
==== Front
Blood
Blood
Blood
0006-4971
1528-0020
The American Society of Hematology

S0006-4971(24)01243-6
10.1182/blood.2024024041
Clinical Trials and Observations
Mass spectrometry–based assessment of M protein in peripheral blood during maintenance therapy in multiple myeloma∗
Kubicki Tadeusz 12
Dytfeld Dominik 2
Barnidge David 3
Sakrikar Dhananjay 3
Przybyłowicz-Chalecka Anna 2
Jamroziak Krzysztof 4
Robak Paweł 5
Czyż Jarosław 6
Tyczyńska Agata 7
Druzd-Sitek Agnieszka 8
Giannopoulos Krzysztof 9
Wróbel Tomasz 10
Nowicki Adam 2
Szczepaniak Tomasz 2
Łojko-Dankowska Anna 2
Matuszak Magdalena 2
Gil Lidia 2
Puła Bartosz 11
Szukalski Łukasz 6
Końska Agnieszka 11
Zaucha Jan Maciej 7
Walewski Jan 8
Mikulski Damian 5
Czabak Olga 12
Robak Tadeusz 5
Jiang Ken 1
Cooperrider Jennifer H. 1
Jakubowiak Andrzej J. 1
Derman Benjamin A. bderman@bsd.uchicago.edu
1∗
1 Section of Hematology/Oncology, University of Chicago, Chicago, IL
2 Poznań University of Medical Sciences, Poznań, Poland
3 Binding Site, part of Thermo Fisher, Rochester, NY
4 Medical University of Warsaw, Warsaw, Poland
5 Medical University of Łódź, Łódź, Poland
6 Nicolaus Copernicus University in Toruń, Ludwik Rydygier Collegium Medicum in Bydgoszcz, Bydgoszcz, Poland
7 Medical University of Gdańsk, Gdańsk, Poland
8 Maria Sklodowska-Curie National Research Institute of Oncology, Warsaw, Poland
9 Department of Experimental Hematooncology, Medical University of Lublin, Lublin, Poland
10 Wrocław Medical University, Wrocław, Poland
11 Institute of Hematology and Blood Transfusion, Warsaw, Poland
12 Medical University of Lublin, Lublin, Poland
∗ Correspondence: Benjamin A. Derman, University of Chicago, 5841 South Maryland Ave, M/C 2115, Chicago, IL 60637; bderman@bsd.uchicago.edu
09 5 2024
29 8 2024
09 5 2024
144 9 955963
13 2 2024
25 4 2024
© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.
2024
American Society of Hematology
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Key Points

• MS provides significant prognostic information in the maintenance setting and complements BM MRD in multiple myeloma.

• Without the aid of a baseline serum sample, MS’s prognostic performance is most significant 18 months after transplant.

Visual Abstract

Abstract

Mass spectrometry (MS) can detect multiple myeloma–derived monoclonal proteins in the peripheral blood (PB) with high sensitivity, potentially serving as a PB assay for measurable residual disease (MRD). This study evaluated the significance of PB MS MRD negativity during posttransplant therapy in patients with newly diagnosed multiple myeloma. Serum samples from 138 patients treated in the phase 3 ATLAS trial of posttransplant maintenance with either carfilzomib, lenalidomide, and dexamethasone, or with lenalidomide alone were analyzed using EXENT MS methodology. We established feasibility of measuring MRD by MS in the PB in the posttransplant setting, despite unavailability of pretreatment calibration samples. There was high agreement between MRD by MS in the PB and paired bone marrow (BM) MRD results at the 10–5 threshold, assessed by either next-generation sequencing (NGS) or multiparameter flow cytometry (MFC) (70% and 67%, respectively). Agreement between PB MS and both BM MRD methods was lowest early after transplant and increased with time. MS negativity was associated with improved progression-free survival (PFS), which, in landmark analysis, reached statistical significance after 18 cycles after transplant. Combined PB/BM MRD negativity by MFC or NGS was associated with superior PFS compared with MRD negativity by only 1 modality. Sustained MS negativity carried similar prognostic performance to sustained BM MRD negativity at the 10–5 threshold. Overall, posttransplant MS assessment was feasible and provided additional prognostic information to BM MRD negativity. Further studies are needed to confirm the role and optimal timing of MS in disease evaluation algorithms. The ATLAS trial is registered at www.clinicaltrials.gov as #NCT02659293.

