Time-Dependent Prognostic Value of Serological and Measurable Residual Disease Assessments after Idecabtagene Vicleucel

The development of chimeric antigen receptor (CAR) T cells has transformed the outcome of triple-class exposed relapsed and refractory multiple myeloma (RRMM) patients (1). Namely, B-cell maturation antigen (BCMA)–directed CAR T cells have shown unprecedented efficacy with a prolonged duration of response in patients with RRMM, leading to the approval of two different BCMA CAR T-cell products (2, 3). That notwithstanding, no event-free plateau has been seen, and relapses continue to occur. Because of their novelty, as well as because of the unique mode of action and schedule of administration of CAR T therapies (i.e., single infusion), there is limited understanding of the tumor burden dynamics throughout initial response, disease resurgence, and progression.

The depth of response including the assessment of measurable residual disease (MRD) is a key prognostic factor in patients with newly diagnosed and RRMM (4). However, there is scarce information about its clinical meaning in patients treated with CAR T cells (2, 5, 6). Furthermore, in the phase I CRB-401 study investigating the B-cell maturation antigen (BCMA)–directed CAR T-cell idecabtagene vicleucel (ide-cel) in patients with RRMM (5), it was observed that serological responses were often delayed compared with MRD negativity. These findings raised uncertainty regarding the optimal time points to assess the efficacy of CAR T therapy in RRMM using each response criterion. Therefore, we aimed at analyzing the longitudinal prognostic value of the depth of serological and MRD responses after ide-cel therapy in the phase II KarMMa clinical trial (NCT03361748).

Before analyzing the longitudinal prognostic value of the depth of serological and MRD responses after ide-cel infusion, we sought to compare the results obtained with next-generation flow (NGF) cytometry and next-generation sequencing (NGS) due to the scarcity of MRD data using these methods after CAR T therapy in RRMM. A total of 336 and 257 MRD assessments in bone marrow (BM) were, respectively, performed using NGF and NGS, at months 1, 3, 6, and 12 within the first year after ide-cel infusion (Supplementary Fig. S1).

MRD status between NGF and NGS was concordant in 67%, 75%, 81.5%, and 73% of BM aspirates analyzed at months 1, 3, 6, and 12 after ide-cel, respectively (Supplementary Table S1). Most discordances were due to BM aspirates classified as hemodiluted by NGF and as MRD negative by NGS, which peaked immediately after ide-cel infusion and persisted until one year after (25%, 13%, 9.3%, and 9.8% at months 1, 3, 6, and 12, respectively). Accordingly, a subanalysis excluding hemodiluted samples showed greater concordance between NGF and NGS (94%, 88%, 90%, and 83% at months 1, 3, 6, and 12 after ide-cel, respectively).

On prognostic grounds, NGF and NGS showed a similar predictive value for progression-free survival (PFS) at all time points (Supplementary Table S2). Undetectable MRD was associated with a significant reduction in the risk of progression and/or death at each landmark regardless of the method, with hazard ratios ranging from 0.05 to 0.265 (Supplementary Table S2). Patients with sustained undetectable MRD at month 12 according to NGF (Fig. 1A, n = 17) or NGS (Fig. 1B, n = 19) did not reach the median PFS. By contrast, patients with persistent MRD progressed soon after the landmark.

Subsequent analyses to investigate the longitudinal prognostic value of the depth of serological and MRD responses after ide-cel infusion were done on the NGF data set because, as noted above, a higher number of samples were tested and the monitoring of hemodilution was intrinsic to the method. The rates of undetectable MRD on the treated patient population (N = 128) at months 1, 3, 6, and 12 after ide-cel were 41%, 46%, 36%, and 19.5%, respectively (Supplementary Table S3). The respective rates at 10⁻⁵ (i.e., considering MRD negative if detectable levels were <1 × 10⁻⁵) were 42%, 48%, 39%, and 22%. Of note, persistent MRD in the 10⁻⁶ logarithmic range was detected in 10 patient samples considered as MRD negative at 10⁻⁵, all of whom progressed shortly thereafter (median 3.6 months; range, 0.3–8.6).

It was interesting to note that the rates of CR on the treated patient population at months 1, 3, 6, and 12 after ide-cel (i.e., 11%, 23%, 26%, and 23%) were lower than those of undetectable MRD in all time points except at month 12 (Supplementary Table S3). These findings are a consequence of the fact that, in this study, undetectable MRD was defined regardless of CR achievement. Accordingly, these results suggest that clearance of BM tumor cells occurred earlier than the M-component, possibly because of the long half-life of some immunoglobulins (7). Alternatively, achievement of a transient MRD-negative status followed by disease progression without ever attaining CR may apply to some patients. Indeed, this was observed in 25 of 53 patients with undetectable MRD at month 1 after ide-cel (Fig. 2). The presence of extramedullary disease is likely associated with these observations. Although positron emission tomography/computed tomography (PET/CT) was not systematically performed in all the patients and at all time points, we observed nonetheless that the presence of extramedullary disease at months 1 (Supplementary Fig. S2A), 3 (Supplementary Fig. S2B), 6 (Supplementary Fig. S2C) and 12 (Supplementary Fig. S2D) after infusion with ide-cel was associated with inferior PFS in patients achieving undetectable MRD.

These discordant patterns of CR and MRD rates further increased the interest in investigating the clinical significance of different serologic and MRD response kinetics after ide-cel infusion. Landmark PFS starting from 1 month after ide-cel infusion was not significantly different between patients in less than CR at the 1-month time point (n = 103) and those in CR (n = 14; median of 8 vs. 11 months, P = 0.09). By contrast, the presence of MRD at month 1 predicted poorer landmark PFS when compared with cases with undetectable MRD in CR or less than CR (Fig. 3A). At months 3, 6, and 12 after ide-cel, patients in CR and undetectable MRD showed significantly longer median landmark PFS than those in less than CR and undetectable MRD (Fig. 3B–D). The median landmark PFS at months 6 and 12 was progressively lower in patients achieving CR/MRDneg vs. <CR/MRDneg vs. CR/MRDpos vs. <CR/MRDpos (Fig. 3C and D).

We hypothesized that the reappearance of normal plasma cells (PC), which are formally analyzed during MRD assessment using NGF, could be used as a surrogate for loss of CAR T-cell persistence and/or functionality as BCMA-directed immunotherapy does not discriminate between tumor and normal PC. In such cases, this information could help identify patients with undetectable MRD at a higher risk of progression due to the loss of CAR T surveillance. Interestingly, the reappearance of normal PC was observed at all time points and often preceded the conversion into an MRD-positive status and disease progression (Fig. 4A). Accordingly, the reappearance of normal PC at months 1, 3, and 12 was associated with inferior PFS (Supplementary Table S4). Although the reduced number of patients precludes definitive conclusions, those with undetectable MRD and reappearance of normal PC at month 1 showed similarly poor PFS to those with positive MRD (Fig. 4B). All cases with the reappearance of normal PC at month 12 had detectable ide-cel levels, as measured by qPCR of vector transgene copies per microgram of genomic DNA (ref. 2; median 355; range, 37.5–23,470), which suggests loss of functionality rather than persistence. None of the three patients showing the absence of normal PC and undetectable MRD at month 12 progressed thus far (Fig. 4C).

Anti-BCMA CAR T cells have been recently approved for the treatment of RRMM (2, 3) and, because of their novelty and unique mode of action and administration, there is uncertainty whether traditional biomarkers remain prognostic. In this prespecified analysis of the KarMMa phase II trial, we showed that deep serological and MRD remission with anti-BCMA CAR T cells resulted in prolonged PFS.

MM is at the forefront of MRD assessment in hematologic malignancies with two next-generation methods achieving a minimum sensitivity of 10⁻⁵ (i.e., NGF and NGS). Although each method has its own set of advantages and disadvantages (8, 9), they have proven to be comparable in terms of detection rates and prognostic value in newly diagnosed MM patients (10). Here, we confirmed and expanded these observations to RRMM patients treated with CAR T cells.

