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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Pott C . Minimal residual disease detection in mantle cell lymphoma: technical aspects and clinical relevance. Semin Hematol 2011; 48: 172–184.
Ferrero S, Drandi D, Mantoan B, Ghione P, Omedè P, Ladetto M . Minimal residual disease detection in lymphoma and multiple myeloma: impact on therapeutic paradigms. Hematol Oncol 2011; 29: 167–176.
Brüggemann M, Gökbuget N, Kneba M . Acute lymphoblastic leukemia: monitoring minimal residual disease as a therapeutic principle. Semin Oncol 2012; 39: 47–57.
Brüggemann M, Raff T, l Kneba M . Has MRD monitoring superseded other prognostic factors in adult ALL? Blood 2012; 120: 4470–4481.
Campana D . Minimal residual disease monitoring in childhood acute lymphoblastic leukemia. Curr Opin Hematol 2012; 19: 313–318.
Conter V, Bartram CR, Valsecchi MG, Schrauder A, Panzer-Grümayer R, Möricke A et al. Molecular response to treatment redefines all prognostic factors in children and adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of the AIEOP-BFM ALL 2000 study. Blood 2010; 115: 3206–3214.
Stow P, Key L, Chen X, Pan Q, Neale GA, Coustan-Smith E et al. Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia. Blood 2010; 115: 4657–4663.
Brüggemann M, Raff T, Flohr T, Gökbuget N, Nakao M, Droese J et al. Clinical significance of minimal residual disease quantification in adult patients with standard-risk acute lymphoblastic leukemia. Blood 2006; 107: 1116–1123.
Raff T, Kaiser M, Gökbuget N, Lüschen S, Ritgen M, Trautmann H et al. Molecular relapse in adult standard-risk ALL patients detected by prospective MRD monitoring during and after maintenance treatment: data from the GMALL 06/99 and 07/03 trials. Blood 2007; 109: 910–915.
Bassan R, Spinelli O, Oldani E, Intermesoli T, Tosi M, Peruta B et al. Improved risk classification for risk-specific therapy based on the molecular study of minimal residual disease (MRD) in adult acute lymphoblastic leukemia (ALL). Blood 2009; 113: 4153–4162.
Campana D . Role of minimal residual disease monitoring in adult and pediatric acute lymphoblastic leukemia. Hematol Oncol Clin North Am 2009; 23: 1083–1098.
Gökbuget N, Kneba M, Raff T, Trautmann H, Bartram CR, Arnold R et al. Adult patients with acute lymphoblastic leukemiaand molecular failure display a poor prognosis and are candidates for stem cell transplantation and targeted therapies. Blood 2012; 120: 1868–1876.
Flohr T, Schrauder A, Cazzaniga G, Panzer-Grümayer R, van der Velden V, Fischer S et al. Minimal residual disease-directed risk stratification using real-time quantitative PCR analysis of immunoglobulin and T-cell receptor gene rearrangements in the international multicenter trial AIEOP-BFM ALL 2000 for childhood acute lymphoblastic leukemia. Leukemia 2008; 22: 771–782.
Pott C, Hoster E, Delfau-Larue MH, Beldjord K, Böttcher S, Asnafi V et al. Molecular remission is an independent predictor of clinical outcome in patients with mantle cell lymphoma after combined immunochemotherapy: a European MCL intergroup study. Blood 2010; 115: 3215–3223.
Ladetto M, De Marco F, Benedetti F, Vitolo U, Patti C, Rambaldi A et al. Prospective, multicenter randomized GITMO/IIL trial comparing in high-risk follicular lymphoma at diagnosis: the superior disease control of R-HDS does not translate into an overall survival advantage. Blood 2008; 111: 4004–4013.
Ladetto M, Pagliano G, Ferrero S, Cavallo F, Drandi D, Santo L et al. Major tumor shrinking and persistent molecular remissions after consolidation with bortezomib, thalidomide, and dexamethasone in patients with autografted myeloma. J Clin Oncol 2010; 28: 2077–2084.
Brown JR, Feng Y, Gribben JG, Neuberg D, Fisher DC, Mauch P et al. Long-term survival after autologous bone marrow transplantation for follicular lymphoma in first remission. Biol Blood Marrow Transplant 2007; 13: 1057–1065.
Paiva B, Gutiérrez NC, Rosiñol L, Vídriales MB, Montalbán MÁ, Martínez-López J et al. High-risk cytogenetics and persistent minimal residual disease by multiparameter flow cytometry predict unsustained complete response after autologous stem cell transplantation in multiple myeloma. Blood 2012; 119: 687–691.
Böttcher S, Ritgen M, Fischer K, Stilgenbauer S, Busch RM, Fingerle-Rowson G et al. Minimal residual disease quantification is an independent predictor of progression-free and overall survival in chronic lymphocytic leukemia: a multivariate analysis from the randomized GCLLSG CLL8 trial. J Clin Oncol 2012; 30: 980–988.
Pott C, Brüggemann M, Ritgen M, van der Velden VH, van Dongen JJ, Kneba M . MRD detection in B-cell non-Hodgkin lymphomas using Ig gene rearrangements and chromosomal translocations as targets for real-time quantitative. Methods Mol Biol Vol. 971. Humana press: Hertfordshire, UK, 2013; pp 175–200.
van der Velden VH, Cazzaniga G, Schrauder A, Hancock J, Bader P, Panzer-Grumayer ER et al. Analysis of minimal residual disease by Ig/TCR gene rearrangements: guidelines for interpretation of real-time quantitative PCR data. Leukemia 2007; 21: 604–611.
