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.

PURPOSE: To evaluate the efficacy of next-generation sequencing (NGS) in minimal-residual-disease (MRD) monitoring in Chinese patients with multiple myeloma (MM). METHODS: This 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. RESULTS: The 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. CONCLUSION: Compared with NGF, NGS exhibits higher sensitivity and reproducibility in MRD detection and can be an effective strategy for MRD monitoring in Chinese MM patients.

Measurable residual disease in acute lymphoblastic leukemia: How low is low enough?

Quantification of measurable residual disease (MRD) in acute lymphoblastic leukemia (ALL) is a well-established clinical tool used to risk stratify patients during the course of chemotherapy, immunotherapy, and/or transplant therapy. As technologies evolve, the sensitivity for quantifying exceptionally low disease burden using either next generation sequencing (NGS) or next generation flow cytometry (NGF) has improved. It is now possible to detect MRD and quantify it precisely in patients who would previously have been deemed MRD negative by older, lower sensitivity methods. Persistence or recurrence of ALL disease burden above 10-4 (0.01%) is accepted as the minimum threshold for making clinical decisions, but with NGS and NGF, clinicians now confront decision-making with disease burdens sometimes quantified to as low as 10-6 (0.0001%, or one leukemia cell in a million leukocytes). Emerging data suggest these higher sensitivity methods are superior for identifying patients at lowest risk for relapse, but it remains controversial whether to institute therapies such as blinatumomab or chimeric antigen receptor (CAR)-T cells or move patients to allogeneic hematopoietic cell transplant (alloHCT) when they have quantifiable disease burden less than 10-4. With additional evidence to facilitate integration of highly sensitive MRD quantification into clinical care and to contextualize MRD within the genotype of individual patients, it will likely be increasingly possible to identify patients able to avoid alloHCT and potentially even de-escalate therapy.

Drug resistance and minimal residual disease in multiple myeloma.

Great progress has been made in improving survival in multiple myeloma (MM) patients over the last 30 years. New drugs have been introduced and complete responses are frequently seen. However, the majority of MM patients do experience a relapse at a variable time after treatment, and ultimately the disease becomes drug-resistant following therapies. Recently, minimal residual disease (MRD) detection has been introduced in clinical trials utilizing novel therapeutic agents to measure the depth of response. MRD can be considered as a surrogate for both progression-free and overall survival. In this perspective, the persistence of a residual therapy-resistant myeloma plasma cell clone can be associated with inferior survivals. The present review gives an overview of drug resistance in MM, i.e., mutation of ß5 subunit of the proteasome; upregulation of pumps of efflux; heat shock protein induction for proteasome inhibitors; downregulation of CRBN expression; deregulation of IRF4 expression; mutation of CRBN, IKZF1, and IKZF3 for immunomodulatory drugs and decreased target expression; complement protein increase; sBCMA increase; and BCMA down expression for monoclonal antibodies. Multicolor flow cytometry, or next-generation flow, and next-generation sequencing are currently the techniques available to measure MRD with sensitivity at 10-5. Sustained MRD negativity is related to prolonged survival, and it is evaluated in all recent clinical trials as a surrogate of drug efficacy.

Clinical Utility of Next-Generation Flow-Based Minimal Residual Disease Assessment in Patients with Multiple Myeloma.

Background: Minimal residual disease (MRD) is an important prognostic factor for evaluating a deeper treatment response in patients with multiple myeloma (MM). We evaluated the clinical utility of next-generation flow (NGF)-based MRD assessment in a heterogeneous MM patient population. Methods: Patients with suspected morphological remission after or during MM treatment were prospectively enrolled. In total, 108 bone marrow samples from 90 patients were analyzed using NGF-based MRD assessment according to the EuroFlow protocol, and progression-free survival (PFS) was evaluated according to the International Myeloma Working Group response status, cytogenetic risk, and MRD status. Results: The overall MRD-positive rate was 31.5% (34/108 samples), and MRD-positive patients showed a lower PFS than MRD-negative patients (P=0.005). MRD-positive patients showed inferior PFS than MRD-negative in patients with stringent complete remission (sCR)/complete remission (P=0.014) and high-risk cytogenetic abnormalities (P=0.016). MRD was assessed twice in 18 patients with a median interval of 12 months. Sustained MRD negativity was only observed in patients with sustained sCR, and their PFS was superior to that of patients who were not MRD-negative (P=0.035). Conclusions: Clinical application of NGF-based MRD assessment can provide valuable information for predicting disease progression in patients with MM in remission, including those with high-risk cytogenetic abnormalities.

Relevance of polyclonal plasma cells and post-therapy immunomodulation in measurable residual disease assessment in multiple myeloma.

