Next-generation Flow Research Articles

The significance of free light-chain ratio in light-chain monoclonal gammopathy of undetermined significance: a flow cytometry sub-study of the iStopMM screening study

Light-chain (LC) monoclonal gammopathy of undetermined significance (MGUS) is a precursor of multiple myeloma (MM) and related conditions. LC-MGUS is characterized by free light-chain (FLC) levels outside defined reference intervals, indirectly indicating underlying plasma cell (PC) monoclonality. Next-generation flow cytometry (NGF) was used to evaluate clonal PC presence in bone marrow (BM) samples from individuals with LC-MGUS in the iStopMM study, aiming to assess the predictive value of the FLC ratio for clonal PC presence and its prognostic implications. BM samples from 61 individuals with LC monoclonal gammopathy were analyzed. Clonal plasma cells were detected in 53.6% of LC-MGUS samples (n = 28) and in all samples from individuals with more advanced conditions (n = 33). The FLC ratio was predictive of clonal PC presence for kappa-involved FLC ratios (p < 0.05; n = 42), with an optimal cutoff of 3.15 (96.7% sensitivity, 91.7% specificity). Of 195 individuals with kappa-involved LC-MGUS in follow-up within the iStopMM study, none with FLC ratios >1.65 to 3.15 progressed to MM (n = 124), whereas 4/71 (5.6%) with FLC ratios >3.15 progressed over median follow-up of 55 months. These findings support using a kappa-involved FLC ratio cutoff of >3.15 to more accurately identify individuals at increased risk of developing symptomatic PC disorders.

Prevalence of measurable residual disease in patients with refractory/relapsed multiple myeloma who reached complete response: a cross-sectional multicentric study

BackgroundMultiple myeloma (MM) is an incurable hematologic malignancy, and even though the complete response (CR) rate has been growing, a high percentage of patients continues to relapse. Recent research showed that most relapses may be related to the persistence of measurable residual disease (MRD). In this study, we intended to evaluate the MRD status in MM patients who reached CR in their second or third lines of treatment.MethodsThis was a cross-sectional, multicentre, non-interventional study to describe the MRD status in patients with relapsed or refractory MM (rrMM), with documented CR; adult male and female patients, from 11 Portuguese sites, in their second or third line of treatment were included. Bone marrow MRD was assessed through next-generation flow cytometry (NGF) technology.ResultsAmong the 68 subjects who gave informed consent, 48 were considered eligible for the study. Of the 48 subjects with confirmed CR, 31 (64.6%) had undetectable MRD levels. The incidence of undetectable MRD was lower in International Staging System (ISS) III patients compared with ISS I/II patients (60% vs. 70.8%; p = 0.45), and lower in patients treated without daratumumab-containing regimens compared with those treated with daratumumab-containing regimens (57.1% vs. 75.0%; p = 0.30). Notably, despite the small sample size, the incidence of undetectable MRD was significantly lower in patients with high-risk cytogenetics compared to those with standard risk (33.3% vs. 76.0%; p = 0.04).DiscussionOur results highlight the possibility of achieving undetectable MRD in the rrMM setting, especially in earlier stages and with highly effective protocols. We expect that this work leverages the implementation of larger real-world evidence studies in rrMM patients, in which MRD may also be defined as a primary endpoint.

Clinical significance of complete remission and measurable residual disease in relapsed/refractory multiple myeloma patients treated with T-cell redirecting immunotherapy.

The impact of measurable residual disease (MRD) in relapse/refractory multiple myeloma (RRMM) patients treated with T-cell redirecting immunotherapy is uncertain. We analyzed MRD dynamics using next-generation flow in 201 patients treated in clinical trials with chimeric antigen receptor (CAR) T cells and T-cell engagers (TCE). Achieving MRD negativity at 10-6 was associated with 89% reduction in the risk of progression and/or death. Survival outcomes were improved in patients with sustained versus transient MRD negativity and were dismal in those who remained MRD positive. The intent-to-treat MRD negative rates were higher in patients treated with CAR T cells versus TCE. However, among patients achieving MRD negativity, there were no differences in survival outcomes when stratified according to treatment with CAR T cells versus TCE. In multivariate analysis including the number of prior lines of treatment, International Staging System, cytogenetic risk, extramedullary disease and type of T-cell redirecting immunotherapy, only the complete remission (CR) and MRD statuses showed independent prognostic value for progression-free and overall survival. In conclusion, our study shows that deep and sustained MRD negative CR is the most relevant prognostic factor and should be considered as the treatment endpoint in RRMM patients treated with CAR T cells and TCE.