Szczepański T, Willemse MJ, van Wering ER, van Weerden JF, Kamps WA, van Dongen JJ . Precursor-B-ALL with D(H)-J(H) gene rearrangements have an immature immunogenotype with a high frequency of oligoclonality and hyperdiploidy of chromosome 14. Leukemia 2001; 15: 1415–1423.
Gawad C, Pepin F, Carlton VE, Klinger M, Logan AC, Miklos DB et al. Massive evolution of the immunoglobulin heavy chain locus in children with B precursor acute lymphoblastic leukemia. Blood 2012; 120: 4407–4417.
Beishuizen A, Verhoeven MA, van Wering ER, Hahlen K, Hooijkaas H, van Dongen JJ . Analysis of Ig and T-cell receptor genes in 40 childhood acute lymphoblastic leukemias at diagnosis and subsequent relapse: implications for the detection of minimal residual disease by polymerase chain reaction analysis. Blood 1994; 83: 2238–2247.
Szczepanski T, Willemse MJ, Brinkhof B, van Wering ER, van der Burg M, van Dongen JJM . Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at relapse of childhood precursor-B -ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease. Blood 2002; 99: 2315–2323.
Logan AC, Zhang B, Narasimhan B, Carlton V, Zheng J, Moorhead M et al. Minimal residual disease quantification using consensus primers and high-throughput IGH sequencing predicts post-transplant relapse in chronic lymphocytic leukemia. Leukemia 2013; 27: 1659–1665.
Faham M, Zheng J, Moorhead M, Carlton VE, Stow P, Coustan-Smith E et al. Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood 2012; 120: 5173–5180.
van Dongen JJ, Langerak AW, Brüggemann M, Evans PA, Hummel M, Lavender FL et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 2003; 17: 2257–2317.
Ladetto M, Donovan JW, Harig S, Trojan A, Poor C, Schlossnan R et al. Real-time polymerase chain reaction of immunoglobulin rearrangements for quantitative evaluation of minimal residual disease in multiple myeloma. Biol Blood Marrow Transplant 2000; 6: 241–253.
Brüggemann M, Droese J, Bolz I, Lüth P, Pott C, von Neuhoff N et al. Improved assessment of minimal residual disease in B cell malignancies using fluorogenic consensus probes for real-time quantitative PCR. Leukemia 2000; 14: 1419–1425.
Böttcher S, Ritgen M, Buske S, Gesk S, Klapper W, Hoster E et al. Minimal residual disease detection in mantle cell lymphoma: methods and significance of four-color flow cytometry compared to consensus IGH-polymerase chain reaction at initial staging and for follow-up examinations. Haematologica 2008; 93: 551–559.
Pott C, Tiemann M, Linke B, Ott MM, von Hofen M, Bolz I et al. Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas. Leukemia 1998; 12: 1630–1637.
Sarasquete ME, García-Sanz R, González D, Martínez J, Mateo G, Martínez P et al. Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry. Haematologica 2005; 90: 1365–1372.
Brüggemann M, Schrauder A, Raff T, Pfeifer H, Dworzak M, Ottmann OG et al. Standardized MRD quantification in European ALL trials: proceedings of the Second International Symposium on MRD assessment in Kiel, Germany, 18–20 September 2008. Leukemia 2010; 24: 521–535.
Andersen NS, Pedersen LB, Laurell A, Elonen E, Kolstad A, Boesen AM et al. Pre-emptive treatment with rituximab of molecular relapse after autologous stem cell transplantation in mantle cell lymphoma. J Clin Oncol 2009; 27: 4365–4370.
Geisler CH, Kolstad A, Laurell A, Andersen NS, Pedersen LB, Jerkeman M et al. Long-term progression-free survival of mantle cell lymphoma after intensive front-line immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma Group. Blood 2008; 112: 2687–2693.
Ferrero S, Capello D, Svaldi M, Boi M, Gatti D, Drandi D et al. Multiple myeloma shows no intra-disease clustering of immunoglobulin heavy chain genes. Haematologica 2012; 97: 849–853.
Hadzidimitriou A, Agathangelidis A, Darzentas N, Murray F, Delfau-Larue MH, Pedersen LB et al. Is there a role for antigen selection in mantle cell lymphoma? Immunogenetic support from a series of 807 cases. Blood 2011; 118: 3088–3095.
Hadzidimitriou A, Stamatopoulos K, Belessi C, Lalayianni C, Stavroyianni N, Smilevska T et al. Immunoglobulin genes in multiple myeloma: expressedand non-expressed repertoires, heavy and light chainpairings and somatic mutation patterns in a series of 101 cases. Haematologica 2006; 91: 781–787.
Corradini P, Ladetto M, Zallio F, Astolfi M, Rizzo E, Sametti S et al. Long-term follow-up of indolent lymphoma patients treated with high-dose sequential chemotherapy and autografting: evidence that durable molecular and clinical remission frequently can be attained only in follicular subtypes. J Clin Oncol 2004; 22: 1460–1468.
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.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
FP, JZ, VC, MF are employees and stock holders at Sequenta Inc. Other authors declare no conflict of interest.
Additional information
Supplementary Information accompanies this paper on the Leukemia website
Supplementary information
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2013.375
Keywords
This article is cited by
-
Have we been qualifying measurable residual disease correctly?
Leukemia (2023)
-
Utility of Measurable Residual Disease (MRD) Assessment in Mantle Cell Lymphoma
Current Treatment Options in Oncology (2023)
-
Measurable Residual Disease Monitoring in Lymphoma
Current Hematologic Malignancy Reports (2023)
-
Clinical application of next-generation sequencing-based monitoring of minimal residual disease in childhood acute lymphoblastic leukemia
Journal of Cancer Research and Clinical Oncology (2023)
-
Minimal residual disease detection by mutation-specific droplet digital PCR for leukemia/lymphoma
International Journal of Hematology (2023)