Minimal residual disease (MRD) is a major prognostic factor in multiple myeloma, although validated technologies are limited.
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.
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.
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.
Taken together, we have shown that LymphoTrack is a suitable strategy for clonality detection and MRD evaluation, with results comparable to gold standard procedures.
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This work was partially supported by the Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness PI15/01956, CIBERONC-CB16/12/00233, and “Una manera de hacer Europa” (Innocampus; CEI-2010-1-0010). García-Álvarez, Prieto-Conde, and Jiménez were supported by the Fundación Española de Hematología y Hemoterapia (FEHH, cofunded by Fundación Cris in the latter case), Medina by the European Social Fund through the University of Salamanca and the ISCIII (FI19/00320), and Sarasquete by the ISCIII (CPII18/00028). All Spanish funding is cosponsored by the European Union FEDER program.
Miller is the CEO of Invivoscribe, Inc, and Jacobsen, Vigil, Hutt, and Huang are Invivoscribe employees. Flores-Montero and Puig are members of the EuroFlow group and participated in the design, standardization, and validation of the next-generation flow approach. Flores-Montero and Orfao report inventorship of the patent “Methods reagents and kits for detecting minimal residual disease” (PCT/NL2013/050420, filed June 14, 2013) by EuroFlow Consortium. The other authors have no relevant financial interest in the products or companies described in this article.
Miller and García-Sanz contributed equally as senior authors.