Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Myeloma

Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma

Leukemia volume 28, pages 1705–1715 (2014)Cite this article

Subjects

Abstract

Although intratumor heterogeneity has been inferred in multiple myeloma (MM), little is known about its subclonal phylogeny. To describe such phylogenetic trees in a series of patients with MM, we perform whole-exome sequencing and single-cell genetic analysis. Our results demonstrate that at presentation myeloma is composed of two to six different major clones, which are related by linear and branching phylogenies. Remarkably, the earliest myeloma-initiating clones, some of which only had the initiating t(11;14), were still present at low frequencies at the time of diagnosis. For the first time in myeloma, we demonstrate parallel evolution whereby two independent clones activate the RAS/MAPK pathway through RAS mutations and give rise subsequently to distinct subclonal lineages. We also report the co-occurrence of RAS and interferon regulatory factor 4 (IRF4) p.K123R mutations in 4% of myeloma patients. Lastly, we describe the fluctuations of myeloma subclonal architecture in a patient analyzed at presentation and relapse and in NOD/SCID-IL2Rγnull xenografts, revealing clonal extinction and the emergence of new clones that acquire additional mutations. This study confirms that myeloma subclones exhibit different survival properties during treatment or mouse engraftment. We conclude that clonal diversity combined with varying selective pressures is the essential foundation for tumor progression and treatment resistance in myeloma.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Hanahan D, Weinberg RA . Hallmarks of cancer: the next generation. Cell 2011; 144: 646–674.

    Article  CAS  PubMed  Google Scholar 

  2. Nowell PC . The clonal evolution of tumor cell populations. Science 1976; 194: 23–28.

    Article  CAS  PubMed  Google Scholar 

  3. Greaves M, Maley CC . Clonal evolution in cancer. Nature 2012; 481: 306–313.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Anderson K, Lutz C, van Delft FW, Bateman CM, Guo Y, Colman SM et al. Genetic variegation of clonal architecture and propagating cells in leukaemia. Nature 2011; 469: 356–361.

    Article  CAS  PubMed  Google Scholar 

  5. Keats JJ, Chesi M, Egan JB, Garbitt VM, Palmer SE, Braggio E et al. Clonal competition with alternating dominance in multiple myeloma. Blood 2012; 120: 1067–1076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Merlo LM, Pepper JW, Reid BJ, Maley CC . Cancer as an evolutionary and ecological process. Nat Rev Cancer 2006; 6: 924–935.

    Article  CAS  PubMed  Google Scholar 

  7. Sottoriva A, Spiteri I, Piccirillo SG, Touloumis A, Collins VP, Marioni JC et al. Intratumor heterogeneity in human glioblastoma reflects cancer evolutionary dynamics. Proc Natl Acad Sci USA 2013; 110: 4009–4014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366: 883–892.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Nik-Zainal S, Van Loo P, Wedge DC, Alexandrov LB, Greenman CD, Lau KW et al. The life history of 21 breast cancers. Cell 2012; 149: 994–1007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Walker BA, Wardell CP, Melchor L, Hulkki S, Potter NE, Johnson DC et al. Intraclonal heterogeneity and distinct molecular mechanisms characterize the development of t(4;14) and t(11;14) myeloma. Blood 2012; 120: 1077–1086.

    Article  CAS  PubMed  Google Scholar 

  12. Campbell PJ, Yachida S, Mudie LJ, Stephens PJ, Pleasance ED, Stebbings LA et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 2010; 467: 1109–1113.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Greaves M . Cancer stem cells as 'units of selection'. Evol Appl 2013; 6: 102–108.

    Article  PubMed  Google Scholar 

  14. Kuehl WM, Bergsagel PL . Molecular pathogenesis of multiple myeloma and its premalignant precursor. J Clin Invest 2012; 122: 3456–3463.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Morgan GJ, Walker BA, Davies FE . The genetic architecture of multiple myeloma. Nat Rev Cancer 2012; 12: 335–348.

    Article  CAS  PubMed  Google Scholar 

  16. Chapman MA, Lawrence MS, Keats JJ, Cibulskis K, Sougnez C, Schinzel AC et al. Initial genome sequencing and analysis of multiple myeloma. Nature 2011; 471: 467–472.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Egan JB, Shi CX, Tembe W, Christoforides A, Kurdoglu A, Sinari S et al. Whole-genome sequencing of multiple myeloma from diagnosis to plasma cell leukemia reveals genomic initiating events, evolution, and clonal tides. Blood 2012; 120: 1060–1066.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Magrangeas F, Avet-Loiseau H, Gouraud W, Lode L, Decaux O, Godmer P et al. Minor clone provides a reservoir for relapse in multiple myeloma. Leukemia 2013; 27: 473–481.

