scholarly journals Interlaboratory Analytical Validation of a Next-Generation Sequencing Strategy for Clonotypic Assessment and Minimal Residual Disease Monitoring in Multiple Myeloma

2021 ◽  
Author(s):  
Alejandro Medina ◽  
Cristina Jiménez ◽  
Noemí Puig ◽  
María Eugenia Sarasquete ◽  
Juan Flores-Montero ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Next Generation Sequencing ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Next Generation ◽  
Paired Samples ◽  
Sequencing Strategy ◽  
Generation Sequencing ◽  
Minimal Residual

Context.— Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited. Objective.— To standardize the performance of the LymphoTrack next-generation sequencing (NGS) assays (Invivoscribe), targeting clonal immunoglobulin rearrangements, in order to reproduce the detection of tumor clonotypes and MRD quantitation in myeloma. Design.— The quantification ability of the assay was evaluated through serial dilution experiments. Paired samples from 101 patients were tested by LymphoTrack, using Sanger sequencing and EuroFlow's next-generation flow (NGF) assay as validated references for diagnostic and follow-up evaluation, respectively. MRD studies using LymphoTrack were performed in parallel at 2 laboratories to evaluate reproducibility. Results.— Sensitivity was set as 1.3 tumor cells per total number of input cells. Clonality was confirmed in 99% and 100% of cases with Sanger and NGS, respectively, showing great concordance (97.9%), although several samples had minor discordances in the nucleotide sequence of rearrangements. Parallel NGS was performed in 82 follow-up cases, achieving a median sensitivity of 0.001%, while for NGF, median sensitivity was 0.0002%. Reproducibility of LymphoTrack-based MRD studies (85.4%) and correlation with NGF (R2 > 0.800) were high. Bland-Altman tests showed highly significant levels of agreement between flow and sequencing. Conclusions.— Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.

A comparison between next-generation sequencing and ASO-qPCR for minimal residual disease detection in multiple myeloma.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 8601-8601
Author(s):  
Hiroyuki Takamatsu ◽  
Ryoichi Murata ◽  
Jianbiao Zheng ◽  
Martin Moorhead ◽  
Yasushi Terasaki ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Next Generation Sequencing ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Survival Rates ◽  
Japanese Patients ◽  
Next Generation ◽  
Universal Primer ◽  
Generation Sequencing ◽  
Minimal Residual

8601 Background: Although molecular CR in multiple myeloma (MM) can be assessed by allele-specific oligonucleotide (ASO)-PCR, this technique requires preparation of clonotype-specific primers for each individual which is laborious and time-consuming. The usage of the LymphoSIGHT method, a next-generation sequencing (NGS)-based platform, may overcome these challenges and increase sensitivity and specificity. We compared the LymphoSIGHT approach with ASO-qPCR for minimal residual disease (MRD) detection in autografts in the autologous peripheral blood stem cell transplantation (ASCT) setting. Methods: Seventeen Japanese patients with newly diagnosed MM who received various induction regimens prior to ASCT were retrospectively analyzed. All patients had achieved a PR or CR after ASCT. Bone marrow (BM) slides from 13 MM patients and fresh BM cells from 4 MM patients at diagnosis as well as autografts were obtained for DNA extraction. Using universal primer sets, we amplified IGH variable (V), diversity (D), and joining (J) gene segments, IGH-DJ, and IGK from genomic DNA. Amplified products were subjected to deep sequencing using NGS. Reads were analyzed using standardized algorithms for clonotype determination. Myeloma-specific clonotypes were identified for each patient based on their high frequency in BM samples. The presence of the myeloma clonotype was then assessed in follow-up samples. Results: MRD in autografts was detected in 6 of 17 (35%) by ASO-qPCR and 13 of 17 (76%) by NGS. When MRD was assessed by NGS, 6 MRD (+) cases received post-ASCT therapy while 4 MRD (-) cases and 7 MRD (+) cases were followed without post-ASCT therapy. The MRD (-) cases tended to show a better PFS than the MRD (+) cases with post-ASCT therapy (P = 0.26) and those without post-ASCT therapy (P = 0.09) although overall survival rates were comparable among the three groups. There was no difference in PFS between MRD (-) and MRD (+) cases when MRD was assessed by ASO-qPCR (P = 0.6). These studies will be extended in 30 additional MM patients, and results will be presented. Conclusions: MRD-negativity in autografts revealed by NGS may be more closely associated with durable remission of MM than that revealed by ASO-qPCR.

scholarly journals Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma

2020 ◽  
Vol 10 (10) ◽  
Author(s):  
Alejandro Medina ◽  
Noemi Puig ◽  
Juan Flores-Montero ◽  
Cristina Jimenez ◽  
M.-Eugenia Sarasquete ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Next Generation Sequencing ◽  
Minimal Residual Disease ◽  
Cox Regression ◽  
Residual Disease ◽  
Next Generation ◽  
Increased Risk ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing ◽  
Minimal Residual

Abstract Detecting persistent minimal residual disease (MRD) allows the identification of patients with an increased risk of relapse and death. In this study, we have evaluated MRD 3 months after transplantation in 106 myeloma patients using a commercial next-generation sequencing (NGS) strategy (LymphoTrack®), and compared the results with next-generation flow (NGF, EuroFlow). The use of different marrow pulls and the need of concentrating samples for NGS biased the applicability for MRD evaluation and favored NGF. Despite that, correlation between NGS and NGF was high (R2 = 0.905). The 3-year progression-free survival (PFS) rates by NGS and NGF were longer for undetectable vs. positive patients (NGS: 88.7% vs. 56.6%; NGF: 91.4% vs. 50%; p < 0.001 for both comparisons), which resulted in a 3-year overall survival (OS) advantage (NGS: 96.2% vs. 77.3%; NGF: 96.6% vs. 74.9%, p < 0.01 for both comparisons). In the Cox regression model, NGS and NGF negativity had similar results but favoring the latter in PFS (HR: 0.20, 95% CI: 0.09–0.45, p < 0.001) and OS (HR: 0.21, 95% CI: 0.06–0.75, p = 0.02). All these results reinforce the role of MRD detection by different strategies in patient prognosis and highlight the use of MRD as an endpoint for multiple myeloma treatment.

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