Monoclonal gammopathy of undetermined significance with multiple paraproteins: A population-based screening study.

Monoclonal gammopathy of undetermined significance (MGUS) is the precursor of multiple myeloma (MM) and related disorders. MGUS is characterized by asymptomatic paraproteinemia. In some cases, multiple paraproteins can be identified but the clinical implications of this phenomenon are poorly understood. In this study, we aim to inform the approach to this challenging MGUS subgroup by utilizing data from iStopMM, a population-based screening study and randomized trial of follow-up strategies. In total, 75,422 Icelanders over the age of 40 were screened for MGUS with 3389 (4.4%) having at least one paraprotein of whom 303 (9%) had multiple paraproteins. IgM paraproteins were more common in those with multiple paraproteins (49% vs. 27% of paraproteins, p < 0.001), and IgM and non-IgM paraproteins frequently co-occurred (60% of cases). Two-thirds of these participants were randomized to active follow-up where only 31% of multiple paraproteins were persistent. Paraprotein concentrations were mostly independent, and although progression events were few, the progression rate was similar between those with multiple paraproteins and a single paraprotein. In a next-generation flow cytometry (NGF) sub-study, two phenotypically distinct aberrant plasma cell populations could be identified in some with multiple paraproteins. The findings suggest that multiple paraproteins often reflect independent ongoing disease processes that should be monitored independently but otherwise treated similarly to other MGUS cases. Specifically, the findings highlight the need for independent monitoring of IgM and non-IgM paraproteins in these individuals. The study provides novel insights into the management of this understudied MGUS subset.

Evaluation of the Prognostic Value of the Mayo Additive Staging System and Minimal Residual Disease in Newly Diagnosed Multiple Myeloma Patients.

This study aimed to evaluate the prognostic value of the Mayo additive staging system (MASS) and minimal residual disease (MRD) in newly diagnosed multiple myeloma (NDMM) patients. A total of 238 NDMM patients were enrolled in Beijing Chaoyang Hospital. Fluorescence in-situ hybridization and next-generation flow cytometry were used to examine cytogenetic abnormalities and MRD, respectively. The patients were classified into three groups to compare the effects on progression-free survival (PFS). Univariate and multivariate analyses were applied to identify the survival-related factors. For MASS group, the PFS was significant difference in MASS I, II, and III patients (p = 0.0006); the patients with sustained MRD-negative, non-sustained MRD-negative, sustained MRD-positive, and non-sustained MRD-positive were divided into Groups 1, 2, 3, and 4, respectively. The Group 1 patients had superior PFS than Groups 2 and 3 patients (p < 0.05), but no difference in PFS was observed for Group 2 versus Group 3, Group 2 versus Group 4, and Group 3 versus Group 4 patients. For the MASS and MRD groups, among Groups 2, 3, and 4, MASS I patients had a superior PFS, while III patients showed the opposite result. Strikingly, no difference in PFS for Group 1 patients regardless of the MASS stage was observed. Despite being in MASS II and III, the PFS of Group 1 patients was longer than those with the other three groups. Response to treatment was an independent prognostic factor for MM patients. Patients with an accumulation of high-risk factors and MRD-positive have poor outcomes. Sustained MRD-negative may improve high-risk patients' prognoses.

Minimally Invasive Assessment of Peripheral Residual Disease During Maintenance or Observation in Transplant-Eligible Patients With Multiple Myeloma.