Mass spectrometry (MS) is a sensitive method for detecting myeloma-derived monoclonal proteins in the peripheral blood to assess measurable residual disease (MRD). Kubicki et al investigated the predictive value of peripheral blood negativity by MS in 138 patients in a phase 3 trial of posttransplant maintenance therapy. MS assessment was feasible and correlated well with bone marrow MRD by next-generation sequencing or multiparameter flow cytometry, though the combination of the 2 provided slightly better predictive value. This raises the possibility that prospective MRD assessment can be obtained without frequent bone marrow assessment.
==== Body
pmcIntroduction

The tremendous progress in therapies for patients living with multiple myeloma has resulted in a significant improvement in prognosis, attributed mostly to increases in the depth and durability of response.1,2 These improvements have increased the need for more sensitive tools for response assessment, particularly in patients who achieve the deepest levels of response. Standard definitions of response to therapy, which rely on measuring myeloma-associated monoclonal protein through serum protein electrophoresis (SPEP) with immunofixation (IFE), assessing serum free light chains ratio and cytomorphological evaluation of bone marrow (BM) biopsies through immunohistochemistry, lack the necessary sensitivity for measuring residual disease beyond the level of complete response.3,4 Evaluation of measurable (minimal) residual disease (MRD) in the BM using either multiparameter flow cytometry (MFC) or next-generation sequencing (NGS) enables the identification of residual myeloma cells in patients who achieve complete response by standard response assessment, with a limit of detection (LoD) as low as 1 myeloma cell per million nucleated cells analyzed.5,6

In addition to providing a more sensitive measure of response, MRD negativity has proven to be 1 of the most important prognostic markers in multiple myeloma, regardless of the disease setting.7 With increasing evidence that MRD negativity is associated with improved progression-free survival (PFS) and overall survival, MRD is now being used as a primary end point in randomized phase 3 trials and as a guide to change treatment as part of MRD-adapted study designs.8, 9, 10, 11, 12, 13 Nevertheless, the assessment of MRD in the BM using current prevailing methods has several important limitations, including a requirement for inconvenient BM biopsies and the possibility of false negative results due to the presence of extramedullary lesions or the patchy nature of the disease.14 These limitations could be overcome by peripheral blood-based assays following the paradigm of liquid biopsy. Recently, sensitive measurement of serum monoclonal (M) protein concentration with mass spectrometry (MS) has emerged as a powerful tool capable of capturing lower concentrations of the M protein than those detectable by SPEP and IFE.15 Several studies have already shown that MS outperforms SPEP and IFE as a more sensitive method of detecting residual M protein, and emerging evidence shows that it can prognostically complement BM-based MRD assessments.16, 17, 18, 19, 20

MS limitations include the potential to falsely indicate the persistence of disease because of delayed clearance of M protein from the serum (particularly for recirculating immunoglobulin G [IgG]21), and the need for a baseline serum sample to enable the tracking of the unique mass-to-charge ratio (m/z) of the myeloma-associated M protein.22 The feasibility and potential utility of disease assessment by MS in patients lacking this baseline spectral signature, particularly after autologous hematopoietic stem cell transplantation (ASCT), has not been studied to date.

Here, we present the results of MS-based assessment of M protein in the peripheral blood without availability of pretreatment samples, as performed on patients with multiple myeloma undergoing maintenance therapy after ASCT in the randomized phase 3 ATLAS trial.

Methods

Patients and study design

ATLAS (clinicaltrials.gov identifier: NCT02659293) is an ongoing, open-label, randomized, phase 3 trial comparing post-ASCT treatment with lenalidomide alone (R) vs MRD-directed, risk-adapted combination of carfilzomib, R, and dexamethasone (KRd). The study, its protocol, and amendments were approved by the institutional review board or ethics committee at each participating institution and the Office for Registration of Medicinal Products, Medical Devices and Biocidal Products in Poland. The study is conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization of Good Clinical Practice guidelines.

The detailed design of the ATLAS trial has been described previously.8 Briefly, patients with multiple myeloma that completed any induction therapy and within 100 days of ASCT were eligible to enroll. Only patients with available stored serum samples and who consented for exploratory testing were included in this analysis.

Treatment

Details of the assigned treatments have been published previously.8 In short, patients were randomized in a 1:1 ratio to either R or KRd. Randomization was stratified by cytogenetic risk (high vs standard), response after ASCT (at least very good partial response vs less than very good partial response), and country of enrollment (Poland vs United States). Patients in the KRd arm who had no detectable MRD at the 10–5 threshold as defined by the International Myeloma Working Group guidelines23 after cycle 6, and had no high-risk cytogenetic features as defined in the protocol (absence of del13, t(4;14), t(14;16), del17p, or hypodiploidy), de-escalated to single-agent R starting with cycle 9. Patients in the R maintenance group received R alone. After 36 cycles of treatment on both arms, all patients were assigned to receive R maintenance. Treatment continued until disease progression, unacceptable toxicity, or patient’s decision.

