scholarly journals Validation of a Sensitive Flow Cytometry Assay to Assess Minimal Residual Disease of Multiple Myeloma Cells in Apheresis Product

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5646-5646
Author(s):  
Nicholas Jones ◽  
Josette William Ragheb ◽  
Brian Ngo ◽  
David Uyeji ◽  
Anselm L. Hii
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Minimal Residual Disease ◽  
Patient Monitoring ◽  
Residual Disease ◽  
Cell Transplant ◽  
Flow Cytometry Assay ◽  
Color Flow ◽  
Minimal Residual

Abstract For treatment of patients with multiple myeloma (MM), flow cytometry has become a widely used and valuable method for the evaluation of minimal residual disease (MRD) in bone marrow. Use of an optimized single-tube, 10-color flow cytometry panel for assessment of MM MRD has shown to be beneficial in patient monitoring and has shown correlation to the acknowledged EuroFlow 8-color, 2-tube method. In order to further correlate levels of MM and relapse in patients, validation of a sensitive MM MRD assay that can be applied to testing for residual disease in apheresis product prior to autologous stem cell transplant, a standard treatment for patients with multiple myeloma. This approach can exhibit great value in patient monitoring and treatment. As new combination therapies are developed for treatment of multiple myeloma in concert with autologous stem cell transplantation, evaluation of MRD in apheresis will likely continue to grow in importance. The purpose of this study is to show the validation and sensitivity of a flow cytometry assay designed to detect Multiple Myeloma (MM) cells in G-CSF and other mobilized apheresis samples from human Multiple Myeloma patients. Using GSM-mobilized apheresis product, spiked with different levels of patient de-identified MM plasma cells, validation of a 10-color MM MRD panel can be evaluated, intended to mimic patient apheresis at varying levels of residual disease post treatment. As with any minimal residual disease assessment, a high number of events are required to ensure sensitivity and precision. Typically, acquisition of 3-5 x 107 events is required to ensure precision of MM MRD levels to 0.001%. Disclosures No relevant conflicts of interest to declare.

scholarly journals Detection of Minimal Residual Disease by Flow Cytometry for Patients with Multiple Myeloma Submitted to Autologous Hematopoietic Stem Cell Transplantation

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Suzane Dal Bó ◽  
Annelise Pezzi ◽  
Bruna Amorin ◽  
Vanessa Valim ◽  
Rosane Isabel Bittencourt ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Minimal Residual Disease ◽  
Cell Transplantation ◽  
Residual Disease ◽  
Hematopoietic Stem ◽  
Minimal Residual

The treatment strategy in multiple myeloma (MM) is to get complete remission followed by high-dose chemotherapy and autologous Hematopoietic Stem Cell Transplantation (HSCT). Neoplastic Plasma Cells (NPCs) are CD45-/dim, CD38+high, CD138+, CD19−, and  CD56+high in most cases. The description of this immunophenotype is of major importance as it leads to the correct identification of minimal residual disease (MRD). Samples from 44 Patients were analyzed prospectively in this study. We analyzed if the presence of MRD at three months after HSCT was predictive of relapse or death. There were 40 evaluable patients of whom 16/40 patients had MRD at three moths after HSCT and there were none in cytological relapse. The mean overall survival (OS) was 34 months and disease-free survival (RFS) was 28 months after HSCT. There was no significant difference in the log rank analysis comparing OS and the presence of MRD (P=0,611) and RFS (P=0,3106). Here, we demonstrate that three color flow cytometry (FCM) is more sensitive for MDR evaluation than cytological analyzes. However, based in our data we can not affirm that MRD is a good predictor of MM relapse or death. In conclusion, our results could be attributed to a short followup, small sample size, and over most to the inability of a three-color FCM to detect the NPC population.

Carfilzomib, Lenalidomide, and Dexamethasone in High-Risk Smoldering Multiple Myeloma: Final Results from the NCI Phase 2 Pilot Study