Clinical trials frequently include multiple end points that mature at different times. The initial report, typically based on the primary end point, may be published when key planned co-primary or secondary analyses are not yet available. Clinical Trial Updates provide an opportunity to disseminate additional results from studies, published in JCO or elsewhere, for which the primary end point has already been reported.In multiple myeloma (MM), measurable residual disease (MRD) is assessed in bone marrow (BM). However, less invasive evaluation of peripheral residual disease (PRD) in blood could be advantageous and less cumbersome. We investigated the prognostic value of PRD monitoring after 24 cycles of maintenance in 138 transplant-eligible patients with MM enrolled in the GEM2012MENOS65/GEM2014MAIN clinical trials. PRD was assessed using next-generation flow (NGF) and mass spectrometry (MS). Positive PRD by NGF in 16/138 (11.5%) patients was associated with a 13-fold increased risk of progression and/or death; median progression-free survival (PFS) and overall survival (OS) were 2.5 and 47 months, respectively. Considering patients' MRD status in BM as the reference, PRD detection using NGF showed positive and negative predictive values of 100% and 73%, respectively. Presence of PRD helped identifying patients at risk of imminent progression among those with positive MRD in BM. Patients with undetectable PRD according to both NGF and MS showed 2-year PFS and OS rates of 97% and 100%, respectively. In multivariate analyses including the Revised International Staging System and the complete remission status, only MRD in BM and PRD by NGF showed independent prognostic value for PFS. This study supports the use of less invasive PRD monitoring during maintenance or observation in transplant-eligible patients with MM.

Real-World Outcomes of Upfront Autologous Hematopoietic Stem Cell Transplantation in Patients With Newly Diagnosed Multiple Myeloma With Deletion 17p

Despite tremendous advancements in multiple myeloma (MM) therapeutics, outcomes remain heterogeneous, heavily influenced by clinical and cytogenetic factors. Among these, deletion of the short arm of chromosome 17 (del(17p)) is a strong predictor of poor prognosis. The aim of this study was to evaluate real-world outcomes in patients with newly diagnosed MM (NDMM) with del(17p) undergoing upfront autologous hematopoietic stem cell transplantation (auto-HCT). We conducted a single-center retrospective analysis of patients with NDMM who underwent upfront auto-HCT at MD Anderson Cancer Center between 2008 and 2018. Primary endpoints were progression-free survival (PFS) and overall survival (OS), with secondary endpoints being hematological response and measurable residual disease (MRD) status postauto-HCT. MRD status in the bone marrow biopsy was evaluated using 8-color next-generation flow cytometry with a sensitivity of 1/10-5 cells. One hundred and fifteen patients were included (55% male). Median age at auto-HCT was 62 years (range 34 to 83). The median del(17p) clone size was 20%, with 51 (53%) patients having clone sizes >20% and 15 (15%) patients having clone sizes >55%. Additional high-risk cytogenetic abnormalities included t(4;14) in 15 (13%) patients, t(14;16) in 8 (7%) patients, and 1q21+ in 25 (22%) patients. After induction, 10% of patients achieved ≥ CR, and 50% achieved ≥ VGPR, with 25% having MRD-negative ≥ VGPR. Post-transplant, 42% achieved ≥ CR, and 83% achieved ≥ VGPR as best response, with 55% (48/87) having MRD-negative ≥ VGPR. With a median follow-up of 31.4 months (range 3.1 to 199.1), median PFS and OS for the entire cohort were 19.9 and 71.5 months, respectively, and 5-year OS was 53%. Concurrent del(17p) and t(4;14) were associated with significantly worse outcomes, with median PFS and OS of 11.5 and 22.4 months, respectively. In multivariable analysis (MVA), female sex was associated with worse PFS (HR [95% CI] 2.87 [1.75 to 4.72], P < .001), while MRD negative CR post-transplant (0.35 [0.18 to 0.68], P = .002) and maintenance therapy (0.46 [0.27 to 0.77], P = 0.003) were associated with better PFS. In MVA for OS, female sex (2.22 [1.18 to 4.17], P = 0.013) and the presence of t(4;14) (2.55 [1.09 to 5.95], P = 0.030) were associated with worse OS, whereas Karnofsky Performance Status of ≥90 (0.47 [0.23 to 0.94], P = 0.034) was associated with better OS. This study affirms del(17p) as a high-risk abnormality with unfavorable outcomes despite modern therapies. The co-occurrence of del(17p) and t(4;14) was associated with particularly poor outcomes. Novel approaches are needed for this high-risk subgroup.