The main discordance between NGF and NGS was observed in samples considered as hemodiluted by the former and MRD negative by the later method. The incidence of hemodilution observed during the first year after ide-cel infusion was greater than that reported in newly diagnosed and RRMM patients treated with other regimens (11). Such a finding could be related to the lymphodepleting chemotherapy prior to CAR T therapy and cytopenias commonly observed thereafter (2, 3, 5). Although the possibility to evaluate hemodilution is an advantage of NGF, the very similar prognostic value of both methods observed in each time point suggests that the (false-)negative results using NGS were inconsequential.

There is growing consensus that the duration and the sensitivity of MRD responses are more important than the methodology used to assess MRD. The rich MRD data set of the KarMMa clinical trial (i.e., paired NGF and NGS MRD assessment in four prespecified time points) presented a unique opportunity to record the nearly identical prognostic value of 12 months sustained MRD negativity using NGF and NGS. On clinical grounds, these results are consistent with the observed impact of sustained MRD negativity on PFS for RRMM patients salvaged with continuous therapy (12).

With regard to sensitivity, we showed that albeit the marginal differences in MRD-negative rates according to the maximum sensitivity achieved by NGF (Supplementary Table S5) and a fixed sensitivity analysis at 10⁻⁵ (i.e., considering MRD negative if detectable levels were <1 × 10⁻⁵), the detection of MRD levels in the 10⁻⁶ logarithmic range was systematically associated with a short time until disease progression. These results suggest that similarly to other treatment scenarios (4, 13–15), MRD should be analyzed with the highest possible sensitivity after CAR T therapy. Indeed, it could be particularly relevant because of their mode of action; in the absence of CAR T-cell persistence and/or functionality, any detectable level of MRD is associated with an imminent risk of relapse.

Beyond the sensitivity of next-generation methods that measure MRD inside the BM, the role of PET/CT to refine depth of response appears to be particularly valuable in RRMM due to the increased incidence of extramedullary disease (16). Accordingly, the presence of extramedullary disease after infusion with ide-cel was associated with inferior PFS in patients achieving undetectable MRD. These results confirm and expand the recent observations in patients treated with CAR T cells at the Mayo Clinic (17). Furthermore, it could help explain the cases in which the achievement of a transient MRD-negative status was followed by disease progression without ever attaining CR.

To our knowledge, we showed here for the first time that assessing the reappearance of normal PC after anti-BCMA CAR T therapy predicts inferior PFS in patients with undetectable MRD. These results suggest highly dynamic responses after CAR T therapy, whereby after initial peak expansion and subsequent tumor depletion (i.e., less than 1 × 10⁻⁶ tumor cells), CAR T cells may no longer persist or lose function, which leads to the reappearance of normal PC before MRD resurgence. Because all cases with reappearance of normal PC at month 12 had detectable ide-cel levels by qPCR, we hypothesized that loss of function is the most plausible explanation. However, the absence of immunophenotypic and functional data to confirm this hypothesis is a limitation of this study. On methodological grounds, monitoring normal cell types expressing target antigens (e.g., BCMA) and tumor burden could provide complementary information.

Another interesting observation of this study (where MRD negativity was defined regardless of CR achievement) was the discordant pattern of CR and MRD response rates observed during the first year after ide-cel infusion. First, CR rates were lower than MRD-negative rates at months 1, 3, and 6. Second, CR rates peaked at month 6 while MRD-negative rates peaked at month 3. Third, CR rates were lower than MRD-negative rates at month 12. Fourth, among patients with undetectable MRD at month 3 and beyond, the PFS was inferior in those with detectable M-protein. These results suggest that because of the long half-life of immunoglobulins, early MRD negativity may better reflect the dynamics of CAR T peak expansion and depth of initial tumor clearance. This is a possible limitation of current response criteria that binds MRD negativity to the achievement of CR (9). Future guidelines could explore new MRD-negative definitions that are independent of the M-protein to overcome this limitation, particularly in clinical trials using early depth of response as an endpoint (e.g., after induction; refs. 18, 19). However, in contrast to newly diagnosed MM (7, 20), achieving CR does matter in MRD-negative RRMM patients with respect to response durability after anti-BCMA CAR T therapy. Therefore, sustained CR and MRD negativity for 12 months may be a reasonable endpoint in CAR T clinical trials (21).