BACKGROUND: Immunophenotypic profile and post-therapy alteration in antigenic expression were evaluated in normal, reactive, and aberrant plasma cells (NPC, RPC, and APC) for impact on measurable residual disease (MRD) assessment in multiple myeloma (MM). METHODS: Samples from non-MM staging marrow (n = 30), Hodgkin's lymphoma (n = 30) and MM (n = 724) were prospectively evaluated for expression profiles of NPC, RPC, and APC using antigens recommended in consensus guidelines. RESULTS: Polyclonal NPC-RPC demonstrated aberrations for all antigens evaluated with a higher frequency of aberrancies in post-therapy samples compared to treatment naïve samples (p < 0.001%). Immunomodulation in APC was observed in 79% of post-therapy samples with a change in expression of 1, 2, and ≥3 antigens in 19.9%, 15.6%, and 43.5% samples, respectively. In 13.4% of samples, APC showed no aberrancy and aberrant status was assigned based on cytoplasmic light chain restriction (cyLCR) alone. 9% samples with an admixture of NPC and APC displayed normal cytoplasmic kappa to lambda ratio (cyKLR) when the percentage of APC of total PC (neoplastic plasma cell index, NPCI), was below 25% and 50% for kappa and lambda restricted cases, respectively. CONCLUSION: The panorama and high frequency of antigenic aberrations on polyclonal PC signify the importance of MRD assay validation on a large cohort under normal and reactive conditions. Frequent Immunophenotypic shifts in APC re-confirm the redundancy of baseline immunophenotype for MRD evaluation. Small clones of APC may be missed by assessment of cyKLR alone and therefore, surface marker aberrancy supported by cyLCR is required for definitive assignment of residual APC.

Contemporary Challenges in Clinical Flow Cytometry: Small Samples, Big Data, Little Time.

BACKGROUND: Immunophenotypic analysis of cell populations by flow cytometry has an established role in primary diagnosis and disease monitoring of many hematologic diseases. A persistent problem in evaluation of specimens is suboptimal cell counts and low cell viability, which results in an undesirable rate of analysis failure. In addition, the increased amount of data generated in flow cytometry challenges existing data analysis and reporting paradigms. CONTENT: We describe current and emerging technological improvements in cell analysis that allow the clinical laboratory to perform multiparameter analysis of specimens, including those with low cell counts and other quality issues. These technologies include conventional multicolor flow cytometry and new high-dimensional technologies, such as spectral flow cytometry and mass cytometry that enable detection of over 40 antigens simultaneously. The advantages and disadvantages of each approach are discussed. We also describe new innovations in flow cytometry data analysis, including artificial intelligence-aided techniques. SUMMARY: Improvements in analytical technology, in tandem with innovations in data analysis, data storage, and reporting mechanisms, help to optimize the quality of clinical flow cytometry. These improvements are essential because of the expanding role of flow cytometry in patient care.

Aberrant Plasma Cell Contamination of Peripheral Blood Stem Cell Autografts, Assessed by Next-Generation Flow Cytometry, Is a Negative Predictor for Deep Response Post Autologous Transplantation in Multiple Myeloma; A Prospective Study in 199 Patients.

High-dose chemotherapy with autologous stem cell support (ASCT) is the standard of care for eligible newly diagnosed Multiple Myeloma (MM) patients. Stem cell graft contamination by aberrant plasma cells (APCs) has been considered a possible predictive marker of subsequent clinical outcome, but the limited reports to date present unclear conclusions. We prospectively estimated the frequency of graft contamination using highly sensitive next-generation flow cytometry and evaluated its clinical impact in 199 myeloma patients who underwent an ASCT. Contamination (con+) was detected in 79/199 patients at a median level 2 × 10-5. Its presence and levels were correlated with response to induction treatment, with 94%, 71% and 43% achieving CR, VGPR and PR, respectively. Importantly, con+ grafts conferred 2-fold and 2.8-fold higher patient-risk of not achieving or delaying reaching CR (4 vs. 11 months) and MRD negativity (5 vs. 18 months) post ASCT, respectively. Our data also provide evidence of a potentially skewed bone marrow (BM) reconstitution due to unpurged grafts, since con+ derived BM had significantly higher prevalence of memory B cells. These data, together with the absence of significant associations with baseline clinical features, highlight graft contamination as a potential biomarker with independent prognostic value for deeper responses, including MRD negativity. Longer follow-up will reveal if this corresponds to PFS or OS advantage.

Next generation flow cytometry for MRD detection in patients with AL amyloidosis.

The treatment of AL amyloidosis aims to eradicate the plasma cell clone and eliminate toxic free light chain production. Only in a minority of patients the plasma cell clone is completely eradicated; residual light chain production may still exist while clonal relapse may occur. We used sensitive next-generation flow cytometry (NGF) to detect minimal residual disease (MRD) in AL amyloidosis patients at complete haematologic response. MRD evaluation was feasible in 51 of 52 (98%) tested patients and at a median sensitivity of 2.3 × 10-6 MRD was undetectable in 23 (45%). An organ response occurred in 86% of MRDneg vs 77% in MRDpos; renal response in 15/17(88%) of MRDneg vs in 14/16(87.5%) of MRDpos and cardiac response in 10/10(100%) of MRDneg vs 11/15(73%) of MRDpos patients. After a median follow-up of 24 months post MRD testing, no MRDneg patient had a haematologic relapse vs 6/28(21%) MRDpos (p = .029). Pooling haematologic and organ progressions, 9 (32%) MRDpos patients had disease progression vs only 1 (4%) MRDneg patient (p = .026). In conclusion, MRD detection using NGF has profound clinical implications, so that AL patients with undetectable MRD have a very high probability of organ response and a very low probability of haematologic relapse.

