Abstract
In our individualized systems medicine program, personalized treatment options are identified and administered to chemorefractory acute myeloid leukemia (AML) patients based on exome sequencing and ex vivo drug sensitivity and resistance testing data. Here, we analyzed how clonal heterogeneity affects the responses of 13 AML patients to chemotherapy or targeted treatments using ultra-deep (average 68 000 × coverage) amplicon resequencing. Using amplicon resequencing, we identified 16 variants from 4 patients (frequency 0.54–2%) that were not detected previously by exome sequencing. A correlation-based method was developed to detect mutation-specific responses in serial samples across multiple time points. Significant subclone-specific responses were observed for both chemotherapy and targeted therapy. We detected subclonal responses in patients where clinical European LeukemiaNet (ELN) criteria showed no response. Subclonal responses also helped to identify putative mechanisms underlying drug sensitivities, such as sensitivity to azacitidine in DNMT3A mutated cell clones and resistance to cytarabine in a subclone with loss of NF1 gene. In summary, ultra-deep amplicon resequencing method enables sensitive quantification of subclonal variants and their responses to therapies. This approach provides new opportunities for designing combinatorial therapies blocking multiple subclones as well as for real-time assessment of such treatments.
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
Ding L, Ley TJ, Larson DE, Miller CA, Koboldt DC, Welch JS et al. Clonal evolution in relapsed acute myeloid leukaemia revealed by whole-genome sequencing. Nature 2012; 481: 506–510.
Estey E, Dohner H . Acute myeloid leukaemia. Lancet 2006; 368: 1894–1907.
Nowell PC . The clonal evolution of tumor cell populations. Science 1976; 194: 23–28.
Parkin B, Ouillette P, Li Y, Keller J, Lam C, Roulston D et al. Clonal evolution and devolution after chemotherapy in adult acute myelogenous leukemia. Blood 2013; 121: 369–377.
Burnett A, Wetzler M, Lowenberg B . Therapeutic advances in acute myeloid leukemia. J Clin Oncol 2011; 29: 487–494.
Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood 2010; 115: 453–474.
Döhner H, Weisdorf DJ, Bloomfield CD . Acute myeloid leukemia. N Engl J Med 2015; 373: 1136–1152.
Grimwade D, Ivey A, Huntly BJ . Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood 2016; 127: 29–41.
Nakao M, Yokota S, Iwai T, Kaneko H, Horiike S, Kashima K et al. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia. Leukemia 1996; 10: 1911–1918.
Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1,612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children's Leukaemia Working Parties. Blood 1998; 92: 2322–2333.
Bacher U, Schnittger S, Haferlach T . Molecular genetics in acute myeloid leukemia. Curr Opin Oncol 2010; 22: 646–655.
Betz BL, Hess JL . Acute myeloid leukemia diagnosis in the 21st century. Arch Pathol Lab Med 2010; 134: 1427–1433.
Cancer Genome Atlas Research Network. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med 2013; 368: 2059–2074.
Ley TJ, Mardis ER, Ding L, Fulton B, McLellan MD, Chen K et al. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature 2008; 456: 66–72.
Mardis ER, Ding L, Dooling DJ, Larson DE, McLellan MD, Chen K et al. Recurring mutations found by sequencing an acute myeloid leukemia genome. N Engl J Med 2009; 361: 1058–1066.
Papaemmanuil E, Gerstung M, Bullinger L, Gaidzik VI, Paschka P, Roberts ND et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med 2016; 374: 2209–2221.
Chan SM, Majeti R . Role of DNMT3A, TET2, and IDH1/2 mutations in pre-leukemic stem cells in acute myeloid leukemia. Int J Hematol 2013; 98: 648–657.
Steensma DP, Bejar R, Jaiswal S, Lindsley RC, Sekeres MA, Hasserjian RP et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood 2015; 126: 9–16.
Xie M, Lu C, Wang J, McLellan MD, Johnson KJ, Wendl MC et al. Age-related mutations associated with clonal hematopoietic expansion and malignancies. Nat Med 2014; 20: 1472–1478.
Corces-Zimmerman MR, Majeti R . Pre-leukemic evolution of hematopoietic stem cells: the importance of early mutations in leukemogenesis. Leukemia 2014; 28: 2276–2282.
Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature 2014; 506: 328–333.
Walter MJ, Shen D, Ding L, Shao J, Koboldt DC, Chen K et al. Clonal architecture of secondary acute myeloid leukemia. N Engl J Med 2012; 366: 1090–1098.
Kronke J, Bullinger L, Teleanu V, Tschurtz F, Gaidzik VI, Kuhn MW et al. Clonal evolution in relapsed NPM1-mutated acute myeloid leukemia. Blood 2013; 122: 100–108.
Landau DA, Carter SL, Getz G, Wu CJ . Clonal evolution in hematological malignancies and therapeutic implications. Leukemia 2014; 28: 34–43.
Landau DA, Carter SL, Stojanov P, McKenna A, Stevenson K, Lawrence MS et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell 2013; 152: 714–726.
Klco JM, Spencer DH, Miller CA, Griffith M, Lamprecht TL, O'Laughlin M et al. Functional heterogeneity of genetically defined subclones in acute myeloid leukemia. Cancer Cell 2014; 25: 379–392.
Pemovska T, Kontro M, Yadav B, Edgren H, Eldfors S, Szwajda A et al. Individualized systems medicine strategy to tailor treatments for patients with chemorefractory acute myeloid leukemia. Cancer Discov 2013; 3: 1416–1429.
Koskela HL, Eldfors S, Ellonen P, van Adrichem AJ, Kuusanmaki H, Andersson EI et al. Somatic STAT3 mutations in large granular lymphocytic leukemia. N Engl J Med 2012; 366: 1905–1913.
Koboldt DC, Zhang Q, Larson DE, Shen D, McLellan MD, Lin L et al. VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 2012; 22: 568–576.
Sulonen AM, Ellonen P, Almusa H, Lepisto M, Eldfors S, Hannula S et al. Comparison of solution-based exome capture methods for next generation sequencing. Genome Biol 2011; 12: R94.
Rubio-Perez C, Tamborero D, Schroeder MP, Antolin AA, Deu-Pons J, Perez-Llamas C et al. In silico prescription of anticancer drugs to cohorts of 28 tumor types reveals targeting opportunities. Cancer Cell 2015; 27: 382–396.
Thiede C, Steudel C, Mohr B, Schaich M, Schakel U, Platzbecker U et al. Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 2002; 99: 4326–4335.
Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, Loeb LA . Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci USA 2012; 109: 14508–14513.
Kinde I, Wu J, Papadopoulos N, Kinzler KW, Vogelstein B . Detection and quantification of rare mutations with massively parallel sequencing. Proc Natl Acad Sci USA 2011; 108: 9530–9535.
Campbell PJ, Pleasance ED, Stephens PJ, Dicks E, Rance R, Goodhead I et al. Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing. Proc Natl Acad Sci USA 2008; 105: 13081–13086.
Faivre S, Demetri G, Sargent W, Raymond E . Molecular basis for sunitinib efficacy and future clinical development. Nat Rev Drug Discov 2007; 6: 734–745.
Fiedler W, Serve H, Dohner H, Schwittay M, Ottmann OG, O'Farrell AM et al. A phase 1 study of SU11248 in the treatment of patients with refractory or resistant acute myeloid leukemia (AML) or not amenable to conventional therapy for the disease. Blood 2005; 105: 986–993.
Yee KW, Schittenhelm M, O'Farrell AM, Town AR, McGreevey L, Bainbridge T et al. Synergistic effect of SU11248 with cytarabine or daunorubicin on FLT3 ITD-positive leukemic cells. Blood 2004; 104: 4202–4209.
Yin B, Morgan K, Hasz DE, Mao Z, Largaespada DA . Nfl gene inactivation in acute myeloid leukemia cells confers cytarabine resistance through MAPK and mTOR pathways. Leukemia 2006; 20: 151–154.
Lauchle JO, Kim D, Le DT, Akagi K, Crone M, Krisman K et al. Response and resistance to MEK inhibition in leukaemias initiated by hyperactive Ras. Nature 2009; 461: 411–414.
Brundage ME, Tandon P, Eaves DW, Williams JP, Miller SJ, Hennigan RH et al. MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1. Oncogene 2014; 33: 5626–5636.