MRD assessment

MRD was assessed by centralized NGS using clonoSEQ (performed at Adaptive Biotechnologies, Seattle, WA; LoD of 6.8 × 10−7 with input of 20 μg DNA) and centralized and standardized MFC (minimum sensitivity of 10−5 based on the method of the EuroFlow Consortium24,25) performed either at the University of Chicago (Chicago, IL) or Poznań University of Medical Sciences (Poznań, Poland). MRD evaluations were performed at screening and after cycles 6, 12, 18, 24, and 36. Unless stated otherwise, MRD positivity by NGS refers to patients with MRD detected above the 10–5 threshold. For MRD by NGS, tracking was only enabled if a baseline sample was available and evaluable for clonotypic tracking. This restriction did not apply to MRD by MFC.

MS

Stored serum samples collected at screening (3 months after transplant) and after cycles 6, 12, 18, 24, and 36 were analyzed using the EXENT Immunoglobulin isotypes (GAM) assay (EXENT Solution, The Binding Site, part of Thermo Fisher Scientific), which uses matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) MS. EXENT is an integrated clinical analyzer under development that consists of an automated liquid handler (EXENT-iP500), a MALDI-TOF mass spectrometer (EXENT-iX500), and instrument control/data analysis software (EXENT-iQ) for the detection and quantification of monoclonal immunoglobulins in serum. The device uses quantitative IgG, IgA, and IgM results (generated on the patient sample with the Optilite IgG, IgA, and IgM assays) combined with the EXENT assay peak areas in the mass spectra to calculate M-protein concentrations. The method, as described previously, allows for identification of M protein with a lower LoD of 0.015 g/L.26, 27, 28 For the current analysis, no baseline diagnostic samples were available, and samples were not available for every patient at every time point. A sample was called positive by MS when the EXENT solution modeled a peak with a concentration higher than that of the LoD, matching the known (diagnostic) isotype of the M protein (supplemental Figure 1, available on the Blood website). If multiple peaks were detected with the same isotype as the M protein at baseline, the peak with the highest concentration was assumed to represent the baseline M protein.

Statistical analysis

Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated, with MRD by NGS or MFC serving as a reference. The numbers are presented with 95% confidence intervals (CIs). Time-to-event analyses such as PFS and overall survival were performed using the Kaplan-Meier method and groups were compared using the log-rank test. Hazard ratios (HRs) and 95% CIs for survival data were derived from Cox proportional hazards regression models. Statistical analysis was performed using GraphPad Prism version 10.1 and R software (version 4.3.0). All survival analyses were conducted using the same data set from the unplanned interim report.8

The study, its protocol, and amendments were approved by the institutional review board or ethics committee at each participating institution and the Office for Registration of Medicinal Products, Medical Devices and Biocidal Products in Poland.

Results

Of 180 patients enrolled in the ATLAS trial, serum samples for MS were evaluable for 138 patients from at least 1 time point. This analysis was performed using clinical data from the previously reported cutoff (31 December 2021), the median follow-up from randomization for these 138 patients was 33.3 months (interquartile range, 9.2-40.7). Baseline characteristics are summarized in Table 1. Overall, M protein was assessed in 585 serum samples across all time points, including 349 paired results for MS and MRD by NGS, as well as 499 paired results for MS and MRD by MFC. A complete breakdown of samples evaluated at different time points can be found in supplemental Table 1. At screening, MS was positive in 66 (55%) patients, decreasing to 46 (39%) after cycle 6, 39 (34%) after cycle 12, 28 (29%) after cycle 18, 24 (30%) after cycle 24, and 12 (22%) after cycle 36. The respective numbers among patients with paired BM MRD and MS results available were 38 (54%), 30 (41%), 23 (64%), 16 (28%), 15 (29%), and 12 (35%) for MRD by NGS; whereas for MRD by MFC, the counts were 34 (32%), 16 (15%), 17 (17%), 10 (13%), 15 (21%), and 7 (18%). Notably, MS was positive in a significant proportion (21%) of IFE-negative samples (supplemental Table 3). This further translated into better outcomes of patients in complete response who achieved MS negativity as a best response, than those who remained MS positive (HR, 0.33 [0.11-0.98]; P = .003; supplemental Figure 7).Table 1. Baseline characteristics

