STIMULUS REPORT Mass spectrometry vs immunofixation for treatment monitoring in multiple myeloma Noemı Puig,1,* Marıa-Teresa Contreras,2,* Cristina Agullo,2 Joaquın Martınez-L opez,3 Albert Oriol,4 Marıa-Jes us Blanchard,5 Rafael Rıos, Jes 6 7 n In us Martın, Marıa-Bele ~igo, Anna Sureda, Miguel-Teodoro Hern 8 9 andez, Javier de la Rubia,11 10 nica Gonzalez-Calle, Isabel Krsnik, Valentın Caban Vero 1 12 ~as, Luis Palomera, Jose 13 14 -Marıa Moraleda,13 Joan Bargay,15 Marıa-Teresa Cedena, Bruno Paiva, Laura Rosin 3 16 ~ol, Joan Blade 17 , Jes 17 us San Miguel,16 Juan-Jose  Lahuerta,18 and Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3234/1899297/advancesadv2021006762.pdf by guest on 06 March 2023 Marıa-Victoria Mateos 1 1 Hematology Department, IBSAL, Instituto de Biologıa Molecular y Celular del Cancer–Consejo Superior de Investigaciones Cientıficas (IBMCC-CSIC), Centro de Investigaci on Biom edica en Red de C ancer (CIBERONC); 2Clinical Biochemistry Department, Hospital Universitario de Salamanca, Salamanca, Spain; 3Hospital Universitario 12 de Octubre, Complutense University, i112, CNIO, CIBERONC CB16/12/00369, Madrid, Spain; 4Institut Catala d’Oncologia, Institut Josep Carreras Hospital Germans i Trials, Barcelona, Spain; 5Hematology Department, Hospital Ram on y Cajal, Madrid, Spain; 6Hematology Department, Hospital Universitario Virgen de las Nieves, Ibs. GRANADA, CIBERESP, Granada, Spain; Hospital Universitario Virgen del Rocıo, Sevilla, Spain; 8Hospital Clınico San Carlos, Madrid, Spain; 9Institut Catala d’Oncologia-l’Hospitalet, IDIBELL, 7 Universitat de Barcelona, Barcelona, Spain; 10Hospital Universitario de Canarias, Santa Cruz de Tenerife, Spain; 11Department of Hematology, University Hospital Le Fe and School of Medicine and Dentistry, Catholic University of Valencia, CIBERONC CB16/12/00284, Valencia, Spain; 12Hospital Puerta de Hierro, Madrid, Spain; 13Hospital Universitario Virgen de la Arrixaca, IMIB, Murcia, Spain; 14Hospital Clınico Universitario Lozano Blesa, Zaragoza, Spain; 15Hospital Son Llatzer, Palma de Mallorca, Spain; 16 Clınica Universidad de Navarra, CIMA, IDISNA, CIBERONC CB16/12/00369, Pamplona, Spain; 17Hospital Clinic, Institut d'Investigacions Biom ediques August Pi I Sunyer (IDIBAPS), Barcelona, Spain; and 18Instituto de Investigaci on del Hospital Universitario 12 de Octubre, CIBERONC, Madrid, Spain Key Points Monitoring of the monoclonal protein (M-protein) by electrophoresis and/or immunofixation (IFE) has long been used to assess treatment response in multiple  EXENT&FLC-MS is myeloma (MM). However, with the use of highly effective therapies, the M-protein becomes more sensitive than frequently undetectable, and more sensitive methods had to be explored. We applied IFE IFE in detecting the and mass spectrometry (EXENT&FLC-MS) in serum samples from newly diagnosed MM M-protein of MM patients enrolled in the PETHEMA/GEM2012MENOS65 obtained at baseline (n 5 223), and patients in serum, both at baseline and after induction (n 5 183), autologous stem cell transplantation (n 5 173), and consolidation during treatment (n 5 173). At baseline, the isotypes identified with both methods fully matched in 82.1% of monitoring. samples; in the rest but 2 cases, EXENT&FLC-MS provided additional information to IFE  with regards to the M-protein(s). Overall, the results of EXENT&FLC-MS and IFE were EXENT&FLC-MS is concordant in .80% of cases, being most discordances due to EXENT&FLC-MS1 but IFE2 more accurate than IFE to predict cases. After consolidation, IFE was not able to discriminate 2 cohorts with different median patients’ outcome. progression-free survival (PFS), but EXENT&FLC-MS did so; furthermore, among IFE2 patients, EXENT&FLC-MS identified 2 groups with significantly different median PFS (P 5 .0008). In conclusion, compared with IFE, EXENT&FLC-MS is more sensitive to detect the M-protein of patients with MM, both at baseline and during treatment, and provides a more accurate prediction of patients’ outcome. This trial was registered at www. clinicaltrials.gov as #NCT01916252. Submitted 3 December 2021; accepted 7 February 2022; prepublished online on Blood The full-text version of this article contains a data supplement. Advances First Edition 14 February 2022; final version published online 31 May 2022. © 2022 by The American Society of Hematology. Licensed under Creative DOI 10.1182/bloodadvances.2021006762. Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC- *N.P. and M.T.C. contributed equally to this study. ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other Requests for data sharing may be submitted to Marıa-Victoria Mateos (mvmateos@ rights reserved. usal.es). 3234 14 JUNE 2022 • VOLUME 6, NUMBER 11 Introduction Results and discussion First, we compared the results obtained with EXENT&FLC-MS and The monoclonal protein (M-protein), secreted by the tumor plasma IFE at baseline (Figure 1A). Both techniques provided fully matched cells in patients with multiple myeloma (MM), has long been used as results in 82.1% (183/223) of cases. Further analysis of the 40 a biomarker to evaluate treatment response.1 In fact, treatment moni- remaining samples showed that: (1) in 24 cases (dark green), both toring in MM is mainly based on the identification and quantification methods concurred with the isotype of the main clone but of the M-protein by electrophoresis and/or immunofixation (IFE) in EXENT&FLC-MS identified minor additional peaks; (2) 4 patients serum and urine samples.2 Although the clinical value of these meth- deemed non-secretory by IFE were found to have an M-protein by ods has been broadly demonstrated, the current use of highly active EXENT&FLC-MS (yellow); (3) in 5 cases (blue), EXENT&FLC-MS therapies has significantly increased the proportion of patients in detected the heavy and light chain of the M-protein but IFE only the whom the M-protein becomes undetectable by IFE during and after corresponding light chain, and in 2 (light green) IFE identified heavy and light chains but EXENT&FLC-MS only identified the light chain; treatment.3-5 In fact, recent data from the PETHEMA/GEM2012ME- Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3234/1899297/advancesadv2021006762.pdf by guest on 06 March 2023 and (4) the 5 cases highlighted in pink corresponded to complete NOS65 trial showed that standard response criteria were no longer isotyping discrepancies. These absolute isotyping discrepancies, prognostically meaningful after consolidation.6 Thus, there is an although difficult to explain, have already been described by Mills urgent need to adjust the sensitivity of the techniques used for et al.8 response assessment to the current treatment efficacy. In this report, we explore the results of mass spectrometry (MS) as an alternative We analyzed the evolution of the results in 12 follow-up samples method to detect the M-protein in serum in patients with MM, by obtained from the 24 cases in which, as compared with IFE, comparing them with those obtained using IFE. EXENT&FLC-MS identified minor additional peaks besides the main clone. Whereas in 6 of them both the main and the minor peaks Methods persisted, in 4 cases only the main peak remained (and the minor peaks disappeared); most importantly, in 2 cases, only the minor We included the first 223 out of the 458 newly diagnosed peaks exclusively identified by EXENT&FLC-MS at diagnosis per- transplant-eligible patients with MM enrolled the PETHEMA/ sisted as a marker of disease. However, further studies are needed GEM2012MENOS65 trial (#NCT01916252), an open-label, phase to ascertain whether these minor peaks represent additional tumor 3 trial encompassing the administration of 6 induction cycles of bor- clones. We identified glycosylated M-proteins in 6.3% of cases tezomib, lenalidomide, and dexamethasone and autologous stem- (n 5 14), a feature more commonly described in certain diagnoses cell transplantation (ASCT) followed by 2 consolidation cycles of (AL amyloidosis9,10 and cold agglutinin disease11) and also associ- bortezomib, lenalidomide, and dexamethasone.7 Each study site’s ated with a higher risk of progression in monoclonal gammopathy of independent ethics committee approved the protocol, and informed unknown significance.12 consent forms were required prior to patient enrollment. The study Then, we analyzed the results of both methods during treatment was conducted according to the Declaration of Helsinki. Patients’ monitoring (Figure 1B). As in previous publications,8,13,14 there was characteristics are detailed in supplemental Table 1. Serum samples substantial concordance between EXENT&FLC-MS and IFE at the were analyzed at baseline (n 5 223) and after induction (n 5 183), 3 time points analyzed: 82% post-induction, 86% post-ASCT, and ASCT (n 5 173), and consolidation (n 5 173). 