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Clinical impact of low-burden BCR-ABL1 mutations detectable by amplicon deep sequencing in Philadelphia-positive acute lymphoblastic leukemia patients

Leukemia volume 30, pages 1615–1619 (2016)Cite this article

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Although the prognosis of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has significantly improved upon the advent of tyrosine kinase inhibitors (TKIs) targeting the deregulated BCR-ABL1 tyrosine kinase activity, disease recurrence remains a major problem.1 The long-term stability of responses is frequently undermined by the selection of BCR-ABL1 mutants that can survive TKI treatment and trigger relapse.2 Imatinib-resistant Ph+ ALL patients frequently harbor the T315I mutation,2 against whom second-generation TKIs (2GTKIs; dasatinib and nilotinib) are ineffective. Even in imatinib-resistant patients who have 2GTKI-sensitive mutants, responses may be short-lived because of the high genetic instability fostering the acquisition of additional mutations, often in a ‘compound’ form (that is, two mutations gained by the same clone).2 Recently, however, effective salvage options for T315I mutation-positive or multi-TKI-resistant cases have become available—including the third-generation TKI ponatinib,3 as well as monoclonal antibodies such as blinatumomab.4 To prevent hematologic relapse with timely and rational therapeutic intervention, minimal residual disease (MRD) monitoring by real time quantitative polymerase chain reaction (RQ-PCR) and BCR-ABL1 mutation screening of patients who do not achieve MRD negativity can thus be expected to have an important role.

The availability of sensitive, amplicon deep sequencing (DS)-based strategies for BCR-ABL1 mutation screening prompted us to explore the clinical significance of low-burden mutations in 151 samples from 56 Ph+ ALL patients treated with TKIs. Patients had been referred to our laboratory for MRD monitoring and for BCR-ABL1 mutation screening by conventional sequencing in case of MRD positivity. Patient demographics is summarized in Supplementary Table S1. RQ-PCR and conventional sequencing of the BCR-ABL1 kinase domain were performed as previously reported.5, 6, 7 DS was performed on a GS Junior instrument (Roche Diagnostics, Mannheim, Germany) using a protocol whose set up and performance characteristics have already been described.8 Mutation detection limit of DS was 1%. Details on the DS procedure and on data analysis can be found in the Supplementary Methods. ‘Low-burden’ mutations are herein defined as BCR-ABL1 sequence variants detectable by DS, but not by conventional sequencing (that is, mutations present in 1–20% of transcripts). According to their sensitivity profile (based on integration of in vitro IC50 data and in vivo response rates), we classified low-burden mutations into (i) ‘resistance-driver’ mutations: mutations known to be insensitive or poorly sensitive to the TKI, the patient was receiving at the time of mutation detection; and (ii) mutations of unknown significance: mutations never reported in association with resistance to any TKI.

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References

  1. Ribera JM . Optimal approach to treatment of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia: how to best use all the available tools. Leuk Lymphoma 2013; 54: 21–27.

    Article  Google Scholar 

  2. Soverini S, De Benedittis C, Papayannidis C, Paolini S, Venturi C, Iacobucci I et al. Drug resistance and BCR-ABL kinase domain mutations in Philadelphia chromosome-positive acute lymphoblastic leukemia from the imatinib to the second-generation tyrosine kinase inhibitor era: The main changes are in the type of mutations, but not in the frequency of mutation involvement. Cancer 2014; 120: 1002–1009.

    Article  CAS  Google Scholar 

  3. Cortes JE, Kantarjian H, Shah NP, Bixby D, Mauro MJ, Flinn I et al. Ponatinib in refractory Philadelphia chromosome-positive leukemias. N Engl J Med 2012; 367: 2075–2088.

    Article  CAS  Google Scholar 

  4. Ribera JM, Ferrer A, Ribera J, Genesca E . Profile of blinatumomab and its potential in the treatment of relapsed/refractory acute lymphoblastic leukemia. Onco Targets Ther 2015; 8: 1567–1574.

    Article  CAS  Google Scholar 

  5. Gabert J, Beillard E, van der Velden VH, Bi W, Grimwade D, Pallisgaard N et al. Standardization and quality control studies of 'real-time' quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia - a Europe Against Cancer program. Leukemia 2003; 17: 2318–2357.

    Article  CAS  Google Scholar 

  6. Bruggemann 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.

    Article  CAS  Google Scholar 

  7. Soverini S, Vitale A, Poerio A, Gnani A, Colarossi S, Iacobucci I et al. Philadelphia-positive acute lymphoblastic leukemia patients already harbor BCR-ABL kinase domain mutations at low levels at the time of diagnosis. Haematologica 2011; 96: 552–557.

    Article  CAS  Google Scholar 

  8. Soverini S, De Benedittis C, Machova Polakova K, Brouckova A, Horner D, Iacono M et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood 2013; 122: 1634–1648.

    Article  CAS  Google Scholar 

  9. Pfeifer H, Lange T, Wystub S, Wassmann B, Maier J, Binckebanck A et al. Prevalence and dynamics of bcr-abl kinase domain mutations during imatinib treatment differ in patients with newly diagnosed and recurrent bcr-abl positive acute lymphoblastic leukemia. Leukemia 2012; 26: 1475–1481.