The outputs of this study could have clinical and laboratory translation since two CAR T-cell products have been approved for the treatment of RRMM (2, 3). For example, persistent MRD around the time of peak expansion likely indicates primary resistance to CAR T cells, and such information can be leveraged to tailor salvage therapy. From the laboratory perspective, this study reinforces the value of highly sensitive MRD assessment (10⁻⁶), as well as the importance of analyzing hemodilution and the reappearance of normal PC after BCMA-directed CAR T therapy. Because of the highly dynamic MRD-negative rates observed during the first year after CAR T-cell infusion, such a comprehensive monitoring should be performed periodically using minimally invasive methods. New and more sensitive flow cytometry protocols empowered to detect MRD below 10⁻⁶ in the peripheral blood (22) could help decrease the number of BM aspirates, particularly at earlier time points when these are frequently uninformative because of hemodilution and transient MRD-negative states. It could also inform on the reappearance of BCMA-positive normal PC that circulate in peripheral blood. Mass spectrometry would probably be the most sensitive method to detect the resurgence of M-proteins at later time points (23), which could be valuable if early rescue intervention before clinical progression is being considered. Additional studies are warranted to confirm these hypotheses, as well as if and how MRD assessments should be performed in RRMM patients treated with CAR T cells.

The design, primary endpoint (i.e., objective response) and patient demographic information for the ongoing KarMMa clinical trial (NCT03361748) were reported previously (2). In brief, eligible patients were 18 years of age or older; had received at least three previous regimens including an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 antibody; had disease that was refractory to their last regimen; had measurable disease; and had adequate organ function (2). Subjects’ median age (range) was 61 (33–78) with slightly more than half of the subjects being male (59%). The majority of subjects were white (80.5%), whereas 2.3% were Asian and 4.7% were Black or African American. The majority of subjects were not Hispanic or Latino (80.5%).

Ide-cel was manufactured and administered as described previously (2, 5). Briefly, it was manufactured from autologous peripheral blood mononuclear cells, stimulated with antibodies to CD3 and CD28, transduced with a lentiviral vector containing the anti-BCMA CAR, and expanded over a period of 10 days. The actual doses delivered were within 20% of the target doses. Bridging therapy was allowed during manufacturing but was stopped at least 14 days before lymphodepletion and was restricted to certain drug classes and to drugs previously received.

The primary endpoint was an overall response (partial response or better), defined according to the International Myeloma Working Group (IMWG) Uniform Response Criteria (9) as assessed by an independent review committee. Secondary endpoints included, among others, CR or better (i.e., comprising conventional and stringent CR), time to and duration of response, PFS, and MRD negativity. Tumor response and disease progression were assessed according to IMWG criteria. The study protocol was approved by local or independent institutional review boards or ethics committees at participating sites. All the patients provided written informed consent. The completion of the clinical study is estimated by December 2024.

MRD was analyzed at months 1, 3, 6, and 12 within the first year after ide-cel infusion regardless of clinical response. Of the 128 patients receiving ide-cel, 125 had at least one MRD assessment (Supplementary Fig. S1). A total of 336 and 257 MRD assessments in BM using NGF and NGS, were respectively evaluable.