    Article  CAS  PubMed  Google Scholar 

  19. Durinck S, Ho C, Wang NJ, Liao W, Jakkula LR, Collisson EA et al. Temporal dissection of tumorigenesis in primary cancers. Cancer Discov 2011; 1: 137–143.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Potter NE, Ermini L, Papaemmanuil E, Cazzaniga G, Vijayaraghavan G, Titley I et al. Single-cell mutational profiling and clonal phylogeny in cancer. Genome Res 2013; 23: 2115–2125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Boyd KD, Ross FM, Chiecchio L, Dagrada GP, Konn ZJ, Tapper WJ et al. A novel prognostic model in myeloma based on co-segregating adverse FISH lesions and the ISS: analysis of patients treated in the MRC Myeloma IX trial. Leukemia 2012; 26: 349–355.

    Article  CAS  PubMed  Google Scholar 

  22. Chiecchio L, Protheroe RK, Ibrahim AH, Cheung KL, Rudduck C, Dagrada GP et al. Deletion of chromosome 13 detected by conventional cytogenetics is a critical prognostic factor in myeloma. Leukemia 2006; 20: 1610–1617.

    Article  CAS  PubMed  Google Scholar 

  23. Walker BA, Wardell CP, Johnson DC, Kaiser MF, Begum DB, Dahir NB et al. Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells. Blood 2013; 121: 3413–3419.

    Article  CAS  PubMed  Google Scholar 

  24. Walker BA, Leone PE, Chiecchio L, Dickens NJ, Jenner MW, Boyd KD et al. A compendium of myeloma-associated chromosomal copy number abnormalities and their prognostic value. Blood 2010; 116: e56–e65.

    Article  CAS  PubMed  Google Scholar 

  25. Walker BA, Leone PE, Jenner MW, Li C, Gonzalez D, Johnson DC et al. Integration of global SNP-based mapping and expression arrays reveals key regions, mechanisms, and genes important in the pathogenesis of multiple myeloma. Blood 2006; 108: 1733–1743.

    Article  CAS  PubMed  Google Scholar 

  26. Weaver S, Dube S, Mir A, Qin J, Sun G, Ramakrishnan R et al. Taking qPCR to a higher level: analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution. Methods 2010; 50: 271–276.

    Article  CAS  PubMed  Google Scholar 

  27. Sims D, Bursteinas B, Gao Q, Jain E, MacKay A, Mitsopoulos C et al. ROCK: a breast cancer functional genomics resource. Breast Cancer Res Treat 2010; 124: 567–572.

    Article  CAS  PubMed  Google Scholar 

  28. Fryer RA, Graham TJ, Smith EM, Walker-Samuel S, Morgan GJ, Robinson SP et al. Characterization of a novel mouse model of multiple myeloma and its use in preclinical therapeutic assessment. PLoS One 2013; 8: e57641.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Scrucca L . GA: A package for genetic algorithms in R. J Stat Softw 2013; 53: 1–37.

    Article  Google Scholar 

  30. Fonseca R, Bailey RJ, Ahmann GJ, Rajkumar SV, Hoyer JD, Lust JA et al. Genomic abnormalities in monoclonal gammopathy of undetermined significance. Blood 2002; 100: 1417–1424.

    CAS  PubMed  Google Scholar 

  31. Walker BA, Wardell CP, Melchor L, Brioli A, Johnson DC, Kaiser MF et al. Intraclonal heterogeneity is a critical early event in the development of myeloma and precedes the development of clinical symptoms. Leukemia 2013; e-pub ahead of print 2 July 2013 doi:10.1038/leu.2013.199.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Park SY, Gonen M, Kim HJ, Michor F, Polyak K . Cellular and genetic diversity in the progression of in situ human breast carcinomas to an invasive phenotype. J Clin Invest 2010; 120: 636–644.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Newburger DE, Kashef-Haghighi D, Weng Z, Salari R, Sweeney RT, Brunner AL et al. Genome evolution during progression to breast cancer. Genome Res 2013; 23: 1097–1108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Merlo LM, Shah NA, Li X, Blount PL, Vaughan TL, Reid BJ et al. A comprehensive survey of clonal diversity measures in Barrett's esophagus as biomarkers of progression to esophageal adenocarcinoma. Cancer Prev Res (Phila) 2010; 3: 1388–1397.