    Article  CAS  Google Scholar 

  10. Liu J, Joha S, Idziorek T, Corm S, Hetuin D, Philippe N et al. BCR-ABL mutants spread resistance to non-mutated cells through a paracrine mechanism. Leukemia 2008; 22: 791–799.

    Article  CAS  Google Scholar 

  11. Jiang X, Forrest D, Nicolini F, Turhan A, Guilhot J, Yip C et al. Properties of CD34+ CML stem/progenitor cells that correlate with different clinical responses to imatinib mesylate. Blood 2010; 116: 2112–2121.

    Article  CAS  Google Scholar 

  12. Grant H, Jiang X, Stebbing J, Foroni L, Craddock C, Griffiths M et al. Analysis of BCR-ABL1 tyrosine kinase domain mutational spectra in primitive chronic myeloid leukemia cells suggests a unique mutator phenotype. Leukemia 2010; 24: 1817–1821.

    Article  CAS  Google Scholar 

  13. Chu S, Xu H, Shah NP, Snyder DS, Forman SJ, Sawyers CL et al. Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment. Blood 2005; 105: 2093–2098.

    Article  CAS  Google Scholar 

  14. Murphy DM, Bejar R, Stevenson K, Neuberg D, Shi Y, Cubrich C et al. NRAS mutations with low allele burden have independent prognostic significance for patients with lower risk myelodysplastic syndromes. Leukemia 2013; 27: 2077–2081.

    Article  CAS  Google Scholar 

  15. Malcikova J, Stano-Kozubik K, Tichy B, Kantorova B, Pavlova S, Tom N et al. Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia. Leukemia 2015; 29: 877–885.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by European Leukemianet, AIRC (Associazione Italiana per la Rierca sul Cancro), AIL (Associazione Italiana contro le Leucemie), Progetto Regione-Università 2010–2012 (L Bolondi), FP7 Next Generation Sequencing for Personalized Therapy of Leukemia (NGS-PTL) project and project for conceptual development of research organization (00023736) from the Ministry of Health of Czech Republic. We would like to thank the Interlaboratory RObustness of Next-generation sequencing (IRON) Phase II study group members for helpful discussions on the deep sequencing assay.

Author contributions

SS designed the research, performed experiments, analyzed and interpreted results, and wrote the paper; CDB performed the experiments, and analyzed and interpreted results; KMP, CV and MM performed experiments; CP, DR, PB, AI, AV, SC, RF, EA and FS provided patient samples and clinical data; AK and TH provided vital technical and bioinformatics support for the development of the DS assay; MC, MB and GM coordinated the clinical and research team activities and supervised the study. All authors gave final approval for submission.

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Authors and Affiliations

  1. Department of Experimental, Diagnostic and Specialty Medicine, Hematology/Oncology ‘L. e A. Seràgnoli’, University of Bologna, Bologna, Italy

    S Soverini, C De Benedittis, C Papayannidis, C Venturi, M Mancini, M Baccarani, M Cavo & G Martinelli

  2. Institute of Hematology and Blood Transfusion, Prague, Czech Republic

    K Machova Polakova

  3. Chair of Hematology, Unit of Blood Diseases and Stem Cell Transplantation, University of Brescia, Brescia, Italy

    D Russo

  4. Department of Oncology and Hematology, Bone Marrow Transplant Unit, University of Modena, Modena, Italy

    P Bresciani

  5. Onco-Hematology Division, IRCCS Ca’ Granda-Maggiore Policlinico Hospital Foundation, Milan, Italy

    A Iurlo

  6. Department of Cellular Biotechnologies and Hematology, Hematology, La Sapienza University, Rome, Italy

    A Vitale, S Chiaretti & R Foà

  7. Hematology, S. Eugenio Hospital, Tor Vergata University, Rome, Italy

    E Abruzzese

  8. Hematology, Università Cattolica del Sacro Cuore, Rome, Italy

    F Sorà

  9. Personalised Healthcare and Biomarkers, AstraZeneca, Cambridge, UK

    A Kohlmann

  10. MLL Munich Leukemia Laboratory, Munich, Germany

    T Haferlach

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  1. S Soverini
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Corresponding author

Correspondence to S Soverini.

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Competing interests

SS served as a consultant for Novartis, Bristol-Myers Squibb and Ariad; KMP received research funding and honoraria from Novartis and Bristol-Myers Squibb; AK was a former employee of the Munich Leukemia Laboratory (MLL), is now an employee of Astra Zeneca, UK, and has received honoraria from Roche diagnostics; TH is part owner of the MLL; MB and GM served as consultants for and received honoraria from Novartis, Bristol-Myers Squibb, Ariad and Pfizer. The remaining authors declare no conflict of interest.

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Soverini, S., De Benedittis, C., Papayannidis, C. et al. Clinical impact of low-burden BCR-ABL1 mutations detectable by amplicon deep sequencing in Philadelphia-positive acute lymphoblastic leukemia patients. Leukemia 30, 1615–1619 (2016). https://doi.org/10.1038/leu.2016.17

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