NGF was performed following the EuroFlow standard operating procedures described elsewhere (14, 24). In brief, the method is based on a (standardized) lyse–wash–stain protocol and an optimized 8-color, 2-tube, antibody panel, for the accurate identification of phenotypically aberrant, clonal PCs: tube 1: CD138-BV421, CD27-BV510, CD38-FITC, CD56-PE, CD45-PerCPCy5.5, CD19-PECy7, CD117-APC, CD81-APCH7; and tube 2: CD138-BV421, CD27-BV510, CD38-FITC, CD56-PE, CD45-PerCPCy5.5, CD19-PECy7, cyKAPPA-APC, cyLAMBDA-APCH7. The two-tube strategy allows the detection of MRD with specific confirmation of light-chain (mono)clonality on phenotypically aberrant PCs, identified by antigen underexpression (CD19, CD27, CD38, CD45, and CD81) or overexpression (CD56, CD117, and CD138) as compared with normal PCs. Data acquisition was performed in a FACSCanto II flow cytometer (BD) using the FACSDiva 6.1 software (BD). Data analysis was performed by experienced operators using the Infinicyt software (Cytognos SL). MRD assessments were performed blinded for clinical outcomes in two laboratories and data were centralized for MRD analyses. The first 8-color antibody combination was additionally used for the enumeration of mast cells (CD117^bright and CD45^dim), nucleated red blood cells (CD45⁻, CD38⁻, CD117^−/+, and SSC^lo), and B-cell precursors (CD19⁺, CD45^dim, CD38^bright, CD81^bright, and CD27⁻). The number of viable nucleated cells was systematically registered, and the limit of detection (LOD) achieved by NGF was determined in each sample according to the formula: (20/viable nucleated cells) × 100. Samples with undetectable MRD, as well as with undetectable levels of all three cell types, were considered hemodiluted (and not as MRD negative). Undetectable MRD was defined regardless of the LOD achieved in that sample, as well as regardless of CR achievement. The number of patient samples where the LOD was inferior to 10⁻⁵ is shown in Supplementary Table S4. Any detectable level of MRD >1 × 10⁻⁶ was considered positive, except in a fixed sensitivity analysis at 10⁻⁵ (i.e., considering MRD negative if detectable levels were <1 × 10⁻⁵).

NGS was performed using clonoSEQ from Adaptive Biotechnologies. Briefly, BM aspirates were collected in K2-EDTA tube, were processed to BM mononuclear cells using red blood cell lysis, and stored as dry cell pellets. Genomic DNA was extracted from frozen cell pellets and processed using the CLIA-validated, FDA-cleared clonoSEQ method by Adaptive Biotechnologies. Any detectable level of MRD >1 × 10⁻⁶ was considered positive. Patients with sustained undetectable MRD at month 12 according to NGF or NGS were defined according to negative MRD results observed at 12 months and any of the previous time points (i.e., months 1, 3, and 6). Persistent MRD at month 12 was defined based on a positive MRD result at this time point, regardless of MRD statuses at previous time points (i.e., months 1, 3, and 6).

PET/CT was not systematically performed in all patients and at all time points. That notwithstanding, a subanalysis investigating the impact of extramedullary disease (defined as paraskeletal soft-tissue masses, soft-tissue masses spreading outside the BM, or both) in the PFS of MRD-negative patients was performed.

Kaplan–Meier methodology was used to estimate PFS distributions, with stratified log-rank tests and Cox models used for between-group comparisons. To address the immortal time bias, PFS analyses were landmarked at months 1, 3, 6, and 12 after ide-cel infusion corresponding to the times of the MRD measurements. Accordingly, PFS was measured from the landmark to the time of progression or death and was restricted to patients who at the landmark time had not progressed or died. Median follow-up was 28.1 months (range, 24.1–34.5).

After the completion of the clinical trial (estimated for December 2024), deidentified patient-level data will be made available through the global clinical research data-sharing platform, Vivli, in compliance with applicable privacy laws, data protection, and requirements for consent and anonymization. A request for data sharing can be made through the portal link at https://www.bms.com/researchers-and-partners/independent-research/data-sharing-request-process.html. Requests are reviewed by an independent review committee (Duke Clinical Research Institute) in accordance with PhRMA and EFPIA Principles for Clinical Trial Data Sharing.

