Correlation of [18F]FMISO autoradiography and pimonodazole immunohistochemistry in human head and neck carcinoma xenografts

  • Esther G. C. TroostEmail author
  • Peter Laverman
  • Mariëlle E. P. Philippens
  • Jasper Lok
  • Albert J. van der Kogel
  • Wim J. G. Oyen
  • Otto C. Boerman
  • Johannes H. A. M. Kaanders
  • Johan Bussink
Original Article



Tumour cell hypoxia is a common feature in solid tumours adversely affecting radiosensitivity and chemosensitivity in head and neck squamous cell carcinomas. Positron emission tomography (PET) using the tracer [18F]fluoromisonidazole ([18F]FMISO) is most frequently used for non-invasive evaluation of hypoxia in human tumours. A series of ten human head and neck xenograft tumour lines was used to validate [18F]FMISO as hypoxia marker at the microregional level.


Autoradiography after injection of [18F]FMISO was compared with immunohistochemical staining for the hypoxic cell marker pimonidazole in the same tumour sections of ten different human head and neck xenograft tumour lines. The methods were compared: first, qualitatively considering the microarchitecture; second, by obtaining a pixel-by-pixel correlation of both markers at the microregional level; third, by measuring the signal intensity of both images; and fourth, by calculating the hypoxic fractions by pimonidazole labelling.


The pattern of [18F]FMISO signal was dependent on the distribution of hypoxia at the microregional level. The comparison of [18F]FMISO autoradiography and pimonidazole immunohistochemistry by pixel-by-pixel analysis revealed moderate correlations. In five tumour lines, a significant correlation between the mean [18F]FMISO and pimonidazole signal intensity was found (range, r 2 = 0.91 to r 2 = 0.99). Comparison of the tumour lines with respect to the microregional distribution pattern of hypoxia revealed that the correlation between the mean signal intensities strongly depended on the microarchitecture. Overall, a weak but significant correlation between hypoxic fractions based on pimonidazole labeling and the mean [18F]FMISO signal intensity was observed (r 2 = 0.18, p = 0.02). For the three tumour models with a ribbon-like microregional distribution pattern of hypoxia, the correlation between the hypoxic fraction and the mean [18F]FMISO signal intensity was much stronger and more significant (r 2 = 0.73, p < 0.001) than for the tumours with a more homogenous, patchy, microregional distribution pattern of hypoxia.


Different patterns of [18F]FMISO accumulation dependent on the underlying microregional distribution of hypoxia were found in ten head and neck xenograft tumours. A weak albeit significant correlation was found between the mean [18F]FMISO signal intensity and the hypoxic fraction of the tumours. In larger clinical tumours, [18F]FMISO–PET provides information on the tumour oxygenation status on a global level, facilitating dose painting in radiation treatment planning. However, caution must be taken when studying small tumour subvolumes as accumulation of the tracer depends on the presence of hypoxia and on the tumour microarchitecture.


[18F]Fluoromisonidazole Pimonidazole Digital autoradiography Immunohistochemistry Hypoxia 



This research was supported by EC FP6 funding (Biocare contract no. LSHC-CT-2004-505785) and by Junior Investigator Grant 2006-38 of the Radboud University Nijmegen Medical Centre, The Netherlands. We thank Dr. J. A. Raleigh for the gift of anti-pimonidazole MAb and Dr. H. J. J. M. Rennen for his kind support with the [18F]FMISO synthesis.

Conflict of interest statement

The authors have no conflict of interest.