84% post-consolidation. Most discordances were due to cases The presence of an M-protein in serum was assessed in parallel by EXENT&FLC-MS1 but IFE2 (15% post-induction, 12% post-ASCT, IFE on the Hydrasys 2 instrument using the Hydragel 9 kit (Sebia and 12% post-consolidation), although there were 16 samples from Inc.) and by MS using the EXENT system (The Binding Site; 14 patients found to be EXENT&FLC-MS2 but IFE1 (5, 4, and 7 Birmingham, UK). Pooled normal serum was used as a negative post-induction, ASCT, and consolidation, respectively). After a control. First, the EXENT-iP500 liquid handler purified the immuno- median follow-up of 5 years, only 2 out of these 14 patients had globulins through paramagnetic beads coated with polyclonal sheep progressed. antibodies specific for human immunoglobulin G (IgG), IgA, or Overall, 14 samples (from 11 patients) were deemed positive based IgM heavy chains, and for total k and l light chains (EXENT-MS). exclusively on the analysis of free light chains by MS (6 post-induc- Paramagnetic beads specifically recognizing free k and free l light tion, 6 post-ASCT, and 2 post-consolidation). Thus, the overall chains were also employed (EXENT&FLC-MS). Then, analysis with results of EXENT&FLC-MS (which includes the analysis of free light the EXENT-iX500 matrix-assisted laser desorption/ionization-time of chains) were almost identical to those of EXENT-MS (that excludes flight device was carried out, and mass spectra from 5000 to them) (supplemental Figure 1A). Of note, 8 of these 14 samples 32 000 mass-to-charge ratio were collected. Spectra were reviewed were obtained from 5 patients with free light chain MM according using the in-house software. The 12 charge state was used for to IFE. interpretation. The mass-to-charge ratio of the M-protein was identi- fied in baseline samples and was used as a patient-specific tumor When we investigated the clinical correlation of the results of both techniques separately, we observed that IFE was able to discrimi- marker in the subsequent ones. nate 2 subgroups of patients with significantly different median PFS Statistical analyses were performed using the GraphPad Prism v.9. after induction (IFE1 5 5.78 years vs IFE2 not reached) and post- Progression-free survival (PFS) was defined as time from sample ASCT (IFE1 5 4.98 years vs IFE2 not reached) but not post-con- collection until disease progression or death from any cause, and solidation (Figure 2A), thus confirming recent data published by curves were constructed using the Kaplan-Meier method and the Jimenez-Ubieto et al.6 In contrast, according to previous (2-sided) log-rank test. reports,15,16 the results of both EXENT-MS (supplemental Figure 14 JUNE 2022 • VOLUME 6, NUMBER 11 MASS-SPEC VS IMMUNOFIXATION FOR MYELOMA MONITORING 3235 A IFE IgGκ IgGλ IgAκ IgAλ IgMκ IgMλ Free κ Free λ Bi/Triclonal NS Total IgGκ 71 2 0 0 0 0 1 0 0 0 74 IgGλ 0 43 0 1 0 0 0 1 0 0 45 IgAκ 0 0 32 0 0 0 1 0 0 0 33 EXENT&FLC-MS IgAλ 1 0 0 18 0 0 0 1 0 0 20 IgMκ 0 0 0 0 0 0 1 0 0 1 2 IgMλ 1 0 0 0 0 0 0 0 0 0 1 Free κ 0 0 0 0 0 0 12 0 0 1 13 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3234/1899297/advancesadv2021006762.pdf by guest on 06 March 2023 Free λ 0 1 0 0 0 1 0 7 0 1 10 Bi/Triclonal 10 3 5 3 0 0 1 2 0 1 25 NS 0 0 0 0 0 0 0 0 0 0 0 Total 83 49 37 22 0 1 16 11 0 4 223 NS: non secretory B EXENT&FLC-MS + / IFE + EXENT&FLC-MS – / IFE – EXENT&FLC-MS + / IFE – EXENT&FLC-MS – / IFE + Post-induction Post-ASCT Post-consolidation 5 (3%) 4 (2%) 7 (4%) 27 (15%) 21 (12%) 21 (12%) 39 (23%) 58 (34%) 88 (48%) 63 (34%) 90 (52%) 106 (61%) Post-induction Post-ASCT Post-consolidation Concordant, n (%) 151 (82.5) 148 (85.5) 145 (83.8) Discordant, n (%) 32 (17.5) 25 (14.5) 28 (16.2) Figure 1. Comparison between EXENT&FLC-MS and IFE results. (A) M-protein(s) isotyping at baseline. (B) Detection of the M-protein post-induction, post-ASCT, and at the end of consolidation. 