Variable	Subcategory	n (%)	
Age (y, median, IQR)		57 (47-63)	
Sex	Male	73 (53%)	
 	Female	65 (47%)	
Study arm	KRd	73 (53%)	
 	R	65 (47%)	
M protein isotype	IgG κ	75 (54%)	
 	IgG λ	27 (19%)	
 	IgA κ	16 (12%)	
 	IgA λ	4 (3%)	
 	IgM κ	1 (1%)	
 	IgM λ	3 (2%)	
 	FLC κ	10 (7%)	
 	FLC λ	2 (2%)	
IMWG response at study entry	<VGPR	16 (12%)	
 	VGPR	73 (53%)	
 	CR	31 (22%)	
 	sCR	18 (13%)	
MRD by NGS at study entry	Negative	35 (25%)	
 	Positive	40 (29%)	
 	Not evaluated	63 (46%)	
MRD by MFC at study entry	Negative	82 (59%)	
 	Positive	39 (29%)	
 	Not evaluated	17 (12%)	
ISS	1	49 (35%)	
 	2	67 (48%)	
 	3	22 (17%)	
Cytogenetic risk	Standard	104 (75%)	
 	High	34 (25%)	
Time from ASCT (d, median, IQR)	 	92.5 (76-114.3)	
CR, complete response; FLC, free light chain; IMWG, International Myeloma Working Group; IQR, interquartile range; ISS, International Staging System; sCR, stringent complete repsonse; VGPR, very good partial response.

When patients with MRD of ≥10–5 were considered MRD positive by NGS, the overall agreement between MS and NGS was 70%. With NGS as the reference standard, sensitivity and specificity of MS was 63% (54%-71%) and 74% (68%-80%), respectively. Discordant cases were split between MS+/NGS– (56 of 349, 16%) and MS–/NGS+ (50 of 349, 14%) cases, with the PPV of 60% (54%-66%) and NPV of 76% (72%-80%). The agreement was lowest at screening (61% agreement) but improved in the subsequent evaluations, reaching the highest level at cycle 18 (74% agreement; Figure 1A). Additionally, the highest values for NPV and specificity were observed at cycle 18, whereas PPV was highest at cycle 6 and sensitivity at screening (supplemental Table 2). We also analyzed the agreement between MS evaluation and NGS results at different cutoffs for MRD positivity (supplemental Figure 2). As expected, when we considered MRD of ≥10–4 as positive, we observed higher proportion of MS+/NGS– samples (82 of 349, 23%) as opposed to MS–/NGS+ (20 of 349, 6%). Accordingly, when we used the more sensitive threshold of 10–6 for MRD negativity, there were less MS+/NGS– samples (28 of 294, 9%) than MS–/NGS+ samples (75 of 294, 26%). The smaller denominator in the latter analysis is because of the fact that not all samples reached the sensitivity of 10–6 for the NGS evaluation.Figure 1. Agreement between peripheral blood MS and MRD in the BM. Rates of concordant and discordant cases assessed at different time points by NGS at the 10–5 threshold (A) and by MFC (B).

With MRD assessment by MFC as the reference standard, the overall agreement with MS was 67%, with a sensitivity of 60% (49%-69%) and specificity of 69% (64%-73%). The discrepancy between MRD by MFC and MS was mostly driven by patients with MS+/MFC– patients (125 of 499, 25%), whereas MS–/MFC+ was less frequent (40 of 499, 8%). The PPV was 32% (28%-37%) and the NPV was 87% (84%-90%). The agreement was lowest at screening (60% agreement) and increased at the subsequent timepoints, with the highest agreement observed after cycle 18 (73% agreement; Figure 1B). Sensitivity and NPV were highest after cycle 6, specificity peaked after cycle 18, and the PPV was at its maximum at screening (supplemental Table 2).

We then analyzed the prognostic implications of M protein detection by MS at different time points, focusing on the first 4 landmarks because of lower sample sizes at the later time points. Landmark analyses did not confirm a statistically significant association of MS status with PFS when assessed at screening and after cycles 6 or 12 (supplemental Figure 3), although the effect size at screening (HR, 0.52; 95% CI, 0.26-1.05; P = .06) and cycle 12 (HR, 0.61 [0.30-1.25]; P = .18) were suggestive of a possible association with PFS. This was, however, not true for cycle 6 (HR, 1.20 [0.6-2.39]; P = .6). Finally, using the landmark method, MS negativity after cycle 18 was associated with significantly superior PFS (HR, 0.39 [0.17-0.91]; P = .025); the direction and magnitude of benefit were similar regardless of the treatment arm. Similarly, C18 BM MRD-negative status was associated with superior PFS for both MFC (HR, 0.13 [0.04-0.35]; P = .0002) and NGS (HR, 0.15 [0.03-0.63]; P = .003; Figure 2).Figure 2. Landmark analysis (after cycle 18) of PFS. PFS differences between positive and negative patients by (A) MS, (B) NGS at the 10–5 threshold, and (C) MFC. Not all patients included in this analysis had the BM MRD testing results available, hence the lower numbers of cases presented in panels B and C.

We then evaluated the effect of combined peripheral blood MS and BM MRD status on PFS (Figure 3; supplemental Figure 4). Using the same landmark analysis after cycle 18, combined peripheral blood and BM negativity (“double negative”) was associated with superior PFS compared with those negative by only 1 modality; this was true for both MS/MFC (PFS: HR, 0.30 [0.11-0.87]; P = .018) and for MS/NGS (PFS: HR, 0.13 [0.01-1.16]; P = .038).Figure 3. Combined impact of peripheral blood MS and BM MRD on PFS. Comparison of PFS in landmark analysis after cycle 18 between those who were double negative (BM and peripheral blood) and those negative in 1 but positive in the other modality. (A) MS and MFC and (B) MS and NGS at the 10–5 threshold.

When we examined the added value of the MS result (positive or negative) among only BM MRD-negative cases at the same specific landmark time point, we observed a potentially favorable PFS benefit for patients with concurrent BM MRD negativity and MS negativity but the difference did not reach statistical significance: NGS−/MS− vs NGS−/MS+ (HR, 0.14 [0.01-1.58]; P = .06) and MFC−/MS− vs MFC−/MS+ (HR, 0.40 [0.13-1.27]; P = .11; supplemental Figure 5). This benefit was further suggested when we analyzed best responses, throughout all analyzed time points. We observed improved PFS for patients who reached BM MRD negativity (at the 10–5 threshold, when both NGS and MFC results were available, at least 1 positive result qualified a sample as positive) and MS negativity as best response compared with MS positivity with BM MRD negativity at the same time points (HR, 0.36 [0.10-1.31]; P = .03; supplemental Figure 6).

Finally, we examined the prognostic implications of sustained (≥12 months) negativity in the peripheral blood and BM at different sensitivity thresholds. This analysis included only patients that achieved 2 consecutive negative test results in landmark time points (screening, and cycles 6, 12, 18, 24, and 36) separated by at least 12 months. Among the 38 patients with sustained MS negativity, 34 also had sustained BM MRD negativity at the 10–5 threshold. PFS was similar for those with sustained BM MRD negativity at 10–5 vs sustained peripheral blood MS negativity (Figure 4). Sustained BM MRD negativity at 10–6 assessed by NGS appeared to be associated with potentially better PFS than sustained MS negativity by EXENT, although it did not yet reach statistical significance at the current data cutoff (Figure 4).Figure 4. Sustained MS and MRD negativity. PFS of patients with sustained (>12 months) undetectable M protein in the peripheral blood by MS and MRD negative in the BM at the 10–5 and 10–6 thresholds.

Discussion

To our knowledge, this is the first report on the feasibility of MS-based assessment of M protein presence in patients with multiple myeloma after ASCT, performed without access to pretreatment baseline serum calibration samples. Consistent with previous reports,17 MS was able to identify the presence of an M protein in a significant proportion of IFE-negative samples. Moreover, our results indicate that peripheral blood-based MRD status determined by EXENT MS tracks closely with BM-based MRD by NGS or MFC at the 10–5 level of sensitivity. In addition, it appears that when added to BM MRD status, MS status further discriminates outcomes in patients with multiple myeloma receiving posttransplant maintenance.

These results show the feasibility of measuring M protein by EXENT MS platform in the posttransplant setting, even without availability of pretreatment calibration samples. Pretreatment samples were also not available in the Mass-Fix analysis in the STAMINA trial.18 However, in that study, the first serum samples were obtained before ASCT, when residual M protein is expected to be higher than after ASCT, and the proportion of patients in very good partial response or better was lower than in our analysis (47% vs 88%), suggesting that there was higher likelihood of sufficient concentration of paraprotein at screening to allow for the assignment of the monoclonal light chain m/z value. Another key difference is that our analysis includes longer serial sampling for MS, extending as far as 36 cycles, compared with only 1 year of serial testing in the STAMINA trial.

MS assessment of residual disease in the blood in the post-ASCT setting investigated in our study by the EXENT platform and by Mass-Fix in the STAMINA trial, both illustrate the emerging value of MS evaluations as a prognostic tool. The results from both studies indicate that procuring samples for calibration in the peri-ASCT period can still allow for MS evaluation of M protein in the posttransplant setting to be feasible. This is important when considering that many patients with myeloma start treatment in nonacademic settings, in which serum samples may not be stored for initial MS evaluation. The clonotypic peptide approach may enable the use of archived SPEP gels for identifying clonotypic peptides and therefore detect M protein in tracking samples.22,29

Our study, to our knowledge, contains 1 of the largest sets of paired MS results along with standard BM MRD assessments published to date. Unsurprisingly, the discrepancies between M protein assessment in the peripheral blood and disease burden in the BM in this study were highest early after ASCT (within 100 days). We attribute these differences to a higher rate of MS positive results, which could be attributed to either prolonged clearance of IgG monoclonal proteins (which were present in 73% of patients) or the presence of posttransplant oligoclonal responses.21,30 False positives from oligoclonal bands can be circumvented if the m/z value for the M protein light chain is derived from a pretreatment serum sample. At later time points, the agreement between the BM and MS methods increased; however, ∼30% were still discrepant, especially when MS was compared with MFC MRD evaluation. This suggests superior sensitivity of MS compared with MFC at the 10–5 threshold, which is aligned with the recent findings by Claveau et al and a large analysis of the STAMINA trial.18,31 These studies, however, did not include MRD analysis by NGS or MRD testing with a similar lower LoD (10–6), which is another unique aspect of this analysis.