  1. 1.
    Bussink J, Kaanders JH, van der Kogel AJ. Tumor hypoxia at the micro-regional level: clinical relevance and predictive value of exogenous and endogenous hypoxic cell markers. Radiother Oncol. 2003;67:3–15.PubMedCrossRefGoogle Scholar
  2. 2.
    Hedley D, Pintilie M, Woo J, Morrison A, Birle D, Fyles A, et al. Carbonic anhydrase IX expression, hypoxia, and prognosis in patients with uterine cervical carcinomas. Clin Cancer Res. 2003;9:5666–74.PubMedGoogle Scholar
  3. 3.
    Nordsmark M, Bentzen SM, Rudat V, Brizel D, Lartigau E, Stadler P, et al. Prognostic value of tumor oxygenation in 397 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol. 2005;77:18–24.PubMedCrossRefGoogle Scholar
  4. 4.
    Nordsmark M, Hoyer M, Keller J, Nielsen OS, Jensen OM, Overgaard J. The relationship between tumor oxygenation and cell proliferation in human soft tissue sarcomas. Int J Radiat Oncol Biol Phys. 1996;35:701–8.PubMedGoogle Scholar
  5. 5.
    Nordsmark M, Overgaard J. A confirmatory prognostic study on oxygenation status and loco-regional control in advanced head and neck squamous cell carcinoma treated by radiation therapy. Radiother Oncol. 2000;57:39–43.PubMedCrossRefGoogle Scholar
  6. 6.
    Kaanders JH, Pop LA, Marres HA, Bruaset I, van den Hoogen FJ, Merkx MA, et al. ARCON: experience in 215 patients with advanced head-and-neck cancer. Int J Radiat Oncol Biol Phys. 2002;52:769–78.PubMedCrossRefGoogle Scholar
  7. 7.
    Overgaard J, Hansen HS, Overgaard M, Bastholt L, Berthelsen A, Specht L, et al. A randomized double-blind phase III study of nimorazole as a hypoxic radiosensitizer of primary radiotherapy in supraglottic larynx and pharynx carcinoma. Results of the Danish Head and Neck Cancer Study (DAHANCA) Protocol 5-85. Radiother Oncol. 1998;46:135–46.PubMedCrossRefGoogle Scholar
  8. 8.
    Bentzen L, Keiding S, Nordsmark M, Falborg L, Hansen SB, Keller J, et al. Tumour oxygenation assessed by 18F-fluoromisonidazole PET and polarographic needle electrodes in human soft tissue tumours. Radiother Oncol. 2003;67:339–44.PubMedCrossRefGoogle Scholar
  9. 9.
    Lartigau E, Vitu L, Haie-Meder C, Cosset MF, Delapierre M, Gerbaulet A, et al. Feasibility of measuring oxygen tension in uterine cervix carcinoma. Eur J Cancer. 1992;28:1354–7.CrossRefGoogle Scholar
  10. 10.
    Nordsmark M, Loncaster J, Aquino-Parsons C, Chou SC, Ladekarl M, Havsteen H, et al. Measurements of hypoxia using pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas. Radiother Oncol. 2003;67:35–44.PubMedCrossRefGoogle Scholar
  11. 11.
    Koch CJ, Evans SM, Lord EM. Oxygen dependence of cellular uptake of EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide]: analysis of drug adducts by fluorescent antibodies vs bound radioactivity. Br J Cancer. 1995;72:869–74.PubMedGoogle Scholar
  12. 12.
    Raleigh JA, Calkins-Adams DP, Rinker LH, Ballenger CA, Weissler MC, Fowler WC Jr., et al. Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res. 1998;58:3765–8.PubMedGoogle Scholar
  13. 13.
    Evans SM, Hahn S, Pook DR, Jenkins WT, Chalian AA, Zhang P, et al. Detection of hypoxia in human squamous cell carcinoma by EF5 binding. Cancer Res. 2000;60:2018–24.PubMedGoogle Scholar
  14. 14.
    Yaromina, et al. Pimonidazole labelling and response to fractionated irradiation of five human squamous cell carcinoma (hSCC) lines in nude mice: the need for a multivariate approach in biomarker studies. Radiother Oncol. 2006;81(2):122–9.Google Scholar
  15. 15.
    Kaanders JH, Wijffels KI, Marres HA, Ljungkvist AS, Pop LA, van den Hoogen FJ, et al. Pimonidazole binding and tumor vascularity predict for treatment outcome in head and neck cancer. Cancer Res. 2002;62:7066–74.PubMedGoogle Scholar
  16. 16.
    Bentzen L, Keiding S, Horsman MR, Gronroos T, Hansen SB, Overgaard J. Assessment of hypoxia in experimental mice tumours by [18F]fluoromisonidazole PET and pO2 electrode measurements. Influence of tumour volume and carbogen breathing. Acta Oncol. 2002;41:304–12.PubMedCrossRefGoogle Scholar
  17. 17.
    Eschmann SM, Paulsen F, Reimold M, Dittmann H, Welz S, Reischl G, et al. Prognostic impact of hypoxia imaging with 18F-misonidazole PET in non-small cell lung cancer and head and neck cancer before radiotherapy. J Nucl Med. 2005;46:253–60.PubMedGoogle Scholar