Measurable residual disease by mass spectrometry and next-generation flow to assess treatment response in myeloma

AbstractQuantitative immunoprecipitation mass spectrometry (QIP-MS) allows the identification of the M-protein in patients with multiple myeloma (MM) otherwise in complete response, and could be considered suitable for measurable residual disease (MRD) evaluation in peripheral blood. In the context of the GEM2012MENOS65 and GEM2014MAIN trials, we compared the performance of QIP-MS in serum with next-generation flow (NGF) cytometry in bone marrow to assess MRD in paired samples obtained postinduction, transplant, consolidation and after 24 cycles of maintenance. At each time point, both NGF and QIP-MS were able to segregate 2 groups of patients with significantly different progression-free survival; when the evolution of the results obtained with either method was considered, maintaining or converting to MRD negativity was associated with longer survival, significantly better when compared with sustaining or converting to MRD positivity. Reemergence of MRD by QIP-MS was associated with high risk of imminent clinical progression. In conclusion, MRD evaluation by NGF and MS achieves similar prognostic value based in single time point assessments and kinetics. Thus, the minimally invasive nature of MRD monitoring by MS represents a breakthrough in highly sensitive response assessment in MM. The trials were registered at www.clinicaltrials.gov as #NCT01916252 (GEM2012MENOS65) and at EudraCT as #2012-005683-10; and as #NCT02406144 (GEM2014MAIN) and at EudraCT as 2014-00055410.

Curative Strategy for High-Risk Smoldering Myeloma: Carfilzomib, Lenalidomide, and Dexamethasone (KRd) Followed by Transplant, KRd Consolidation, and Rd Maintenance.

Early treatment of high-risk smoldering myeloma has been shown to delay progression to multiple myeloma (MM). We conducted this trial with curative intention using a treatment approach employed for newly diagnosed patients with MM. Patients with high-risk smoldering myeloma (>50% progression risk at 2 years) and transplant candidates were included and received induction therapy with carfilzomib, lenalidomide, and dexamethasone (KRd), six cycles, followed by high-dose melphalan (200 mg/m2) autologous stem-cell transplantation (HDM-ASCT), two KRd consolidation cycles, and Rd maintenance for 2 years. The primary end point was undetectable measurable residual disease (uMRD) rate by next-generation flow after ASCT. Sustained uMRD 4 years after ASCT was the secondary end point. Between June 2015 and June 2017, 90 patients were included, and 31% met at least one SixtyLightchain MRI (SLiM)-hypercalcemia, renal impairment, anemia, bone disease (CRAB) criterion. After a median follow-up of 70.1 months, 3 months after ASCT, in the intention-to-treat population, 56 (62%) of 90 patients had uMRD, and 4 years later, it was sustained in 29 patients (31%). Five patients progressed to MM, and the 70-month progression rate was 94% (95% CI, 84 to 89). The presence of any SLiM CRAB criteria predicted progression to MM (four of the five patients; hazard ratio, 0.12; 95% CI, 0.14 to 1.13; P = .03). Thirty-six patients showed biochemical progression, and failure to achieve uMRD at the end of treatment predicted it. The 70-month overall survival was 92% (95% CI, 82 to 89). Neutropenia and infections were the most frequent adverse events during treatment, resulting in one treatment-related death. Three second primary malignancies have been reported. Although a longer follow-up is needed, this curative approach is encouraging and more effective than active MM, with 31% of the patients maintaining the uMRD 4 years after HDM-ASCT.

Serum mass spectrometry for treatment monitoring in patients with multiple myeloma receiving ARI0002h CAR T-cells.

Chimeric antigen receptor (CAR) T-cell therapies have increased the patients with relapsed/refractory multiple myeloma (RRMM) in whom standard electrophoretic techniques fail to detect the M-protein. Quantitative immunoprecipitation mass spectrometry (QIP-MS) can accurately measure serum M-protein with high sensitivity, and identify interferences caused by therapeutic monoclonal antibodies. Here, we investigate the outcome of QIP-MS in 33 patients treated with the academic BCMA-directed CAR T-cell ARI0002h (Cesnicabtagene Autoleucel). QIP-MS offered more detailed insights than serum immunofixation (sIFE), identifying glycosylated M-proteins and minor additional peaks. Moreover, the potential interferences owing to daratumumab or tocilizumab treatments were successfully detected. When analysing different assay platforms during patient's monitoring after ARI0002h administration, we observed that QIP-MS showed a high global concordance (78.8%) with sIFE, whereas it was only moderate (55.6%) with bone marrow (BM)-based next-generation flow cytometry (NGF). Furthermore, QIP-MS consistently demonstrated the lowest negativity rate across the different timepoints (27.3% vs. 60.0% in months 1 and 12, respectively). Patients with QIP-MS(+)/BM-based NGF(-) showed a non-significant shorter median progression free survival than those with QIP-MS(-)/BM-based NGF(-). In summary, we show the first experience to our knowledge demonstrating that QIP-MS could be particularly useful as a non-invasive technique when evaluating response after CAR T-cell treatment in MM.