The observed significant difference in PFS by MS status using a landmark analysis after cycle 18 is consistent with observations regarding delayed clearance of paraprotein after tumor lysis in the BM. This finding corroborates the findings of a previous study, in which MS status after KRd induction, ASCT, and KRd consolidation was associated with PFS,16 along with results from several other studies in which the prognostic significance of MS evaluation increased with time.18,19 The seemingly paradoxical results observed after cycle 6 may be, at least in part, related to the study design which includes de-escalation of the therapy in the subset of standard risk patients after cycle 8, based on BM MRD status assessed after cycle 6. This landmark analysis may be particularly susceptible to confounding factors arising from different treatment. Importantly, in the landmark analysis of MS status after cycle 18, HRs for PFS were similar in both ATLAS study arms.

Our results further support the potential role of multimodal response assessment as evidenced by the superior prognosis for patients with “double MRD negativity” (negative in peripheral blood and in BM) compared with single modality MRD negativity. Although this complementarity has already been reported when calibration samples have been available,19,32 it is encouraging that the same principle applies in the post-ASCT setting without available calibration samples. Although our results suggest a complementarity of blood-based MS and BM-based NGS or MFC MRD evaluations, they do not justify forgoing a BM evaluation at this time. The impact of sustained BM and blood-based MRD negativity on PFS support future evaluations of the prognostic impact of other blood-based MRD methodologies, including more sensitive MS techniques along with alternative methods such as blood-based NGS. For the time being, our data show that sustained BM MRD negativity at the 10–6 threshold still carries the greatest prognostic significance and remains the most desirable goal of therapy. However, this may be challenged in future by applying more sensitive methods of MS-based disease assessment, using liquid chromatography MS and clonotypic peptide approach that are ∼10 and 100 times more sensitive than the EXENT platform.28,33,34 Nonetheless, these methods are time consuming, labor intensive, and not automated. Their widespread applicability requires further studies.

Perhaps the most significant limitation of this study is the retrospective nature of the MS assessment, in which all stored serum samples were analyzed in the same batch and were not available for every patient at every time point. However, based on the criteria used to classify a sample as negative via MS, the subsequent identification of the specific m/z value for the M protein light chain for later time points resulted in a shift from positive to negative in only a small subset of patients (6 of 138). This alteration was observed exclusively during the early time point screening and cycle 6. Another limitation may lie in the fact that, for the landmark analyses, time of follow-up is relatively short and the numbers of patients at later time points preclude definitive conclusions. Consistent with other similar studies,18,19 we refrained from adjusting P value for comparisons at multiple time points, guided by our primary focus on avoiding type 2 rather than type 1 errors in this exploratory analysis,35 the kinetics of MRD that lead to a different composition of patients at each time point, and the aforementioned biological plausibility that later time points are more reliable because of immunoglobulin recycling.21 It is also essential to acknowledge that MALDI-TOF intact immunoglobulin MS for detecting low concentrations of M proteins is most effective when a baseline sample is available. Future studies on multimodal disease assessment should strive to include the calibration step, as, for example, in the currently ongoing prospective EMN33 TAURUS study.

In summary, this analysis provides rationale for MS-based disease evaluation even in the posttransplant setting without a pretreatment calibration sample. In this study posttransplant cycle 18 appeared to be an optimal time point for prognostic assessment by MS. Further implementation of this technique requires subsequent prospective investigations to confirm the optimal timing for MS assessment to best inform prognosis; clinical practice; and, potentially, 1 day, to guide treatment intensity and duration.

Conflict-of-interest disclosure: T.K. reports financial support for attending meetings from Janssen. D.D. reports speaker honoraria and participation in advisory boards for Amgen and Celgene (Bristol Myers Squibb); and had conference fees paid by Amgen. D.B. is a current employee of The Binding Site. D.S. is a current employee of The Binding Site. B.P. served as a consultant for AbbVie, Roche, and Sandoz; and received honoraria and research funding from AbbVie, Amgen, Gilead, Celgene, and Janssen. J.W. reports consultancy for AbbVie, Gilead, Novartis, Roche, and Takeda; reports research or clinical trial funding from GlaxoSmithKline, Novartis, and Roche; and received honoraria from AbbVie, Amgen, Gilead, GlaxoSmithKline, Novartis, Roche, and Takeda. A.J.J. reports consulting fees and honoraria for lectures and presentations from AbbVie, Amgen, Celgene (Bristol Myers Squibb), Gracell, GlaxoSmithKline, Janssen, and Sanofi-Aventis. B.A.D. reports consulting fees from COTA Healthcare, Janssen, and Sanofi; and reports payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from MJH Life Sciences and Plexus Communications. A.D.-S. reports honoraria for lectures and presentations, travel grants, and participation in advisory boards for Amgen and Bristol Myers Squibb. The remaining authors declare no competing financial interests.