Cai J, Damaraju VL, Groulx N, Mowles D, Peng Y, Robins MJ et al. Two distinct molecular mechanisms underlying cytarabine resistance in human leukemic cells. Cancer Res 2008; 68: 2349–2357.
Galmarini CM, Thomas X, Calvo F, Rousselot P, Rabilloud M, El Jaffari A et al. In vivo mechanisms of resistance to cytarabine in acute myeloid leukaemia. Br J Haematol 2002; 117: 860–868.
Metzeler KH, Walker A, Geyer S, Garzon R, Klisovic RB, Bloomfield CD et al. DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia. Leukemia 2012; 26: 1106–1107.
Traina F, Visconte V, Elson P, Tabarroki A, Jankowska AM, Hasrouni E et al. Impact of molecular mutations on treatment response to DNMT inhibitors in myelodysplasia and related neoplasms. Leukemia 2014; 28: 78–87.
Whittaker SR, Theurillat JP, Van Allen E, Wagle N, Hsiao J, Cowley GS et al. A genome-scale RNA interference screen implicates NF1 loss in resistance to RAF inhibition. Cancer Discov 2013; 3: 350–362.
Acknowledgements
We are grateful to all the patients for donating their samples for research. Kirsi Autio (HUSLAB, Helsinki, Finland) and Tuija Lundan (TYKSLAB, Turku, Finland) are thanked for providing sample materials from clinical specimens. Alun Parsons and Minna Suvela are acknowledged for their efforts to process the samples and staff of the FIMM Sequencing Lab (Maija Lepistö, Tiina Hannunen Aino Palva and Sari Hannula) are thanked for exome sequencing. Jani Saarela, Laura Turunen, Katja Suomi and staff of FIMM High Throughput Biomedicine Unit are thanked for drug sensitivity and resistance testing (DSRT) experiments and analysis. Disha Malani is thanked for her help in retrieving DSRT data. The research leading to these results was supported by Academy of Finland (Center of Excellence for Translational Cancer Biology), Cancer Society of Finland, Sigrid Juselius Foundation, EU-Systems Microscopy (FP7) and TEKES. PNO is supported by the Doctoral Program in Biomedicine (DPBM) from the University of Helsinki. MK is supported by Doctoral Programme in Clinical Research, University of Helsinki. DT is supported by the People Programme (Marie Curie Actions) and Agency of Competitiveness for Companies of the Government of Catalonia, ACCIO.
Author contributions
PNO, HE, MW and OK designed the study and provided critical inputs; MK and KP coordinated clinical sample collection and administered therapies, obtained ethical permits and provided key biological and clinical insights; PNO, HE and SE performed data analysis and interpretation of sequencing data; PE, SL, TM, PNO and HA performed amplicon resequencing experiments, analysis and data interpretation; DT performed driver gene analysis and cancer cell fraction estimation; CH collected samples, coordinated sample management, preparation, processing and analysis; PNO, MW and OK wrote the paper; KW, HE, DT MK, KP and CH provided critical insights into the paper.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Competing interests
The authors declare no conflict of interest. The senior authors have received collaborative research grants for other projects as listed. OK has received research funding from Pfizer, Roche and the IMI Predect consortium and is a board member and co-founder of bioinformatics company Medisapiens Ltd, Helsinki, Finland. KP received honoraria and research funding from Bristol-Myers Squibb, Celgene, Novartis and Pfizer. MW received research funding from Pfizer and Bayer Pharma.
Additional information
Supplementary Information accompanies this paper on the Leukemia website
Rights and permissions
About this article
Cite this article
Ojamies, P., Kontro, M., Edgren, H. et al. Monitoring therapy responses at the leukemic subclone level by ultra-deep amplicon resequencing in acute myeloid leukemia. Leukemia 31, 1048–1058 (2017). https://doi.org/10.1038/leu.2016.286
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/leu.2016.286
This article is cited by
-
The Contribution of Evolutionary Game Theory to Understanding and Treating Cancer
Dynamic Games and Applications (2022)
-
Leukemia’s Clonal Evolution in Development, Progression, and Relapse
Current Stem Cell Reports (2019)
-
Multicenter validation of cancer gene panel-based next-generation sequencing for translational research and molecular diagnostics
Virchows Archiv (2018)
-
The evolutionary landscape of chronic lymphocytic leukemia treated with ibrutinib targeted therapy
Nature Communications (2017)