Chinese expert consensus on minimal residual disease detection in multiple myeloma based on bone marrow samples (2024)

Multiple myeloma (MM) is the second most common hematologic malignant tumor. Standard holistic treatment model and new drug-based regimens have greatly improved the survival of patients with MM; however, minimal residual disease (MRD) causes relapse in most patients. Therefore, combining MRD testing on the basis of traditional serological efficacy evaluation is necessary to achieve a more accurate assessment of the patient's disease status. At present, next-generation flow cytometry (NGF) and next-generation sequencing (NGS) are the mainstream technologies for detecting MRD based on bone marrow samples. To standardize and normalize MRD detection, the expert group discussed and formulated Chinese expert consensus on the application of NGF and NGS technology for bone marrow MRD detection in patients with MM.

Low circulating tumor cell levels correlate with favorable outcomes and distinct biological features in multiple myeloma.

There is growing interest in multiple myeloma (MM) circulating tumor cells (CTCs), but their rareness in peripheral blood (PB) and inconsistency in cutoffs question their clinical utility. Herein, we applied next-generation flow cytometry in 550 bone marrow (BM) and matched PB samples to define an optimal CTC cutoff for both transplant-eligible and transplant-ineligible newly diagnosed MM (NDMM) patients. Deep phenotyping was performed to investigate unique microenvironmental features associated with CTC dissemination. CTCs were detected in 90% of patients (median 0.01%; range: 0.0002%-12.6%) and increased levels associated with adverse features. Correlations were observed between high CTC percentages and a diffused MRI pattern, a distinct BM composition characterized by altered B-cell differentiation together with an expansion of effector cells and tumor-associated macrophages, as well as a greater phenotypic dissimilarity between BM and PB clonal cells. Progression-free survival (PFS) and overall survival (OS) gradually worsened with each logarithmic increment of CTCs. Conversely, NDMM patients without CTCs showed unprecedented outcomes, with 5-year PFS and OS rates of 83% and 97%, respectively. A cutoff of 0.02% CTCs was independent of the ISS, LDH, and cytogenetics in a multivariate analysis of risk factors for PFS. The 0.02% CTC cutoff synergized with the MGUS-like phenotype and the R-ISS for improving the risk stratification systems. MRD negativity was less frequent if CTCs were ≥0.02% at diagnosis, but whenever achieved, the poor prognosis of these patients was abrogated. This study shows the clinical utility of CTC assessment in MM and provides evidence toward a consensus cutoff for risk stratification.

Long-term follow-up of ARI0002h (cesnicabtagene autoleucel), an academic point-of-care B-cell maturation antigen (BCMA)-directed chimeric antigen receptor (CAR) T-cell strategy: Activity and safety after fractionated initial therapy and booster dose in 60 patients with relapsed/refractory multiple myeloma (RRMM).

7544 Background: The activity and safety ofARI0002h, an academic autologous CAR T-cell product with a humanized single chain variable fragment targeting BCMA has been reported in a pilot multicenter clinical trial (CARTBCMA-HCB-01) treating 30 patients (pts) with RRMM (NCT04309981) (Oliver-Caldés, Lancet Onc 2023). Here, we describe results of the final cohort of 60 pts with longer follow-up. Methods: Patients aged 18-75 years old with RRMM were eligible if they had measurable disease, were refractory to the last line of treatment and received ≥2 prior regimens, including a proteasome inhibitor, an immunomodulatory drug and an anti-CD38 antibody. The target dose (3x106/kg CAR+cells) was administered in a fractionated manner (10%/30%/60%). A second dose of up to 3x106 CAR+ cells/kg was planned at least 3 months after the first dose in pts with any kind of response and no limiting side effects. Primary endpoints were overall response rate (ORR) within the first 3 months and rate of cytokine release syndrome (CRS) and/or neurotoxicity in the first 30 days. Bone marrow minimal residual disease (MRD) was analyzed by next-generation flow at a sensitivity of 10-6. Results: As of December 18th 2023, 72 pts with RRMM were screened, 69 underwent apheresis and 61 received LD, with 60 pts finally receiving ARI0002h. The ORR in the first 3 months was 95% (≥ very good partial response (VGPR) in 77%). Median time to first response was one month. Responses deepened over time, achieving 58% complete response (CR) (55% stringent CR). MRD-negative rates on evaluable samples on days 28 and 100 were 98% and 96%, respectively. With a median follow-up of 24 months (95%CI 9.8-39.9), estimated median progression-free survival (PFS) was 20 months (95% CI 13.2-26.8). Median overall survival (OS) was not reached with OS rate at 24 months of 63%. CRS was observed in 90% with 5% grades ≥ 3. Median time to CRS was 7 days (1-14) with a median duration of 4.5 days. Mild acute neurotoxicity was reported in only 2 pts (3%) with no late neurologic events. 6 pts (10%) developed a macrophage activation syndrome (4 grades 1-2, 1 grade 3, 1 grade 5). Seven patients (11.7%) developed second primary malignancies after ARI0002h infusion, including 3 skin cancers (one in situ melanoma), 3 solid tumors and one acute myeloid leukemia. 80% (44 out of 5) eligible pts had already received the booster dose, with no relevant toxicities. Median time after first infusion was 4.4 months. Response was evaluable in 42 pts; 45% (n=19) were already in sCR, 29% (n=12) maintained the response and 26% (n=11) improved the response. Conclusions: Results from 60 pts and a longer follow-up confirm the safety profile and the deep and durable responses obtained after ARI0002h infusion. Clinical trial information: NCT04309981 .