Minimal Residual Disease in Multiple Myeloma: State of the Art and Applications in Clinical Practice.

Novel drugs have revolutionized multiple myeloma therapy in the last 20 years, with median survival that has doubled to up to 8-10 years. The introduction of therapeutic strategies, such as consolidation and maintenance after autologous stem cell transplants, has also ameliorated clinical results. The goal of modern therapies is becoming not only complete remission, but also the deepest possible remission. In this context, the evaluation of minimal residual disease by techniques such as next-generation sequencing (NGS) and next-generation flow (NGF) is becoming part of all new clinical trials that test drug efficacy. This review focuses on minimal residual disease approaches in clinical trials, with particular attention to real-world practices.

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Technical and Clinical Advances.

Introduction: Acute lymphoblastic leukemia (ALL) is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring has proven to be a fundamental tool to guide therapeutic choices. The most standardized methods to study MRD in ALL are multi-parametric flow cytometry (MFC) and polymerase chain reaction (PCR) amplification-based methods. Emerging technologies hold the promise to improve MRD detection in ALL patients. Moreover, novel therapies, such as monoclonal antibodies, bispecific T-cell engagers, and chimeric antigen receptor T cells (CART) represent exciting advancements in the management of B-cell precursor (BCP)-ALL. Aims: Through a review of the literature and in house data, we analyze the current status of MRD assessment in ALL to better understand how some of its limitations could be overcome by emerging molecular technologies. Furthermore, we highlight the future role of MRD monitoring in the context of personalized protocols, taking into account the genetic complexity in ALL. Results and Conclusions: Molecular rearrangements (gene fusions and immunoglobulin and T-cell receptor-IG/TR gene rearrangements) are widely used as targets to detect residual leukemic cells in ALL patients. The advent of novel techniques, namely next generation flow cytometry (NGF), digital-droplet-PCR (ddPCR), and next generation sequencing (NGS) appear important tools to evaluate MRD in ALL, since they have the potential to overcome the limitations of standard approaches. It is likely that in the forthcoming future these techniques will be incorporated in clinical trials, at least at decisional time points. Finally, the advent of new powerful compounds is further increasing MRD negativity rates, with benefits in long-term survival and a potential reduction of therapy-related toxicities. However, the prognostic relevance in the setting of novel immunotherapies still needs to be evaluated.

[Comparison of minimal residual disease in multiple myeloma patients detected by 8-color panels and next generation flow cytometry].

Objective: To compare the sensitivity of 8-color panels and next generation flow cytometry (NGF) for detecting minimal residual disease of multiple myeloma patients. Methods: 8-color-membrane antigens (8C-Mem) panel was built including CD45, CD38, CD138, CD19, CD56, CD81, CD27 and CD117 to identify the plasma cells, while 8-color-cytoplasmic antigens (8C-Cyto) panel was built including CD45, CD38, CD138, CD19, CD56, CD81, cKappa (cK) and cLambda (cλ) , and 8-color-two-tubes (8C-2tubes) panel were built including 8C-Mem and 8C-Cyto panels, the data of three groups was analyzed by Diva software. NGF uses Infinicyt software to fuse 8C-2tubes data to further analyze the expression of plasma antigens. Bone marrow aspiration obtained from 20 controls and 76 multiple myeloma patients who achieved complete remission were measured and analyzed. Results: Positive MRD samples were discriminated in 88.2% of the specimen evaluated through either abnormal plasma cells (aPCs) or clonal plasma cells (cPCs) by NGF antigens panel, Among of them, consistency was 94.7%. The median percentage of cPCs was 0.3530%, The lowest sensitivity of NGF was 0.0003%. In 8-color panels, the positive MRD rates of 8C-Mem, 8C-Cyto and 8C-2tubes panels were 84.2%, 85.5% and 86.8%, respectively, which lower than that of NGF (P<0.001) . The positive MRD rate of 8C-Mem and 8C-Cyto panels were lower than that of 8C-2tubes panel (P<0.001) , and the positive MRD rate of 8C-Mem panel was lower than that of 8C-Cyto panel (P<0.001) . Sensitivity and specificity of NGF was higher than that of 8-color panels. 8C-2tubes panel has the best sensitivity, accuracy, negative predicted value, positive predicted value and specificity than other 8-color panels. However, huge data and low efficiency for analysis is the disadvantage. 8C-Cyto panel was the second choice, and 8C-Mem panel was the last. Conclusions: Membrane and cytoplasmic light chain is a better method for multiple myeloma-MRD detection and NGF panel is an ideal approach. 8C-Cyto panel is recommended in 8-MFC groups.