Comparison of Next-Generation Flow Cytometry (NGF) and Next-Generation Sequencing (NGS) in the Assessment of Minimal Residual Disease in Multiple Myeloma

Abstract

Background:Next generation flow cytometry (NGF) and next generation sequencing (NGS) are standard methods for detection of minimal residual disease (MRD) in multiple myeloma (MM) according to domestic and international guidelines. However, due to the inability to standardize laboratory technology among hospitals, the results are unreliable. In order to meet the challenges of data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment，and to further confirm the role of MRD in evaluating deep remission，we provide a approach to evaluate and compare the efficacy of each patient after treatment by NGF and NGS concurrently. Methods: In a VRD clinical study in our center, a cohort analysis was performed on 82 patients with newly diagnosed multiple myeloma in the First Affiliated Hospital of Soochow University from September 01, 2019 to January 31, 2022.Bone marrow (BM) sample from 82 MM patients were analyzed by NGS and NGF MM MRD methods during treating procedure. Limit of detection (LOD) and tumor load (TL) were calculated, and the concept of MRD negative was defined. At the same time, step-by-step statistics and analysis of MRD results were carried out to confirm its role in evaluating the depth of remission, and to analyze and compare whether the two methods were consistent and the difference in detection rate, evaluating the prediction of patient survival by MRD detection. Results:Among the 82 patients, 100.00% of NGF specimens and 96.34% of NGS specimens met the LOD criteria of 1.0e-5. Non-detection of tumor cells at the 1.0e-5 level was defined as NGF-MRD-negative or NGS-MRD-negative. After induction therapy, the positive detection rate of NGS was higher than that of NGF (81.01% vs 51.22%). The patients who did not achieve VGPR serological response were all MRD positive. The positive rate of NGF-MRD was 42.25%, and the positive rate of NGS-MRD was 78.26% in patients with serological response reaching VGPR or above. With the deepening of serological efficacy: from PR to VGPR to CR and sCR, the positive rate of MRD gradually reduced（NGF：100.00%-70.37%-25.00%；NGS：100.00%-96.15%-67.44%). The median tumor load was progressively decreased (NGF: 10-3-10-4-10-6 level; NGS: 10-1-10-2-10-4 level). Compared with NGF, NGS not only has a higher positive detection rate, but also can detect more tumor load. Pairing the NGF-MRD and NGS-MRD results, the agreement between the two methods (NGF+NGS+ and NGF-NGS-) was 63.77%, and the Pearson correlation coefficient indicated a certain correlation in TL quantification (n=54 , r=0.4371, P=0.001). Conclusion: An MRD assessment (at least a 10-5 level) should be performed after obtaining a serologic response to VGPR and above. When analyzing MM-MRD, the LOD value should be marked and the definition of MRD negative should be stated. Compared with NGF, NGS not only has a higher positive detection rate of MRD (67.44% vs 25.00%), but also can detect more tumor load and has higher sensitivity in patients with CR or above after induction therapy. NGF MRD positivity predicts shorter PFS and earlier relapse after induction therapy. NGF and NGS MRD negativity predicts 100.00% in 2-year PFS. Keywords: multiple myeloma (MM); minimal residual disease (MRD); next-generation flow cytometry (NGF); next-generation gene sequencing (NGS); line of detection (LOD); tumor load (TL) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal