin heavy/light chain ratio are more sensitive methods of detecting residual disease and low level M-Ig than traditional methods; therefore, when used in parallel they offer a sensitive method of detecting subtle changes. Figure 5 shows an example of clonal changes identified during monitoring of an IgAκ MM patient. The patient presents with clones expressing IgAκ and FLCκ M-Ig. Following treatment, he achieves a CR after ~500 days, at which time both IgAκ/IgAλ and FLCκ/λ ratios normalise. The patient undergoes a clonal change after ~750 days, where a clone emerges that expresses monoclonal IgAκ, but not FLCκ. Following treatment, commenced after ~1100 days, the IgAκ clone is reduced, but not eliminated, and the patient subsequently undergoes a second clonal event after ~2000 days and relapses with clones expressing both monoclonal IgAκ and monoclonal FLCκ (Prof. Mark Drayson, personal communication). prognostic value Baseline Freelite κ/λ ratios have been reported to be prognostic in MM (21, 22) and are required to assign stringent CR (6). Therefore, it is perhaps unsurprising that Ig κ/ig λ ratios have also been analysed with respect to outcome at baseline and at maximum response. Some authors have reported that Ig κ/ig λ ratios provide prognostic information at baseline, although the cut-offs reported by each study that identified patients at high risk were not identical (5, 14). Perhaps the most intriguing aspect of the prognostic value of Ig κ/ig λ ratios is the observation that it is the degree of suppression that dictates the outcome rather than the degree of production. In two studies the degree of uninvolved suppression was prognostic for progression, free survival and transformation from MGUS to MM, respectively (14, 15). Therefore, rather than the production of the neoplastic plasma cell, it seems the impact of the cell on its surroundings that more accurately reflects the course of the patient s disease. CONCLUSIONS Intact immunoglobulin heavy/light chain ratios can be used to identify M-Ig production in MGUS and MM and in both of these settings the quantitative determination of either the ratio or of the uninvolved isotype matched immunoglobulin provides prognostic information. The use of a paired Ig κ/ig λ test is allowing us to investigate a hitherto hidden impact of monoclonal plasma cells, 368 biochimica clinica, 2013, vol. 37, n. 5

5 which suggests that there may be selective suppression of polyclonal immunoglobulin production. It is too early to understand how these observations may be used to guide the physician, but increasingly we understand that a multi-parametric algorithm approach to monitoring is likely to benefit patients. CONFLICTS OF INTeReST Stephen Harding, Philip Young and Richard Hughes are employees of The Binding Site Group (UK). Marina di Fazio is employee of The Binding Site Italy. ReFeReNCeS 1. Kyle RA, Therneau TM, Rajkumar SV, Prevalence of monoclonal gammopathy of undetermined significance. N Engl J Med 2006;354: Dispenzieri A, Kyle RA. Multiple myeloma: clinical features and indications for therapy. Best Pract Res Clin Haemat 2005;18: Pinney JH, Smith CJ, Taube JB. Systemic amyloidosis in England: an epidemiological study. Br J Haematol 2013;161: Tseng CH, Chang CY, Liu KS, et al. Accuracy of serum IgM and IgA monoclonal protein measurements by densitometry. Ann Clin Lab Sci 2003;33: Ludwig H, Milosavljevic D, Zojer N, et al. Immunoglobulin heavy/light chain ratios improve paraprotein detection and monitoring, identify residual disease and correlate with survival in multiple myeloma patients. Leukemia 2013;27: Dimopoulos M, Kyle R, Fermand JP, et al. Consensus recommendations for standard investigative workup: report of the International Myeloma Workshop Consensus Panel 3. Blood 2011;117: Murray DL, Ryu E, Snyder MR, et al. Quantitation of serum monoclonal proteins: relationship between agarose gel electrophoresis and immunonephelometry. Clin Chem 2009;55: Katzmann JA, Snyder MR, Rajkumar SV, et al. Long-term biologic variation of serum protein electrophoresis M- spike, urine M-spike, and monoclonal serum free light chain quantification: implications for monitoring monoclonal gammopathies. Clin Chem 2011;57: Waldmann TA, Strober W. Metabolism of immunoglobulins. Prog Allergy 1969;13: Abraham RS, Charlesworth MC, Owen BA, et al. Trimolecular complexes of lambda light chain dimers in serum of a patient with multiple myeloma. Clin Chem 2002;48: Palladini G, Russo P, Bosoni T, et al. Identification of amyloidogenic light chains requires the combination of serum-free light chain assay with immunofixation of serum and urine. Clin Chem 2009;55: Katzmann JA, Kyle RA, Benson J, et al. Screening panels for detection of monoclonal gammopathies. Clin Chem 2009;55: Bradwell AR, Harding SJ, Fourrier NJ, et al. Assessment of monoclonal gammopathies by nephelometric measurement of individual immunoglobulin kappa/lambda ratios. Clin Chem 2009;55: Bradwell A, Harding S, Fourrier NJ, et al. Prognostic utility of intact immunoglobulin Ig'κ/Ig'λ ratios in multiple myeloma patients. Leukemia 2013;27: Katzmann JA, Clark R, Kyle RA, et al. Suppression of uninvolved immunoglobulins defined by heavy/light-chain pair suppression is a risk factor for progression of MGUS. Leukemia 2013;27: Donato LJ, Zeldenrust SR, Murray DL, et al. A 71-year-old woman with multiple myeloma status after stem cell transplantation. Clin Chem 2011;57: Hobbs JR. Growth rates and responses to treatment in human myelomatosis. Br J Haematol 1969;16: Keats JJ, Chesi M, Egan JB, et al. Clonal competition with alternating dominance in multiple myeloma. Blood 2012;120: Zamarin D, Giralt S, Landau H, et al. Patterns of relapse and progression in multiple myeloma patients after auto- SCT: implications for patients' monitoring after transplantation. Bone Marrow Transplant 2013;48: Magrangeas F, Avet-Loiseau H, Gouraud W, et al. Minor clone provides a reservoir for relapse in multiple myeloma. Leukemia 2013;27: Kyrtsonis MC, Vassilakopoulos TP, Kafasi N, et al. Prognostic value of serum free light chain ratio at diagnosis in multiple myeloma. Br J Haematol 2007;137: Snozek CL, Katzmann JA, Kyle RA, et al. Prognostic value of the serum free light chain ratio in newly diagnosed myeloma: proposed incorporation into the international staging system. Leukemia 2008;22: biochimica clinica, 2013, vol. 37, n