Supplementary Material

Supplemental Tables and Figures

Acknowledgments

The authors thank Gabriella Lakos (The Binding Site, part of Thermo Fisher, Rochester, NY) for her feedback on the manuscript text and study design.

The ATLAS study was funded by Amgen and Celgene.

The sponsors had no role in data collection, analysis, or interpretation.

Authorship

Contribution: D.D. and A.J.J. conceptualized, designed, and acquired funding, and were responsible for administration and supervision of the 10.13039/501100014713 ATLAS trial; T.K., D.D., A.J.J., and B.A.D. designed the study; D.B. and D.S. performed laboratory evaluations; T.K., D.D., D.B., D.S., A.J.J., and B.A.D. analyzed the results; T.K. made the figures; T.K., D.D., D.B., A.J.J., and B.A.D. drafted the first version of the manuscript; and all authors were involved in data collection and edited and approved the final version of the manuscript.

∗ A.J.J. and B.A.D. are joint senior authors and contributed equally to this study.

Presented in abstract form at the 65th annual meeting of the American Society of Hematology, San Diego, CA, 9 December 2023.

Deidentified participant data will be made available to investigators upon reasonable request from the corresponding author, Benjamin A. Derman (bderman@bsd.uchicago.edu).

The online version of this article contains a data supplement.

There is a Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.
==== Refs
References