Detection of clonal plasma cells in POEMS syndrome using multiparameter flow cytometry

POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein [M-protein], and skin changes) is a rare systemic disorder characterized by various symptoms caused by underlying plasma cell (PC) dyscrasia. Detection of monoclonal PCs is mandatory for the diagnosis of POEMS syndrome; however, the usefulness of EuroFlow-based next-generation flow cytometry (EuroFlow-NGF) in POEMS syndrome for detecting monoclonal PCs in bone marrow (BM) and the gating strategy suitable for flow cytometry study of POEMS syndrome remain unknown. We employed EuroFlow-NGF-based single-tube eight-color multiparameter flow cytometry (MM-flow) and established a new gating strategy (POEMS-flow) to detect the monoclonal PCs in POEMS syndrome, gating CD38 broadly from dim to bright and CD45 narrowly from negative to dim compared to MM-flow. MM-flow detected monoclonal PCs in 9/25 (36.0%) cases, including 2/2 immunofixation electrophoresis (IFE)-negative cases (100%). However, POEMS-flow detected monoclonal PCs in 18/25 cases (72.0%), including 2/2 IFE-negative cases (100%). POEMS-flow detected monoclonal PCs with immunophenotypes of CD19− in 17/18 (94.4%). In six cases where post-treatment samples were available, the size of the clones was significantly reduced after the treatment (P = 0.031). POEMS-flow can enhance the identification rate of monoclonal PCs in POEMS syndrome and become a valuable tool for the diagnosis of POEMS syndrome.

The Evolving Landscape of Flowcytometric Minimal Residual Disease Monitoring in B-Cell Precursor Acute Lymphoblastic Leukemia.

Detection of minimal residual disease (MRD) is a major independent prognostic marker in the clinical management of pediatric and adult B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), and risk stratification nowadays heavily relies on MRD diagnostics. MRD can be detected using flow cytometry based on aberrant expression of markers (antigens) during malignant B-cell maturation. Recent advances highlight the significance of novel markers (e.g., CD58, CD81, CD304, CD73, CD66c, and CD123), improving MRD identification. Second and next-generation flow cytometry, such as the EuroFlow consortium's eight-color protocol, can achieve sensitivities down to 10-5 (comparable with the PCR-based method) if sufficient cells are acquired. The introduction of targeted therapies (especially those targeting CD19, such as blinatumomab or CAR-T19) introduces several challenges for flow cytometric MRD analysis, such as the occurrence of CD19-negative relapses. Therefore, innovative flow cytometry panels, including alternative B-cell markers (e.g., CD22 and CD24), have been designed. (Semi-)automated MRD assessment, employing machine learning algorithms and clustering tools, shows promise but does not yet allow robust and sensitive automated analysis of MRD. Future directions involve integrating artificial intelligence, further automation, and exploring multicolor spectral flow cytometry to standardize MRD assessment and enhance diagnostic and prognostic robustness of MRD diagnostics in BCP-ALL.