1B) and EXENT&FLC-MS (Figure 2B) retained clinical value at the of 3.32 years in the 21 cases EXENT&FLC-MS1 vs not reached; 3 time points, so that patients with undetectable disease by either P 5 .0008). This result, in accordance with Nandakumar et al,17 of them displayed a statistically significantly longer PFS as com- confirms that the higher sensitivity of EXENT&FLC-MS is also asso- pared with positive cases. ciated with a meaningful clinical value. Finally, we analyzed the clinical value of the combined results of IFE Overall, these data show that, as compared with IFE, EXENT&FLC- and EXENT&FLC-MS at the end of consolidation (Figure 2C). MS is able to better identify and characterize the M-protein of Importantly, among IFE2 patients (ie, in complete response or bet- patients with MM in baseline samples and detects residual disease ter; n 5 127), EXENT&FLC-MS was able to segregate 2 groups in a higher proportion of cases during treatment monitoring. This with significantly different PFS from consolidation (median PFS translates to a meaningful clinical value in terms of PFS throughout 3236 PUIG et al 14 JUNE 2022 • VOLUME 6, NUMBER 11 A Post-induction Post-ASCT Post-consolidation p=.0016 p=.0228 p=.1830 100 100 100 80 80 80 % Patients 60 60 60 40 40 40 IFE + IFE + IFE + 20 20 20 IFE – IFE – IFE – 0 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3234/1899297/advancesadv2021006762.pdf by guest on 06 March 2023 Time (years) Time (years) Time (years) HR (95% IC) 0.45 (0.28–0.73) 0.57 (0.35–0.96) 0.70 (0.40–1.23) %PFS (5ys) IFE + 54.6 47.4 53.5 %PFS (5ys) IFE – 79.7 68.0 65.4 B Post-induction Post-ASCT Post-consolidation p=.0013 p=.0170 p=.0007 100 100 100 80 80 80 % Patients 60 60 60 40 40 40 EXENT&FLC-MS + EXENT&FLC-MS + EXENT&FLC-MS + 20 20 20 EXENT&FLC-MS – EXENT&FLC-MS – EXENT&FLC-MS – 0 0 0 0 2 4 6 8 0 2 4 6 8 0 2 4 6 8 Time (years) Time (years) Time (years) HR 0.40 (0.25–0.66) 0.56 (0.35–0.91) 0.44 (0.26–0.75) %PFS (5ys) MS + 56.7 49.9 46.3 %PFS (5ys) MS – 83.7 69.3 68.4 C Post-consolidation 100 IFE + / EXENT&FLC-MS + (n=39) 80 % Patients 60 IFE – / EXENT&FLC-MS + (n=21) p=.0193 p=.0008 40 IFE + / EXENT&FLC-MS – (n=7) 20 IFE – / EXENT&FLC-MS – (n=106) 0 0 2 4 6 8 Time (years) Figure 2. PFS after induction, post-ASCT, and at the end of consolidation. (A) IFE status. (B) EXENT&FLC-MS status. (C) combined IFE and EXENT&FLC-MS status. HR, hazard ratio. 14 JUNE 2022 • VOLUME 6, NUMBER 11 MASS-SPEC VS IMMUNOFIXATION FOR MYELOMA MONITORING 3237 the treatment that IFE fails to show post-consolidation, and further- as consultancy and served on a speakers bureau for Celgene and more, the results of EXENT&FLC-MS identify 2 groups of patients Amgen and served as consultancy for Janssen, Sanofi, and GSK. among those IFE2 with significantly different PFS. Further studies R.R. has received honoraria from or or served in an advisory role for comparing the results of MS with bone marrow–based MRD meth- Becton-Dickinson, Sanofi, and The Binding Site. J.M. has received ods, such as next-generation flow and next-generation sequencing, registration fees and travel and accommodation fees from Janssen- are warranted. Cilag, Amgen, Sandoz, and Bio-Rad Laboratories. M.