1 Moore KLF Turesson I Genell A Improved survival in myeloma patients–a nationwide registry study of 4,647 patients ≥75 years treated in Denmark and Sweden Haematologica 108 6 2023 1640 1651 36300775
2 Rajkumar SV Multiple myeloma: 2022 update on diagnosis, risk-stratification and management Am J Hematol 97 8 2022 1086 1107 35560063
3 Jiménez-Ubieto A Paiva B Puig N Validation of the International Myeloma Working Group standard response criteria in the PETHEMA/GEM2012MENOS65 study: are these times of change? Blood 138 19 2021 1901 1905 34329372
4 Paiva B San-Miguel J Avet-Loiseau H MRD in multiple myeloma: does CR really matter? Blood 140 23 2022 2423 2428 35560160
5 Paiva B Puig N Cedena M-T Measurable residual disease by next-generation flow cytometry in multiple myeloma J Clin Oncol 38 8 2020 784 792 31770060
6 Perrot A Lauwers-Cances V Corre J Minimal residual disease negativity using deep sequencing is a major prognostic factor in multiple myeloma Blood 132 23 2018 2456 2464 30249784
7 Munshi NC Avet-Loiseau H Anderson KC A large meta-analysis establishes the role of MRD negativity in long-term survival outcomes in patients with multiple myeloma Blood Adv 4 23 2020 5988 5999 33284948
8 Dytfeld D Wróbel T Jamroziak K Carfilzomib, lenalidomide, and dexamethasone or lenalidomide alone as maintenance therapy after autologous stem-cell transplantation in patients with multiple myeloma (ATLAS): interim analysis of a randomised, open-label, phase 3 trial Lancet Oncol 24 2 2023 139 150 36642080
9 Costa LJ Chhabra S Medvedova E Daratumumab, carfilzomib, lenalidomide, and dexamethasone with minimal residual disease response-adapted therapy in newly diagnosed multiple myeloma J Clin Oncol 40 25 2022 2901 2912 34898239
10 Burgos L Puig N Cedena M-T Measurable residual disease in multiple myeloma: ready for clinical practice? J Hematol Oncol 13 1 2020 82 32571377
11 Rosiñol L Oriol A Ríos R Lenalidomide and dexamethasone maintenance with or without ixazomib, tailored by residual disease status in myeloma Blood 142 18 2023 1518 1528 37506339
12 Gay F Roeloffzen W Dimopoulos MA Results of the phase III randomized Iskia trial: isatuximab-carfilzomib-lenalidomide-dexamethasone vs carfilzomib-lenalidomide-dexamethasone as pre-transplant induction and post-transplant consolidation in newly diagnosed multiple myeloma patients [abstract] Blood 142 suppl 1 2023 4 37410508
13 Derman BA Kansagra A Zonder J Elotuzumab and weekly carfilzomib, lenalidomide, and dexamethasone in patients with newly diagnosed multiple myeloma without transplant intent: a phase 2 measurable residual disease-adapted study JAMA Oncol 8 9 2022 1278 1286 35862034
14 Kubicki T Derman BA Dytfeld D Jakubowiak AJ Measurable residual disease in peripheral blood in myeloma: dream or reality Curr Opin Oncol 35 6 2023 574 580 37621165
15 Murray DL Puig N Kristinsson S Mass spectrometry for the evaluation of monoclonal proteins in multiple myeloma and related disorders: an International Myeloma Working Group Mass Spectrometry Committee report Blood Cancer J 11 2 2021 24 33563895
16 Derman BA Stefka AT Jiang K Measurable residual disease assessed by mass spectrometry in peripheral blood in multiple myeloma in a phase II trial of carfilzomib, lenalidomide, dexamethasone and autologous stem cell transplantation Blood Cancer J 11 2 2021 19 33563912
17 Puig N Contreras M-T Agulló C Mass spectrometry vs immunofixation for treatment monitoring in multiple myeloma Blood Adv 6 11 2022 3234 3239 35157768
18 Dispenzieri A Krishnan A Arendt B Mass-fix better predicts for PFS and OS than standard methods among multiple myeloma patients participating on the STAMINA trial (BMT CTN 0702 /07LT) Blood Cancer J 12 2 2022 27 35145071
19 Mai EK Huhn S Miah K Implications and prognostic impact of mass spectrometry in patients with newly-diagnosed multiple myeloma Blood Cancer J 13 1 2023 1 36599831
20 Eveillard M Rustad E Roshal M Comparison of MALDI-TOF mass spectrometry analysis of peripheral blood and bone marrow-based flow cytometry for tracking measurable residual disease in patients with multiple myeloma Br J Haematol 189 5 2020 904 907 32026474
21 Roopenian DC Akilesh S FcRn: the neonatal Fc receptor comes of age Nat Rev Immunol 7 9 2007 715 725 17703228
22 Zajec M Langerhorst P VanDuijn MM Mass spectrometry for identification, monitoring, and minimal residual disease detection of M-proteins Clin Chem 66 3 2020 421 433 32031591
23 Kumar S Paiva B Anderson KC International Myeloma Working Group consensus criteria for response and minimal residual disease assessment in multiple myeloma Lancet Oncol 17 8 2016 e328 e346 27511158
24 Kalina T Flores-Montero J van der Velden VHJ EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols Leukemia 26 9 2012 1986 2010 22948490
25 Krzywdzińska A Puła B Czyż A Harmonization of flow cytometric minimal residual disease assessment in multiple myeloma in centers of polish myeloma consortium Diagnostics 11 10 2021 1872 34679569
26 Barnidge DR Dasari S Botz CM Using mass spectrometry to monitor monoclonal immunoglobulins in patients with a monoclonal gammopathy J Proteome Res 13 3 2014 1419 1427 24467232
27 Barnidge DR Krick TP Griffin TJ Murray DL Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to detect monoclonal immunoglobulin light chains in serum and urine Rapid Commun Mass Spectrom 29 21 2015 2057 2060 26443406
28 Barnidge D Troske D North S Wallis G Perkins M Harding S Endogenous monoclonal immunoglobulins analyzed using the EXENT® solution and LC-MS J Mass Spectrom Adv Clin Lab 32 2024 31 40 38405412
29 Zajec M Jacobs JFM de Kat Angelino CM Integrating serum protein electrophoresis with mass spectrometry, a new workflow for M-protein detection and quantification J Proteome Res 19 7 2020 2845 2853 31895568
30 Fernández de Larrea C Tovar N Cibeira MT Emergence of oligoclonal bands in patients with multiple myeloma in complete remission after induction chemotherapy: association with the use of novel agents Haematologica 96 1 2011 171 173 20884709
31 Claveau J-S Murray DL Dispenzieri A Value of bone marrow examination in determining response to therapy in patients with multiple myeloma in the context of mass spectrometry-based M-protein assessment Leukemia 37 1 2023 1 4 36482129
32 Abeykoon JP Murray DL Murray I Implications of detecting serum monoclonal protein by MASS-fix following stem cell transplantation in multiple myeloma Br J Haematol 193 2 2021 380 385 33216966
33 Noori S Wijnands C Langerhorst P Dynamic monitoring of myeloma minimal residual disease with targeted mass spectrometry Blood Cancer J 13 1 2023 30 36828828
34 Liyasova M McDonald Z Taylor P A personalized mass spectrometry–based assay to monitor M-protein in patients with multiple myeloma (EasyM) Clin Cancer Res 27 18 2021 5028 5037 34210683
35 Feise RJ Do multiple outcome measures require p-value adjustment? BMC Med Res Methodol 2 1 2002 8 12069695