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4746-4746 ◽  
Author(s):  
Ola Landgren ◽  
Mark Roschewski ◽  
Sham Mailankody ◽  
Mary Kwok ◽  
Elisabet E. Manasanch ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
High Risk ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Color Flow ◽  
Smoldering Multiple Myeloma ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract BACKGROUND: Early treatment with lenalidomide and dexamethasone delays progression and increases overall survival in patients with high-risk smoldering multiple myeloma. The addition of the selective proteasome inhibitor carfilzomib to a lenalidomide and dexamethasone backbone has proven effective in patients with newly-diagnosed multiple myeloma; this combination may allow patients with high-risk smoldering multiple myeloma to obtain deep and durable responses. METHODS: In this phase 2 pilot study, patients with high-risk smoldering multiple myeloma received eight 28-day cycles of induction therapy with carfilzomib (at a dose of 20/36 mg per square meter on days 1, 2, 8, 9, 15, and 16), lenalidomide (at a dose of 25 mg on days 1–21), and dexamethasone (at a dose of 10 or 20 mg on days 1, 2, 8, 9, 15, 16, 22, and 23). Patients achieving stable disease or better after combination therapy received 2 years of maintenance therapy with lenalidomide. Minimal residual disease was assessed with multi-color flow cytometry, next-generation sequencing by the LymphoSIGHT method, and fluorodeoxyglucose-positron emission tomography-computed tomography (FDG-PET/CT). Myeloma clonotypes were identified in genomic DNA obtained from CD138+ bone marrow cell lysate or cell-free bone marrow aspirate at baseline for each patient based on their high frequency within the B-cell repertoire. Per study protocol, minimal residual disease assessment by next-generation sequencing, multi-color flow cytometry and FDG-PET/CT was repeated when patients achieved a complete response or completed 8 cycles of induction treatment. A sample size of 12 evaluable patients was calculated as being minimally necessary based on the following probability calculations: If the true probability of a very good partial response was 20% or 50%, we calculated that there would be a 7.3% or 80.6% probability, respectively, if 5 or more patients exhibiting a very good partial response (VGPR). Thus, if 5 or more patients out of 12 achieved a very good partial response, there would be strong evidence that the true probability of a VGPR was 50% or more. RESULTS: Twelve patients were enrolled. All 11 patients (100%) who completed 8 cycles of combination therapy obtained VGPR or better (primary end point). Minimal residual disease assessment by next-generation sequencing was performed on bone marrow supernatant to detect cell-free myeloma clonotypes, while flow cytometry analysis utilized bone marrow cells. Overall (N=12), 100% of patients achieved a complete response or better over the study period, including 11 patients (92%) negative for minimal residual disease based on multi-color flow cytometry. Based on next-generation sequencing, two of the 12 patients were positive for minimal residual disease in the bone marrow supernatant; one of these two patients was also positive for minimal residual disease based on multi-color flow cytometry in the bone marrow cells. Information regarding longitudinal minimal residual disease status will be available and presented at the meeting. Adverse events were manageable. CONCLUSIONS: Early treatment with carfilzomib, lenalidomide, and dexamethasone was associated with high rates of complete response and minimal residual disease negativity by multi-color flow cytometry, next-generation sequencing, and FDG-PET/CT in patients with high-risk smoldering multiple myeloma. Disclosures Landgren: Onyx Pharmaceuticals: Consultancy; Medscape: Consultancy; Millennium Pharmaceuticals: Independent Data Monitoring Committee (IDMC), Independent Data Monitoring Committee (IDMC) Other. Off Label Use: Carfilzomib and lenalidomide for high-risk smoldering multiple myeloma.

scholarly journals Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1625-1625
Author(s):  
Anwar Khan ◽  
Nagehan Pakasticali ◽  
Omar Fathalla ◽  
Taiga Nishihori ◽  
Mohammad O Hussaini
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Correlation Coefficient ◽  
Research Funding ◽  
Residual Disease ◽  
High Sensitivity ◽  
Spearman Correlation ◽  
Color Flow ◽  
Minimal Residual

Abstract Introduction: Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices. Methods: We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review. 10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter). clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences Statistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis. Results: 192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1). Conclusion: Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. Figure 1 Figure 1. Disclosures Nishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

Assessment of Minimal Residual Disease in Multiple Myeloma patients undergoing Autologous Stem Cell Transplant using Multiparameter Flow Cytometry

QJM ◽  
2021 ◽  
Vol 114 (Supplement_1) ◽  
Author(s):  
NN Mostafa ◽  
WA El-Salakawy ◽  
HM Abdelbary ◽  
RA Radwan ◽  
RK Fathy ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Limit Of Detection ◽  
Multiparameter Flow Cytometry ◽  
P Value ◽  
Cell Transplant ◽  
Post Transplant ◽  
Minimal Residual

Abstract BACKGROUND Multiple Myeloma (MM) is still considered an incurable relapsing disease, despite major advances in multidrug combinations, incorporation of autologous transplant and achieving higher rates of CR. This suggests persistent presence of residual disease, not detected by current techniques. Minimal residual disease (MRD) monitoring by multiparameter flow cytometry (MFC) is a powerful tool to quantitatively measure residual disease and allow tailoring of the management plan on individual basis. AIM To assess the value of MRD by MFC in determining the efficacy of treatment, monitoring depth of remission and predicting impending relapse. PATIENTS AND METHODS We included 33MM patients, who were candidates for ASCT in this prospective study. MFC (with a 0.01% limit of detection) on a BM aspirate obtained before transplant and at day 100 post-transplant, was used to measure MRD for all patients. Patients were then later assessed for progression 1-year post-transplant. RESULTS MRD status at 100 days post-transplant was strongly related to the risk of 1-year post-transplant progression (p-value 0.001), and by using a ROC curve, we found that MRD (%) at 100 days post-transplant can predict progression after 1 year with a best cutoff value ≥ 0.04 achieving a 75% sensitivity and 81.2% specificity. On dividing the patients according to their MRD status before and after transplant, group 3 who were MRD positive pre- and post-transplant, had a high risk of progression 1year post-transplant (p-value 0.003). The use of bortezomib-based regimens resulted in a deeper response and a more negative MRD pre-transplant (p-value 0.01), that was maintained post-transplant. Also, ASCT resulted in the development of deeper remission (p-value 0.004) and more MRD negativity (p-value 0.02). CONCLUSION we recommend the use of MRD by MFC as a powerful prognostic tool that should be incorporated in routine myeloma workup.

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