    Article  Google Scholar 

  35. Forrester K, Almoguera C, Han K, Grizzle WE, Perucho M . Detection of high incidence of K-ras oncogenes during human colon tumorigenesis. Nature 1987; 327: 298–303.

    Article  CAS  PubMed  Google Scholar 

  36. Farr CJ, Saiki RK, Erlich HA, McCormick F, Marshall CJ . Analysis of RAS gene mutations in acute myeloid leukemia by polymerase chain reaction and oligonucleotide probes. Proc Natl Acad Sci USA 1988; 85: 1629–1633.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Itzykson R, Kosmider O, Renneville A, Morabito M, Preudhomme C, Berthon C et al. Clonal architecture of chronic myelomonocytic leukemias. Blood 2013; 121: 2186–2198.

    Article  CAS  PubMed  Google Scholar 

  38. Andrulis M, Lehners N, Capper D, Penzel R, Heining C, Huellein J et al. Targeting the BRAF V600E mutation in multiple myeloma. Cancer Discov 2013; 3: 862–869.

    Article  CAS  PubMed  Google Scholar 

  39. Lu R . Interferon regulatory factor 4 and 8 in B-cell development. Trends Immunol 2008; 29: 487–492.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Lamy L, Ngo VN, Emre NC, Shaffer AL 3rd, Yang Y, Tian E et al. Control of autophagic cell death by caspase-10 in multiple myeloma. Cancer Cell 2013; 23: 435–449.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Shaffer AL, Emre NC, Lamy L, Ngo VN, Wright G, Xiao W et al. IRF4 addiction in multiple myeloma. Nature 2008; 454: 226–231.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lopez-Girona A, Mendy D, Ito T, Miller K, Gandhi AK, Kang J et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia 2012; 26: 2326–2335.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Zhang LH, Kosek J, Wang M, Heise C, Schafer PH, Chopra R . Lenalidomide efficacy in activated B-cell-like subtype diffuse large B-cell lymphoma is dependent upon IRF4 and cereblon expression. Br J Haematol 2013; 160: 487–502.

    Article  CAS  PubMed  Google Scholar 

  44. Zhu YX, Braggio E, Shi CX, Bruins LA, Schmidt JE, Van Wier S et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood 2011; 118: 4771–4779.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Havelange V, Pekarsky Y, Nakamura T, Palamarchuk A, Alder H, Rassenti L et al. IRF4 mutations in chronic lymphocytic leukemia. Blood 2011; 118: 2827–2829.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Swanton C . Intratumor heterogeneity: evolution through space and time. Cancer Res 2012; 72: 4875–4882.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We acknowledge Professor Mel Greaves for his scientific input and the Tumour Profiling Unit at the Institute of Cancer Research for their support and technical expertise in this study. This study was supported by a Cancer Research UK sample collection grant (CTAAC), grants from the National Institutes of Health Biomedical Research Centre at the Royal Marsden Hospital and a Myeloma UK programme grant (LM, CPW, RAF, DCJ and BAW). MFK is supported by a Research Fellowship by Deutsche Forschungsgemeinschaft (KA 3338/1-1). FED is a Cancer Research UK Senior Clinical Fellow.

Author information

Authors and Affiliations

  1. Division of Molecular Pathology, Centre for Myeloma Research, The Institute of Cancer Research, London, UK

    L Melchor, A Brioli, C P Wardell, A Murison, M F Kaiser, R A Fryer, D C Johnson, D B Begum, S Hulkki Wilson, F E Davies, B A Walker & G J Morgan

  2. Istituto di Ematologia Seràgnoli, Università degli Studi di Bologna, Policlinico S. Orsola-Malpighi, Bologna, Italy

    A Brioli & M Cavo

  3. Division of Molecular Pathology, Molecular Haematology, Haemato-Oncology Research Unit, The Institute of Cancer Research, London, UK

    N E Potter & I Titley

  4. Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK

    G Vijayaraghavan

Authors
  1. L Melchor
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A Brioli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. C P Wardell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A Murison
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. N E Potter
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M F Kaiser
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R A Fryer
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. D C Johnson
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. D B Begum
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. S Hulkki Wilson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. G Vijayaraghavan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. I Titley
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. M Cavo
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. F E Davies
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. B A Walker
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. G J Morgan
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G J Morgan.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Melchor, L., Brioli, A., Wardell, C. et al. Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma. Leukemia 28, 1705–1715 (2014). https://doi.org/10.1038/leu.2014.13

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2014.13

Keywords

This article is cited by

Search

Advanced search

Quick links