Relapsed childhood acute lymphoblastic leukemia (ALL) carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance1,2. The biological pathways that mediate resistance are unknown. Here, we report the transcriptome profiles of matched diagnosis and relapse bone marrow specimens from ten individuals with pediatric B-lymphoblastic leukemia using RNA sequencing. Transcriptome sequencing identified 20 newly acquired, novel nonsynonymous mutations not present at initial diagnosis, with 2 individuals harboring relapse-specific mutations in the same gene, NT5C2, encoding a 5′-nucleotidase. Full-exon sequencing of NT5C2 was completed in 61 further relapse specimens, identifying additional mutations in 5 cases. Enzymatic analysis of mutant proteins showed that base substitutions conferred increased enzymatic activity and resistance to treatment with nucleoside analog therapies. Clinically, all individuals who harbored NT5C2 mutations relapsed early, within 36 months of initial diagnosis (P = 0.03). These results suggest that mutations in NT5C2 are associated with the outgrowth of drug-resistant clones in ALL.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Sequence Read Archive

Referenced accessions


  1. 1.

    et al. Reinduction platform for children with first marrow relapse in acute lymphoblastic lymphoma. J. Clin. Oncol. 26, 3971–3978 (2008).

  2. 2.

    et al. In vitro cellular drug resistance in children with relapsed/refractory acute lymphoblastic leukemia. Blood 86, 3861–3868 (1995).

  3. 3.

    , , , & Cancer incidence among children and adolescents in the United States, 2001–2003. Pediatrics 121, e1470–e1477 (2008).

  4. 4.

    et al. Improved survival for children and adolescents with acute lymphoblastic leukemia between 1990 and 2005: a report from the children's oncology group. J. Clin. Oncol. 30, 1663–1669 (2012).

  5. 5.

    & Treatment of acute lymphoblastic leukemia. N. Engl. J. Med. 354, 166–178 (2006).

  6. 6.

    et al. Long-term follow-up of relapsed childhood acute lymphoblastic leukaemia. Br. J. Haematol. 123, 396–405 (2003).

  7. 7.

    et al. Outcomes after HLA-matched sibling transplantation or chemotherapy in children with B-precursor acute lymphoblastic leukemia in a second remission: a collaborative study of the Children's Oncology Group and the Center for International Blood and Marrow Transplant Research. Blood 107, 4961–4967 (2006).

  8. 8.

    et al. Bone marrow transplantation versus prolonged intensive chemotherapy for children with acute lymphoblastic leukemia and an initial bone marrow relapse within 12 months of the completion of primary therapy: Children's Oncology Group study CCG-1941. J. Clin. Oncol. 24, 3150–3156 (2006).

  9. 9.

    et al. Integrated genomic analysis of relapsed childhood acute lymphoblastic leukemia reveals therapeutic strategies. Blood 118, 5218–5226 (2011).

  10. 10.

    et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

  11. 11.

    et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

  12. 12.

    et al. Genome remodelling in a basal-like breast cancer metastasis and xenograft. Nature 464, 999–1005 (2010).

  13. 13.

    et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011).

  14. 14.

    et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 39, D945–D950 (2011).

  15. 15.

    et al. Exome sequencing identifies GRIN2A as frequently mutated in melanoma. Nat. Genet. 43, 442–446 (2011).

  16. 16.

    Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 474, 609–615 (2011).

  17. 17.

    et al. Patterns of somatic mutation in human cancer genomes. Nature 446, 153–158 (2007).

  18. 18.

    et al. CREBBP mutations in relapsed acute lymphoblastic leukaemia. Nature 471, 235–239 (2011).

  19. 19.

    et al. Mutational evolution in a lobular breast tumour profiled at single nucleotide resolution. Nature 461, 809–813 (2009).

  20. 20.

    & Mammalian 5′-nucleotidases. J. Biol. Chem. 278, 46195–46198 (2003).

