Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders

Abstract

In this study, we compared immunoglobulin heavy-chain-gene-based minimal residual disease (MRD) detection by real-time quantitative PCR (RQ-PCR) and next-generation sequencing (NGS) to assess whether NGS could overcome some limitations of RQ-PCR and further increase sensitivity, specificity, accuracy and reproducibility. In total, 378 samples from 55 patients with acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) or multiple myeloma (MM) were investigated for clonotype identification, clonotype identity and comparability of MRD results. Forty-five clonotypes were identified by RQ-PCR and 49 by NGS. Clonotypes identified by both tools were identical or >97% homologous in 96% of cases. Both tools were able to routinely reach a sensitivity level of 1 × E-05. A good correlation of MRD results was observed (R=0.791, P<0.001), with excellent concordance in 79.6% of cases. Few discordant cases were observed across all disease subtypes. NGS showed at least the same level of sensitivity as allele-specific oligonucleotides-PCR, without the need for patient-specific reagents. We conclude that NGS is an effective tool for MRD monitoring in ALL, MCL and MM. Prospective comparative analysis of unselected cases is required to validate the clinical impact of NGS-based MRD assessment.

Minimal residual disease in mantle cell lymphoma: insights into biology and impact on treatment

“…20 In 4 patients, HTS quantified MRD whereas no suitable MRD marker could be developed for RQ-PCR because of the failure of marker sequencing or an unsuitable junctional region for primer design. Of 156 follow-up samples, 82% were concordant between the 2 methods.…”

Critical methodological factors in diagnosing minimal residual disease in hematological malignancies using quantitative PCR

“…Finally, nextgeneration sequencing is seen as a promising tool for other targets in both leukemia and lymphoma and reports on sensitivities show that this technique is highly competitive and in some cases superior to the present qPCR technology [32,33]. However, the next-generation sequencing is not yet standardized enough within the MRD territory, which is required, when patients, as matters stand, are stratified to specific treatment protocols.…”

Stem cell transplantation in multiple myeloma and other plasma cell disorders (report from an EBMT preceptorship meeting)

“…Three approaches are currently available and include allele-specific oligonucleotide PCR (ASO-PCR) and its most recent derivatives digital droplet PCR, MFC, and next-generation sequencing (NGS). [54][55][56][57][58][59] Characteristics of the three methods are compared in Table 2. Potential advantages of the recent introduction of NGS include a higher rate of target identification, non-reliance on patient-specific primers, possibility of performing sub-clone analyses, and possibly improved sensitivity.…”

“…Potential advantages of the recent introduction of NGS include a higher rate of target identification, non-reliance on patient-specific primers, possibility of performing sub-clone analyses, and possibly improved sensitivity. [56,57,60] However, it has not yet been standardized and compared with standard PCR-based MRD methods. Moreover, its clinical validation is still in progress.…”