Comparison of Waldenstrom Macroglobulinemia Responses Using Immunoglobulin Heavy / Light Chain Analysis and Conventional Electrophoresis Techniques
Authors: Boyle Eileen M , Lejeune Julie , Manier Salomon , Musset Lucile , Bories Claire , Dulery Remy , Guidez Stéphanie , Bonnet Sarah , Fouquet Guillemette , Onraed Brigitte , Faucompré Jean-Luc , Tricot Sabine , Poulain Stephanie , Leblond Veronique , Leleu Xavier , Harding Stephen 
Published: October 2019

Abstract
Background. Waldenstrom's macroglobulinaemia (WM) is a B cell lymphoproliferative disorder which is characterised by the production of monoclonal IgM (m-IgM). Response assessment is reliant upon reductions in the m-IgM as measured by serum protein electrophoresis (SPEP) or nephelometric/turbidimetric total IgM (tIgM) measurements alongside lymphadenopathy / splenomegaly assessment. Response assessment guidelines include reductions of <25% m-IgM (stable disease, SD), 25-50% (minor response, MR), 50-90% (partial response, PR), >90% (very good partial response, VGPR), 100% with negative immunofixation (complete response, CR) with progressive disease being described as a >25% increase in m-IgM. However, quantitation of m-IgM is not without limitations. The pentameric form of IgM has a molecular weight of >900 kDa, which may be responsible for its tendency to self-aggregate which causes issues in the accuracy of quantitation by SPE. tIgM values are systematically greater than measurement by SPEP densitometry and unable to discriminate between m-IgM and polyclonal IgM as serum concentrations approach the normal range (~2 g/L). Recently novel immunoassays quantifying the heavy / light chain partnership for IgM (IgMκ / IgMλ) have become available, here we report on the performance of the assay in a population of Waldenstrom's patients.
Methods. Diagnostic serum was available for 78 WM patients (48IgMκ / 30IgMλ); longitudinal samples were available for 25 patient (median 9 samples, range 3-20), median 1146 days (min 62, max 2220). Sera were analysed retrospectively using the IgM Hevylite immunoassay (Binding Site, UK) on a SPAPLUS (Binding Site, UK) analyser. Results were compared to SPEP / Immunofixation (Sebia, Hydrasys II), tIgM (Binding Site, UK) and established normal ranges (IgMκ: 0.19-1.63 g/L; IgMλ: 0.12-1.01 g/L; IgMk/IgMl ratio: 1.18-2.74; total IgM: 0.5-2 g/L). dHLC (monoclonal isotype IgM HLC – uninvolved isotype IgM HLC) was used as an assessment of m-IgM. Substantial equivalence was assessed by Weighted Kappa (WK, >0.81 considered to be agreement between methods)
Results. At presentation all WM patients had an abnormal IgMκ / IgMλ ratio (IgMκ median 140, range 9-2850; IgMλ median 0.1, 0.001-0.9), 2/48 IgMκ and 9/30 IgMλ patients were not quantifiable by SPEP and 4 patients had m-IgM within the normal tIGM normal range. There were considerable differences in the m-IgM concentration as measured by the three tests. IgMκ SPEP (median 13.52g/L, range 0-52.0g/L), tIgM (18.15g/L, 3.28-65.4g/L) and IgMκ HLC (21.2g/L, 2.82-126g/L); IgMl SPEP (median 10.62/L, range 0-39.88/L), tIgM (11.30/L, 1.40-65.40/L) and IgMl HLC (13.4g/L, 0.77-67.4g/L). However, responses assigned by the test during follow-up were remarkably consistent (see table 1) SPEP v dHLC WK 0.84 (s.e. 0.045 95% c.i. 0.75-0.93), tIgM v dHLC WK 0.86 (s.e. 0.04 95% c.i. 0.78-0.94),. Interestingly, IgMκ / IgMλ ratios indicated residual disease in 5/25 patients in whom electrophoresis indicated a CR, in 2/5 of these patients the abnormal ratio was driven by a suppression of the uninvolved HLC IgM. Furthermore, in 7/25 patients tIgM values fell within the normal range during monitoring, and IFE was required to confirm m-IgM. In 7/7 patients IgMκ / IgMλ ratios remained abnormal.
Conclusion. Quantification of m-IgM can be problematic not assisted by the poor agreement between methods and method specific limitations. To adequately evaluate patients SPEP, IFE and tIgM are all required to overcome these issues; in our study these could be replaced using IgM HLC. Furthermore the sensitivity to detect residual disease appeared to be greater when looking at IgMκ / IgMλ ratios than when using electrophoretic techniques. This may be due isotype matched pair suppression which has been shown to be prognostic in other B cell disorders including MGUS and Multiple Myeloma. Further studies are required to thoroughly evaluate the role of IgM HLC and pair suppression in WM.
Table 1: Comparison of response assignment using electrophoretic techniques (SPEP/IFE), total IgM / IFE (tIgM/IFE) to IgM HLC. 25 patients were followed for up to 2220 days, with multiple responses / relapses assigned. Weighted Kappa (WK) was used to compare response assignments, WK>0.81 is considered agreement. WK SPEP/IFE v IgM HLC 0.84 (s.e. 0.045 95% c.i. 0.75-0.93), tIgM v dHLC WK 0.86 (s.e. 0.04 95% c.i. 0.78-0.94).

Disclosures
Leblond: Roche: Honoraria, Speakers Bureau.