  21. 21.

    et al. Nucleoside phosphotransferase activity of human colon carcinoma cytosolic 5′-nucleotidase. Arch. Biochem. Biophys. 291, 212–217 (1991).

  22. 22.

    et al. Cytosolic 5′-nucleotidase/phosphotransferase of human colon carcinoma. Adv. Exp. Med. Biol. 309B, 173–176 (1991).

  23. 23.

    et al. Crystal structure of human cytosolic 5′-nucleotidase II: insights into allosteric regulation and substrate recognition. J. Biol. Chem. 282, 17828–17836 (2007).

  24. 24.

    , , & ATP and phosphate reciprocally affect subunit association of human recombinant high Km 5′-nucleotidase. Role for the C-terminal polyglutamic acid tract in subunit association and catalytic activity. Eur. J. Biochem. 259, 851–858 (1999).

  25. 25.

    et al. Relation of 5′-nucleotidase and phosphatase activities with immunophenotype, drug resistance and clinical prognosis in childhood leukemia. Leuk. Res. 16, 873–880 (1992).

  26. 26.

    et al. Expression of high Km 5′-nucleotidase in leukemic blasts is an independent prognostic factor in adults with acute myeloid leukemia. Blood 98, 1922–1926 (2001).

  27. 27.

    et al. Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine. Br. J. Haematol. 122, 53–60 (2003).

  28. 28.

    et al. Cytosolic and mitochondrial deoxyribonucleotidases: activity with substrate analogs, inhibitors and implications for therapy. Biochem. Pharmacol. 66, 471–479 (2003).

  29. 29.

    et al. Genome-wide copy number profiling reveals molecular evolution from diagnosis to relapse in childhood acute lymphoblastic leukemia. Blood 112, 4178–4183 (2008).

  30. 30.

    et al. Somatic deletions of genes regulating MSH2 protein stability cause DNA mismatch repair deficiency and drug resistance in human leukemia cells. Nat. Med. 17, 1298–1303 (2011).

  31. 31.

    et al. Structural insights into the inhibition of cytosolic 5′-nucleotidase II (cN-II) by ribonucleosidse 5′-monophosphate analogues. PLOS Comput. Biol. 7–e1002295 (2011).

  32. 32.

    et al. Identification and characterization of inhibitors of cytoplasmic 5′-nucleotidase cN-II issued from virtual screening. Biochem. Pharmacol. 85, 497–506 (2013).

  33. 33.

    et al. Uniform approach to risk classification and treatment assignment for children with acute lymphoblastic leukemia. J. Clin. Oncol. 14, 18–24 (1996).

  34. 34.

    & Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

  35. 35.

    1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  36. 36.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011).

  37. 37.

    et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).

  38. 38.

    , & Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073–1081 (2009).

  39. 39.

    & The contour-buildup algorithm to calculate the analytical molecular surface. J. Struct. Biol. 116, 138–143 (1996).

  40. 40.

    & Rapid boundary element solvation electrostatics calculations in folding simulations: successful folding of a 23-residue peptide. Biopolymers 60, 124–133 (2001).

  41. 41.

    et al. HPLC determination of thiopurine nucleosides and nucleotides in vivo in lymphoblasts following mercaptopurine therapy. Clin. Chem. 48, 61–68 (2002).

Download references


We would like to thank the members of the Carroll laboratory as well as L.B. Gardner, M. Karajannis and I. Osman for their critical review of the manuscript. We gratefully acknowledge the Children's Oncology Group (COG) for patient specimens, the New York University Genome Technology Center for expert assistance with Illumina (B. Baysa) and Roche 454 (E. Venturini) deep-sequencing experiments (supported in part by US National Institutes of Health/National Center for Research Resources (NIH/NCRR) grant S10 RR026950-01), P. Grace, J.D. Ernst and M.R. Phillips (New York University School of Medicine) for expression and lentiviral vectors and F. Tsai, M.R. Phillips and S.M. Brown for technical guidance. This work was supported by US NIH grants R01 CA140729 and R21 CA152838-02 to W.L.C. and New York University Cancer Center Support Grant 5 P30 CA16087-30 in collaboration with the New York University Genome Technology Center. Additional support was provided by grants from the National Cancer Institute to COG, including U10 CA98543 (COG Chair's grant), U10 CA98413 (COG Statistical Center) and U24 CA114766 (COG Specimen Banking). J.A.M. is supported by NIH grant T32 CA009161. L.E.H. was supported by the American Society of Hematology and St. Baldrick's Foundation. S.P.H. is the Ergen Family Chair in Pediatric Cancer.

