Cancer Chemotherapy and Pharmacology

, Volume 80, Issue 3, pp 645–652 | Cite as

A phase I/II trial and pharmacokinetic study of mithramycin in children and adults with refractory Ewing sarcoma and EWS–FLI1 fusion transcript

  • Patrick J. Grohar
  • John GlodEmail author
  • Cody J. Peer
  • Tristan M. Sissung
  • Fernanda I. Arnaldez
  • Lauren Long
  • William D. Figg
  • Patricia Whitcomb
  • Lee J. Helman
  • Brigitte C. Widemann
Clinical Trial Report



In a preclinical drug screen, mithramycin was identified as a potent inhibitor of the Ewing sarcoma EWS–FLI1 transcription factor. We conducted a phase I/II trial to determine the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), and pharmacokinetics (PK) of mithramycin in children with refractory solid tumors, and the activity in children and adults with refractory Ewing sarcoma.

Patients and methods

Mithramycin was administered intravenously over 6 h once daily for 7 days for 28 day cycles. Adult patients (phase II) initially received mithramycin at the previously determined recommended dose of 25 µg/kg/dose. The planned starting dose for children (phase I) was 17.5 µg/kg/dose. Plasma samples were obtained for mithramycin PK analysis.


The first two adult patients experienced reversible grade 4 alanine aminotransferase (ALT)/aspartate aminotransferase (AST) elevation exceeding the MTD. Subsequent adult patients received mithramycin at 17.5 µg/kg/dose, and children at 13 µg/kg/dose with dexamethasone pretreatment. None of the four subsequent adult and two pediatric patients experienced cycle 1 DLT. No clinical responses were observed. The average maximal mithramycin plasma concentration in four patients was 17.8 ± 4.6 ng/mL. This is substantially below the sustained mithramycin concentrations ≥50 nmol/L required to suppress EWS–FLI1 transcriptional activity in preclinical studies. Due to inability to safely achieve the desired mithramycin exposure, the trial was closed to enrollment.


Hepatotoxicity precluded the administration of a mithramycin at a dose required to inhibit EWS–FLI1. Evaluation of mithramycin in patients selected for decreased susceptibility to elevated transaminases may allow for improved drug exposure.


Ewing sarcoma Mithramycin EWS–FLI1 



We thank the patients and their families who participated in this trial, Linda Ellison-Dejewski for research nursing support, Natasha Brunson for assistance with data management, Dr. Seth Steinberg for assistance with the statistical design of the study, and Jeff Roth for technical support in the performance of pharmacokinetic assays.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest relating to this study.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study.