Evaluation of next-generation sequencing versus next-generation flow cytometry for minimal-residual-disease detection in Chinese patients with multiple myeloma

PurposeTo evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM).MethodsThis study analyzed 60 Chinese MM patients. During MRD monitoring in these patients’ post-therapy, clonal immunoglobulin heavy chain (IGH) rearrangements were detected via NGS using LymphoTrack assays. MRD monitoring was performed using NGS or next-generation flow cytometry (NGF), and the results were compared. Additionally, the sensitivity and reproducibility of the NGS method were assessed.ResultsThe MRD detection range of the NGS method was 10–6–10–1, which suggested good linearity, with a Pearson correlation coefficient of 0.985 and a limit of detection of 10–6. Intra- and inter-assay reproducibility analyses showed that NGS exhibited 100% reproducibility with low variability in clonal cells. At diagnosis, unique clones were found in 42 patients (70.0%) with clonal IGH rearrangements, which were used as clonality markers for MRD monitoring post-therapy. Comparison of NGS and NGF for MRD monitoring showed 79.1% concordance. No samples that tested MRD-positive via NGF were found negative via NGS, indicating the higher sensitivity of NGS. MRD could be detected using NGS in 6 of 7 samples before autologous hematopoietic stem-cell transplantation, and 5 of them tested negative post-transplantation. In contrast, the NGF method could detect MRD in only 1 sample pre-transplantation.ConclusionCompared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

Biomarkers of Efficacy and Safety of the Academic BCMA-CART ARI0002h for the Treatment of Refractory Multiple Myeloma.

B-cell maturation antigen (BCMA)-chimeric antigen receptor T-cells (CART) improve results obtained with conventional therapy in the treatment of relapsed/refractory multiple myeloma. However, the high demand and expensive costs associated with CART therapy might prove unsustainable for health systems. Academic CARTs could potentially overcome these issues. Moreover, response biomarkers and resistance mechanisms need to be identified and addressed to improve efficacy and patient selection. Here, we present clinical and ancillary results of the 60 patients treated with the academic BCMA-CART, ARI0002h, in the CARTBCMA-HCB-01 trial. We collected apheresis, final product, peripheral blood and bone marrow samples before and after infusion. We assessed BCMA, T-cell subsets, CART kinetics and antibodies, B-cell aplasia, cytokines, and measurable residual disease by next-generation flow cytometry, and correlated these to clinical outcomes. At cut-off date March 17, 2023, with a median follow-up of 23.1 months (95% CI, 9.2-37.1), overall response rate in the first 3 months was 95% [95% confidence interval (CI), 89.5-100]; cytokine release syndrome (CRS) was observed in 90% of patients (5% grades ≥3) and grade 1 immune effector cell-associated neurotoxicity syndrome was reported in 2 patients (3%). Median progression-free survival was 15.8 months (95% CI, 11.5-22.4). Surface BCMA was not predictive of response or survival, but soluble BCMA correlated with worse clinical outcomes and CRS severity. Activation marker HLA-DR in the apheresis was associated with longer progression-free survival and increased exhaustion markers correlated with poorer outcomes. ARI0002h kinetics and loss of B-cell aplasia were not predictive of relapse. Despite deep and sustained responses achieved with ARI0002h, we identified several biomarkers that correlate with poor outcomes.

Endogenous monoclonal immunoglobulins analyzed using the EXENT® solution and LC-MS

IntroductionThe EXENT® Solution, a fully automated system, is a recent advancement for identifying and quantifying monoclonal immunoglobulins in serum. It combines immunoprecipitation with MALDI-TOF mass spectrometry. Compared to gel-based methods, like SPEP and IFE, it has demonstrated the ability to detect monoclonal immunoglobulins in serum at lower levels. In this study, samples that tested negative using EXENT® were reflexed to LC-MS to determine if the more sensitive LC-MS method could identify monoclonal immunoglobulins missed by EXENT®. ObjectivesTo assess whether monoclonal immunoglobulins that are not detected by EXENT® can be detected by LC-MS using a low flow LC system coupled to a Q-TOF mass spectrometer. MethodsSamples obtained from patients confirmed to have multiple myeloma (MM) were diluted with pooled polyclonal human serum and analyzed using EXENT®. If a specific monoclonal immunoglobulin was not detected by EXENT®, the sample was then subjected to analysis by LC-MS. For the LC-MS analysis, the sample eluate, obtained after the MALDI-TOF MS spotting step, was collected and transferred to an autosampler tray for subsequent analysis using LC-MS. ConclusionLC-MS has the capability to detect monoclonal immunoglobulins that are no longer detected by EXENT®. Reflexing samples to LC-MS for analysis does not involve additional sample handling, allowing for a faster time-to-result compared to current approaches, such as Next-Generation Sequencing, Next-Generation Flow, and clonotypic peptide methods. Notably, LC-MS offers equivalent sensitivity in detecting these specific monoclonal immunoglobulins.