-T.H. served in a consulting or advisory role for Celgene, Janssen, Amgen, Takeda, Acknowledgments and GSK and served on a speakers bureau for Janssen, Celgene, and Amgen. J.d.l.R. has served as a consultant and provided expert This study was supported by grants from the Centro de Inves- testimony within the past 2 years for Amgen, Celgene, GSK, tigaci on Biom  edica en Red–Area de Oncologıa–del Instituto de Takeda, Janssen, and Sanofi. V.G.C. has received honoraria from Salud Carlos III CIBERONC, CB16/12/00369, CB16/12/00400, Janssen and Celgene and research funding from Janssen (BECA CB16/12/00233, and CB16/12/00284, Instituto de Salud Carlos SEHH-JANSSEN ESTANCIAS DE FORMACION  EN EL EXTRA- Downloaded from http://ashpublications.org/bloodadvances/article-pdf/6/11/3234/1899297/advancesadv2021006762.pdf by guest on 06 March 2023 III/Subdirecci on General de Investigaci on Sanitaria FIS no. PI15/ NJERO 2016-2017) and served in a consulting or advisory role for 01956, PI15/02049, PI15/02062, PI18/01709, PI18/01673, and Prothena and Janssen. J.M.M. served in a consultancy or advisory PI19/01451, the Cancer Research UK, FCAECC, and AIRC role for Novartis, Gilead, Roche, Sanofi, Jazz, and Takeda. L.R. has under the Accelerator Award Program (EDITOR). received honoraria from Janssen, Celgene, Amgen, and Takeda; J.B. has received honoraria from Janssen, Celgene, Takeda, Amgen, and Authorship Oncopeptides. J.S.M. has received consultancy or served in an advi- sory role for Abbvie, Amgen, Bristol Myers Squibb, Celgene, Glax- Contribution: N.P., M.T.C., and M.-V.M. conceived the analysis; N.P., oSmithKline, Janssen, Karyopharm, MSD, Novartis, Roche, Sanofi, M.T.C., M.-V.M., L.R., J.B., J.S.M., and J.-J.L. designed the analysis pro- SecuraBio, and Takeda. J.-J.L. served in a consulting or advisory tocol; N.P. and M.T.C. analyzed the MS data and the M-protein kinet- role for Celgene, Takeda, Amgen, Janssen, and Sanofi and has ics; N.P., M.T.C., and M.-V.M. analyzed and interpreted data; N.P. and received travel accommodations and expenses for Celgene. M.-V.M. M.T.C. performed statistical analysis; N.P., M.T.C., and M.-V.M. wrote has received honoraria from and served as membership on an the manuscript; and all authors provided study material or patients entity’s Board of Directors or advisory committees for Janssen, Cel- and reviewed and approved the manuscript. gene, Takeda, Amgen, Adaptive, GSK, Sanofi, and Oncopeptides Conflict-of-interest disclosure: N.P. has received honoraria from and has received honoraria from membership in Board of Directors Amgen, Celgene, Janssen, Takeda, and The Binding Site; served in or advisory committees for Abbvie, Roche, Pfizer, Regeneron, and a consulting or advisory role for Amgen, Celgene, Janssen, and Seattle Genetics. The remaining authors declare no competing Takeda; served on a speakers bureau for Celgene; received financial interests. research funding from Celgene, Janssen, Amgen, and Takeda; ORCID profiles: J.M.-L., 0000-0001-7908-0063; A.O., 0000- received travel, accommodations, and expense fees from Amgen, 0001-6804-2221; R.R., 0000-0001-8193-1402; M-T.H., 0000- Celgene, Janssen, and Takeda. B.P. has served as consultancy and 0002-6576-7881; J.d.l.R., 0000-0002-8354-768X; V.G-C., 0000- received honoraria and research funding and served on a speakers 0002-5493-6707; I.K., 0000-0002-2912-5072; V.C., 0000-0002- bureau for Amgen, Bristol Myers Squibb, Celgene, Janssen, Novar- 6248-2549; J-M.M., 0000-0001-9080-1466; M-T.C., 0000-0001- tis, Roche, and Sanofi; received unrestricted grants from Celgene, 5851-3720; B.P., 0000-0003-1977-3815; J.S.M., 0000-0002- EngMab, and Takeda; and served as consultancy for Celgene, Jans- 9183-4857; J-J.L., 0000-0002-3393-9570; M-V.M., 0000-0003- sen, and Sanofi. M.T.C. has received honoraria from Janssen, Cel- 2390-1218. gene, and Abbvie. J.M.-L. served as consultancy, has received honoraria and research funding, and served on a speakers bureau Correspondence: Marıa-Victoria Mateos, Hospital Universi- for Amgen, Astellas, Bristol Myers Squibb, Janssen, Novartis, Roche, tario de Salamanca, Paseo de San Vicente 58-182, CP 37007, and Sanofi, and received unrestricted grants from BMS. A.O. served Salamanca, Spain; e-mail: mvmateos@usal.es. References 1. Tate JR. The paraprotein—an enduring biomarker. Clin Biochem Rev. 2019;40(1):5-22. 2. Kumar S, Paiva B, Anderson KC, et al. International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma. Lancet Oncol. 2016;17(8):e328-e346. 3. Lahuerta J-J, Paiva B, Vidriales M-B, et al; GEM (Grupo Espa~ nol de Mieloma)/PETHEMA (Programa para el Estudio de la Terap eutica en Hemopatıas Malignas) Cooperative Study Group. 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