  1. 1.
    Esiashvili N, Goodman M, Marcus RB Jr (2008) Changes in incidence and survival of Ewing sarcoma patients over the past 3 decades: surveillance epidemiology and end results data. J Pediatr Hematol Oncol 30:425–430. doi: 10.1097/MPH.0b013e31816e22f3 CrossRefPubMedGoogle Scholar
  2. 2.
    Gaspar N et al (2015) Ewing sarcoma: current management and future approaches through collaboration. J Clin Oncol 33:3036–3046. doi: 10.1200/JCO.2014.59.5256 CrossRefPubMedGoogle Scholar
  3. 3.
    Womer RB et al (2012) Randomized controlled trial of interval-compressed chemotherapy for the treatment of localized Ewing sarcoma: a report from the Children’s Oncology Group. J Clin Oncol 30:4148–4154. doi: 10.1200/JCO.2011.41.5703 CrossRefPubMedCentralGoogle Scholar
  4. 4.
    Delattre O et al (1994) The Ewing family of tumors—a subgroup of small-round-cell tumors defined by specific chimeric transcripts. N Engl J Med 331:294–299. doi: 10.1056/NEJM199408043310503 CrossRefPubMedGoogle Scholar
  5. 5.
    Delattre O et al (1992) Gene fusion with an ETS DNA-binding domain caused by chromosome translocation in human tumours. Nature 359:162–165. doi: 10.1038/359162a0 CrossRefPubMedGoogle Scholar
  6. 6.
    Bailly RA et al (1994) DNA-binding and transcriptional activation properties of the EWS–FLI-1 fusion protein resulting from the t(11;22) translocation in Ewing sarcoma. Mol Cell Biol 14:3230–3241CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Kauer M et al (2009) A molecular function map of Ewing’s sarcoma. PLoS One 4:e5415. doi: 10.1371/journal.pone.0005415 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Riggi N et al (2014) EWS–FLI1 utilizes divergent chromatin remodeling mechanisms to directly activate or repress enhancer elements in Ewing sarcoma. Cancer Cell 26:668–681. doi: 10.1016/j.ccell.2014.10.004 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Gangwal K et al (2008) Microsatellites as EWS/FLI response elements in Ewing’s sarcoma. Proc Natl Acad Sci USA 105:10149–10154. doi: 10.1073/pnas.0801073105 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Riggi N, Stamenkovic I (2007) The biology of Ewing sarcoma. Cancer Lett 254:1–10. doi: 10.1016/j.canlet.2006.12.009 CrossRefPubMedGoogle Scholar
  11. 11.
    Hu-Lieskovan S et al (2005) EWS–FLI1 fusion protein up-regulates critical genes in neural crest development and is responsible for the observed phenotype of Ewing’s family of tumors. Cancer Res 65:4633–4644. doi: 10.1158/0008-5472.CAN-04-2857 CrossRefPubMedGoogle Scholar
  12. 12.
    May WA et al (1997) EWS/FLI1-induced manic fringe renders NIH 3T3 cells tumorigenic. Nat Genet 17:495–497. doi: 10.1038/ng1297-495 CrossRefPubMedGoogle Scholar
  13. 13.
    Maksimenko A, Malvy C (2005) Oncogene-targeted antisense oligonucleotides for the treatment of Ewing sarcoma. Expert Opin Ther Targets 9:825–830. doi: 10.1517/14728222.9.4.825 CrossRefPubMedGoogle Scholar
  14. 14.
    Grohar PJ et al (2011) Identification of an inhibitor of the EWS–FLI1 oncogenic transcription factor by high-throughput screening. J Natl Cancer Inst 103:962–978. doi: 10.1093/jnci/djr156 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Mendiola M et al (2006) The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors. Int J Cancer 118:1381–1389. doi: 10.1002/ijc.21578 CrossRefPubMedGoogle Scholar
  16. 16.
    Kinsey M, Smith R, Lessnick SL (2006) NR0B1 is required for the oncogenic phenotype mediated by EWS/FLI in Ewing’s sarcoma. Mol Cancer Res 4:851–859. doi: 10.1158/1541-7786.MCR-06-0090 CrossRefPubMedGoogle Scholar
  17. 17.
    Nishimori H et al (2002) The Id2 gene is a novel target of transcriptional activation by EWS–ETS fusion proteins in Ewing family tumors. Oncogene 21:8302–8309. doi: 10.1038/sj.onc.1206025 CrossRefPubMedGoogle Scholar
  18. 18.
    Osgood CL et al (2016) Identification of mithramycin analogues with improved targeting of the EWS–FLI1 transcription factor. Clin Cancer Res 22:4105–4118. doi: 10.1158/1078-0432.CCR-15-2624 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Kofman S, Perlia CP, Economou SG (1973) Mithramycin in the treatment of metastatic Ewing’s sarcoma. Cancer 31:889–893CrossRefPubMedGoogle Scholar
  20. 20.
    Kennedy BJ, Torkelson JL (1995) Long-term follow-up of stage III testicular carcinoma treated with mithramycin (plicamycin). Med Pediatr Oncol 24:327–328CrossRefPubMedGoogle Scholar
  21. 21.
    Curreri AR, Ansfield FJ (1960) Mithramycin-human toxicology and preliminary therapeutic investigation. Cancer Chemother Rep 8:18–22Google Scholar
  22. 22.
    Spear PW (1963) Clinical trial with mithramycin. Cancer Chemother Rep 29:109–110Google Scholar
  23. 23.
    Mithramycin (mithracin) (1971) For intravenous use. Clin Pharmacol Ther 12:310–313CrossRefGoogle Scholar
  24. 24.
    Kofman S, Eisenstein R (1963) Mithramycin in the treatment of disseminated cancer. Cancer Chemother Rep 32:77–96PubMedGoogle Scholar
  25. 25.
    Bilezikian JP (1992) Management of acute hypercalcemia. N Engl J Med 326:1196–1203. doi: 10.1056/NEJM199204303261806 CrossRefPubMedGoogle Scholar
  26. 26.
    Perlia CP et al (1970) Mithramycin treatment of hypercalcemia. Cancer 25:389–394CrossRefPubMedGoogle Scholar
  27. 27.
    Roth J et al (2014) Quantitative determination of mithramycin in human plasma by a novel, sensitive ultra-HPLC-MS/MS method for clinical pharmacokinetic application. J Chromatogr B Analyt Technol Biomed Life Sci 970:95–101. doi: 10.1016/j.jchromb.2014.08.021 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Eisenhauer EA et al (2009) New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45:228–247. doi: 10.1016/j.ejca.2008.10.026 CrossRefGoogle Scholar
  29. 29.
    Fraisse F, Marche C, Gibert C, Coquin Y, Vachon F (1980) Acute hepatic necrosis and hemorrhagic syndrome leading to a fatal outcome during treatment of hypercalcemia with mithramycin (author’s transl). Ann Med Interne (Paris) 131:281–284Google Scholar
  30. 30.
    Fang K et al (1992) Determination of plicamycin in plasma by radioimmunoassay. Ther Drug Monit 14:255–260CrossRefPubMedGoogle Scholar
  31. 31.
    Grosso F et al (2006) Steroid premedication markedly reduces liver and bone marrow toxicity of trabectedin in advanced sarcoma. Eur J Cancer 42:1484–1490. doi: 10.1016/j.ejca.2006.02.010 CrossRefPubMedGoogle Scholar
  32. 32.
    Osgood CL et al (2016) 18F-FLT positron emission tomography (PET) is a pharmacodynamic marker for EWS–FLI1 activity and Ewing sarcoma. Sci Rep 6:33926. doi: 10.1038/srep33926 CrossRefPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany (outside the USA) 2017

Authors and Affiliations

  • Patrick J. Grohar
    • 1
    • 2
  • John Glod
    • 1
    Email author
  • Cody J. Peer
    • 3
  • Tristan M. Sissung
    • 2
  • Fernanda I. Arnaldez
    • 1
  • Lauren Long
    • 1
  • William D. Figg
    • 3
  • Patricia Whitcomb
    • 1
  • Lee J. Helman
    • 1
  • Brigitte C. Widemann
    • 1
  1. 1.Pediatric Oncology Branch, Center for Cancer ResearchNational Cancer InstituteBethesdaUSA
  2. 2.Department of Pediatrics, Van Andel Research Institute, Helen DeVos Children’s HospitalMichigan State UniversityEast LansingUSA
  3. 3.Clinical Pharmacology ProgramNational Cancer InstituteBethesdaUSA

Personalised recommendations