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Minimal Residual Disease

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.

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Acknowledgements

The authors would like to thank Ariane Stuhr for excellent technical assistance and the FIL secretariat, Antonella Fiorillo and Franca Trotto Gatta for excellent secretarial support. Funding: This work was supported by Progetto di Rilevante Interesse Nazionale (PRIN 2009) from Ministero Italiano dell'Università e della Ricerca (MIUR), Roma, Italy (code: 7.07.02.60 AE01); Progetti di Ricerca Finalizzata 2008 (head unit: IRCCS Centro di Riferimento Oncologico della Basilicata (CROB), Rionero in Vulture (Potenza), Italy) (code: 7.07.08.60 P49), Progetto di Ricerca Sanitaria Finalizzata 2008, (head unit: Divisione di Ematologia, A. O. S. Maurizio, Bolzano/Bozen, Italy) (code: 7.07.08.60 P51), Progetto di Ricerca Sanitaria Finalizzata 2009 (head unit: Divisione di Ematologia, A. O. S. Maurizio, Bolzano/Bozen, Italy) (code: RF-2009-1469205), Progetto di Ricerca Sanitaria Finalizzata 2010 (head unit: Divisione di Ematologia, A. O. S. Maurizio, Bolzano/Bozen, Italy) (code: RF-2010-2307262), Fondi di Ricerca Locale, Università degli Studi di Torino, Torino, Italy, and by Fondazione Neoplasie del sangue (FO. NE. SA), Torino, Italy.

Author Contributions

ML, MB, LM, DB, MF and CP designed the research, analyzed and interpreted data, and wrote the manuscript. ML, MB, LM, SF, DD, DB, HT, CP, MR and NG were responsible for samples, RQ-PCR MRD analysis, data collection and interpretation. FP, JZ, VC and MF performed the NGS assays and analyzed and interpreted data. MB and AP provided critical organizational support. RP conducted the statistical analysis. All authors approved the final version of the manuscript. This work has been presented as oral presentation at the American Society of Hematology annual meeting 7–12 December 2012 and as poster presentation at the European Hematology Association annual meeting 13–16 June 2013.

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Correspondence to M Ladetto.

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FP, JZ, VC, MF are employees and stock holders at Sequenta Inc. Other authors declare no conflict of interest.

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Ladetto, M., Brüggemann, M., Monitillo, L. et al. Next-generation sequencing and real-time quantitative PCR for minimal residual disease detection in B-cell disorders. Leukemia 28, 1299–1307 (2014). https://doi.org/10.1038/leu.2013.375

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