Monitoring Minimal Residual Disease in Patients with Multiple Myeloma by Targeted Tracking Serum M-Protein Using Mass Spectrometry (EasyM).

We investigated both the clinical utilities and the prognostic impacts of the clonotypic peptide mass spectrometry (MS)-EasyM, a blood-based minimal residual disease (MRD) monitoring protocol in multiple myeloma. A total of 447 sequential serum samples from 56 patients with multiple myeloma were analyzed using EasyM. Patient-specific M-protein peptides were sequenced from diagnostic samples; sequential samples were quantified by EasyM to monitor the M-protein. The performance of EasyM was compared with serum immunofixation electrophoresis (IFE), bone marrow multiparameter flow cytometry (MFC), and next-generation flow cytometry (NGF) detection. The optimal balance of EasyM sensitivity/specificity versus NGF (10-5 sensitivity) was determined and the prognostic impact of MS-MRD status was investigated. Of the 447 serum samples detected and measured by EasyM, 397, 126, and 92 had time-matching results for comparison with serum IFE, MFC-MRD, and NGF-MRD, respectively. Using a dotp >0.9 as the MS-MRD positive, sensitivity was 99.6% versus IFE and 100.0% versus MFC and NGF. Using an MS negative cutoff informed by ROC analysis (<1.86% of that at diagnosis), EasyM sensitivity remained high versus IFE (88.3%), MFC (85.1%), and NGF (93.2%), whereas specificity increased to 90.4%, 55.8%, and 93.2%, respectively. In the multivariate analysis, older diagnostic age was an independent predictor for progression-free survival [PFS; high risk (HR), 3.15; 1.26-7.86], the best MS-MRD status (MS-MRD negative) was independent predictor for both PFS (HR, 0.25; 0.12-0.52) and overall survival (HR, 0.16; 0.06-0.40). EasyM is a highly sensitive and minimal invasive method of MRD monitoring in multiple myeloma; MS-MRD had significant predictive ability for survival outcomes.

Impact of pretransplant minimal residual disease in patients with multiple myeloma and a very good partial response or better receiving autologous hematopoietic stem cell transplantation.

The prognostic significance of minimal residual disease (MRD) status before autologous hematopoietic stem cell transplantation (autoHCT) in patients with multiple myeloma (MM) has not been clearly elucidated. Retrospective single-center study of adult MM patients who achieved ≥very good partial response (VGPR) after induction therapy from 2015 to 2021 received upfront autoHCT and had available pretransplant MRD status by next-generation flow cytometry. The cohort was divided into pretransplant MRD-negative (MRDneg) and MRD-positive (MRDpos) groups. A total of 733 patients were included in our analysis; 425 were MRDneg and 308 MRDpos at autoHCT. In the MRDpos group, more patients had high-risk cytogenetic abnormalities (48% vs. 38%, respectively; p=.025), whereas fewer patients achieved ≥CR before autoHCT (14% vs. 40%; p<.001). At day 100 after autoHCT, 37% of the MRDpos versus 71% of the MRDneg achieved ≥CR, and at best posttransplant response 65% versus 88% achieved ≥CR, respectively. After a median follow-up of 27.6months (range, 0.7-82.3), the median PFS was significantly shorter for patients in the MRDpos group compared to the MRDneg group: 48.2months (95% confidence interval [CI], 0.3-80.5) versus 80.1months (95% CI, 0.5-80.1), respectively (p<.001). There was no significant difference in overall survival between the two groups (p=.41). Pretransplant MRDpos status was predictive of shorter PFS in multivariate analysis (hazard ratio, 1.80; 95% CI, 1.31-2.46; p<.001). The impact of pretransplant MRD status was retained in most of the examined subgroups. In patients achieving ≥VGPR to induction, pretransplant MRDpos status was associated with a lower CR rate after autoHCT and a shorter PFS.