Author information

Author notes

    • Laura E Hogan

    Present address: Department of Pediatrics, Stony Brook University, Stony Brook, New York, USA.


  1. New York University Cancer Institute, New York University Langone Medical Center, New York, New York, USA.

    • Julia A Meyer
    • , Jinhua Wang
    • , Laura E Hogan
    • , Smita Dandekar
    • , Jiri Zavadil
    • , Elizabeth A Raetz
    • , Debra J Morrison
    •  & William L Carroll
  2. Department of Pathology, New York University Langone Medical Center, New York, New York, USA.

    • Julia A Meyer
    •  & William L Carroll
  3. New York University Center for Health Informatics and Bioinformatics, New York University Langone Medical Center, New York, New York, USA.

    • Jinhua Wang
    • , Zuojian Tang
    •  & Jiri Zavadil
  4. Department of Pediatrics, New York University Langone Medical Center, New York, New York, USA.

    • Laura E Hogan
    • , Smita Dandekar
    • , Elizabeth A Raetz
    • , Debra J Morrison
    •  & William L Carroll
  5. Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA.

    • Jun J Yang
    •  & William E Evans
  6. Human Oncology and Pathogenesis Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Jay P Patel
    •  & Ross L Levine
  7. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, USA.

    • Paul Zumbo
    • , Sheng Li
    •  & Christopher E Mason
  8. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College of Cornell University, New York, New York, USA.

    • Paul Zumbo
    • , Sheng Li
    •  & Christopher E Mason
  9. Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, New York, USA.

    • Ross L Levine
  10. Department of Pharmacology, New York University Langone Medical Center, New York, New York, USA.

    • Timothy Cardozo
  11. University of Colorado School of Medicine, Aurora, Colorado, USA.

    • Stephen P Hunger
  12. Children's Hospital Colorado, Aurora, Colorado, USA.

    • Stephen P Hunger


  1. Search for Julia A Meyer in:

  2. Search for Jinhua Wang in:

  3. Search for Laura E Hogan in:

  4. Search for Jun J Yang in:

  5. Search for Smita Dandekar in:

  6. Search for Jay P Patel in:

  7. Search for Zuojian Tang in:

  8. Search for Paul Zumbo in:

  9. Search for Sheng Li in:

  10. Search for Jiri Zavadil in:

  11. Search for Ross L Levine in:

  12. Search for Timothy Cardozo in:

  13. Search for Stephen P Hunger in:

  14. Search for Elizabeth A Raetz in:

  15. Search for William E Evans in:

  16. Search for Debra J Morrison in:

  17. Search for Christopher E Mason in:

  18. Search for William L Carroll in:


J.A.M., L.E.H., J.J.Y., J.Z., R.L.L., T.C., W.E.E., D.J.M., C.E.M. and W.L.C. planned experiments. J.A.M., L.E.H., J.J.Y., S.D., J.P.P. and D.J.M. performed experiments and analyzed data. J.W., Z.T., P.Z., S.L. and C.E.M. performed sequencing and analyzed sequence data. T.C. performed molecular modeling. S.P.H. and E.A.R. provided patient samples and clinical data. J.A.M. and W.L.C. wrote the manuscript. W.L.C. coordinated the study. All authors discussed the results and reviewed the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to William L Carroll.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Note, Supplementary Figures 1–8 and Supplementary Tables 1–4

About this article

Publication history






Further reading