B. Paiva reports other support from BMS-Celgene during the conduct of the study; grants and personal fees from BMS, GSK, Sanofi, Takeda, Roche, grants from BeiGene, personal fees from ­Janssen, Adaptive, Amgen, and Becton Dickinson outside the submitted work. J. Rytlewski reports personal fees from Bristol Myers Squibb and ­Bristol Myers Squibb during the conduct of the study; personal fees from Adaptive Biotechnologies outside the submitted work; in addition, J. Rytlewski has a patent for multiple CAR T–related patents pending to Bristol Myers Squibb. T. Campbell reports other support from Bristol Myers Squibb during the conduct of the study; other support from Bristol Myers Squibb outside the submitted work. ­C.C. Kazanecki reports personal fees from Bristol Myers Squibb during the conduct of the study; personal fees and other support from Bristol Myers Squibb outside the submitted work. N. Martin reports personal fees from BMS during the conduct of the study; personal fees from BMS outside the submitted work. L.D. Anderson reports personal fees from Janssen, Celgene, BMS, Amgen, GSK, AbbVie, Beigene, Cellectar, Sanofi, and Prothena outside the submitted work. J.G. Berdeja reports grants from BMS during the conduct of the study; grants and personal fees from CRISPR Therapeutics, grants from 2 Seventy Bio, AbbVie, Amgen, C4 Therapeutics, CARsgen, Cartesian, Celularity, Fate Therapeutics, Genentech, GSK, Ichnos Sciences, Incyte, Karyopharm, Sanofi, Lilly, Novartis, Poseida, Teva, and Zentalis, personal fees from Kite Pharma, Legend Biotech, Roche, Secura Bio, grants and personal fees from Janssen, Takeda outside the submitted work. S. Lonial reports personal fees from Amgen, BMS, Takeda, Celgene, Novartis, Janssen, Genentech, AbbVie, Pfizer, Regeneron, and GSK outside the submitted work; and trial support from Takeda, Novartis, Janssen, BMS; member of the board of directors with stock in TG Therapeutics (no cancer agents currently). N.S. Raje reports personal fees from BMS during the conduct of the study; grants and personal fees from Pfizer, grants from 2Seventybio, personal fees from Janssen, Immuneel, Caribou Biosciences, Sanofi, GSK, and AbbVie outside the submitted work. Y. Lin reports other support from BMS during the conduct of the study; other support from Janssen, Kite/Gilead, Caribou Biosciences, Pfizer, Takeda, Chimeric Therapeutics, NekTar, NexImmune, and Sanofi outside the submitted work. P. Moreau reports personal fees from Celgene, Janssen, AbbVie, Sanofi, Amgen, and Pfizer outside the submitted work. J.F. San-Miguel reports personal fees from AbbVie, Amgen, BMS-Celgene, Roche, GSK, Janssen, Karyopharm, Merck, Novartis, Sanofi, and Takeda outside the submitted work. N.C. Munshi reports personal fees from BMS during the conduct of the study; personal fees from Pfizer, Novartis, DCT, Janssen, and GSK outside the submitted work; in addition, N.C. Munshi has a patent for Oncopep issued and licensed. S. Kaiser reports personal fees from Bristol Myers Squibb outside the submitted work; in addition, S. Kaiser has a patent for uses of anti-BCMA chimeric antigen receptors pending to Celgene/Bristol Myers Squibb. No disclosures were reported by the other authors.

B. Paiva: Conceptualization, data curation, formal analysis, supervision, investigation, visualization, methodology, writing–original draft, writing–review and editing. I. Manrique: Data curation, formal analysis, investigation, methodology, writing–review and editing. J. Rytlewski: Conceptualization, data curation, formal analysis, investigation, visualization, methodology, writing–review and editing. T. Campbell: Data curation, investigation, writing–review and editing. C.C. Kazanecki: Data curation, investigation, writing–review and editing. N. Martin: Data curation, investigation, writing–review and editing. L.D. Anderson: Investigation, writing–review and editing. J.G. Berdeja: Investigation, writing–review and editing. S. Lonial: Investigation, writing–review and editing. N.S. Raje: Investigation, writing–review and editing. Y. Lin: Investigation, writing–review and editing. P. Moreau: Investigation, writing–review and editing. J.F. San-Miguel: Investigation, writing–review and editing. N.C. Munshi: Investigation, writing–review and editing. S. Kaiser: Conceptualization, resources, formal analysis, writing–original draft, writing–review and editing.

We thank all the study participants and their families, as well as the clinical staff at participating centers. This study was supported by 2seventy bio (formerly bluebird bio) and Celgene, a Bristol Myers Squibb company.

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