  18. 18.
    Koh WJ, Rasey JS, Evans ML, Grierson JR, Lewellen TK, Graham MM, et al. Imaging of hypoxia in human tumors with [F-18]fluoromisonidazole. Int J Radiat Oncol Biol Phys. 1992;22:199–212.PubMedGoogle Scholar
  19. 19.
    Rasey JS, Koh WJ, Evans ML, Peterson LM, Lewellen TK, Graham MM, et al. Quantifying regional hypoxia in human tumors with positron emission tomography of [18F]fluoromisonidazole: a pretherapy study of 37 patients. Int J Radiat Oncol Biol Phys. 1996;36:417–28.PubMedGoogle Scholar
  20. 20.
    Sorger D, Patt M, Kumar P, Wiebe LI, Barthel H, Seese A, et al. [18F]Fluoroazomycinarabinofuranoside (18FAZA) and [18F]fluoromisonidazole (18FMISO): a comparative study of their selective uptake in hypoxic cells and PET imaging in experimental rat tumors. Nucl Med Biol. 2003;30:317–26.PubMedCrossRefGoogle Scholar
  21. 21.
    Troost EG, Laverman P, Kaanders JHAM, Philippens M, Lok J, Oyen WJG, van der Kogel AJ, Boerman OC, Bussink J. Imaging hypoxia after oxygenation-modification: comparing [18F]FMISO autoradiography with pimonidazole immunohistochemistry in human xenograft tumors. Radiother Oncol 2006;80:157–64.PubMedCrossRefGoogle Scholar
  22. 22.
    Wyss MT, Honer M, Schubiger PA, Ametamey SM. NanoPET imaging of [(18)F]fluoromisonidazole uptake in experimental mouse tumours. Eur J Nucl Med Mol Imaging 2005;33:311–8.PubMedCrossRefGoogle Scholar
  23. 23.
    Hicks RJ, Rischin D, Fisher R, Binns D, Scott AM, Peters LJ. Utility of FMISO PET in advanced head and neck cancer treated with chemoradiation incorporating a hypoxia-targeting chemotherapy agent. Eur J Nucl Med Mol Imaging. 2005;32:1384–91.PubMedCrossRefGoogle Scholar
  24. 24.
    Rischin D, Hicks RJ, Fisher R, Binns D, Corry J, Porceddu S, et al. Prognostic significance of [18F]-misonidazole positron emission tomography-detected tumor hypoxia in patients with advanced head and neck cancer randomly assigned to chemoradiation with or without tirapazamine: a substudy of Trans-Tasman Radiation Oncology Group Study 98.02. J Clin Oncol. 2006;24:2098–104.PubMedCrossRefGoogle Scholar
  25. 25.
    Thorwarth D, Eschmann SM, Holzner F, Paulsen F, Alber M. Combined uptake of [18F]FDG and [18F]FMISO correlates with radiation therapy outcome in head-and-neck cancer patients. Radiother Oncol. 2006;80:151–6.PubMedCrossRefGoogle Scholar
  26. 26.
    Allemann KWM, Wergin M, Ohlerth S, Rohrer-Bley C, Evans SM, Schubiger AP, Ametamey SM, Kaser-Hotz B. Measurements of hypoxia ([18F]-FMISO, [18F]-EF5) with positron emission tomography (PET) and perfusion using PET ([15O]-H2O) and power Doppler ultrasonography in feline fibrosarcomas. Vet Comp Oncol. 2005;3:211–21.CrossRefGoogle Scholar
  27. 27.
    Pugachev A, Claus F, Sun X, Cai S, Koziorowsky J, Finn R, et al. Validation of PET hypoxia tracers by autoradiography and fluorescent microscopy. Med Phys. 2005;32:2055.CrossRefGoogle Scholar
  28. 28.
    Tanaka T, Furukawa T, Fujieda S, Kasamatsu S, Yonekura Y, Fujibayashi Y. Double-tracer autoradiography with Cu-ATSM/FDG and immunohistochemical interpretation in four different mouse implanted tumor models. Nucl Med Biol. 2006;33:743–50.PubMedCrossRefGoogle Scholar
  29. 29.
    Yuan H, Schroeder T, Bowsher JE, Hedlund LW, Wong T, Dewhirst MW. Intertumoral differences in hypoxia selectivity of the PET imaging agent 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone). J Nucl Med. 2006;47:989–98.PubMedGoogle Scholar
  30. 30.
    Ljungkvist AS, Bussink J, Rijken PF, Kaanders JH, van der Kogel AJ, Denekamp J. Vascular architecture, hypoxia, and proliferation in first-generation xenografts of human head-and-neck squamous cell carcinomas. Int J Radiat Oncol Biol Phys. 2002;54:215–28.PubMedGoogle Scholar
  31. 31.
    Lim JL, Berridge MS. An efficient radiosynthesis of [18F]fluoromisonidazole. Appl Radiat Isotopes 1993;44:1085–91.CrossRefGoogle Scholar
  32. 32.
    Rijken PF, Bernsen HJ, Peters JP, Hodgkiss RJ, Raleigh JA, van der Kogel AJ. Spatial relationship between hypoxia and the (perfused) vascular network in a human glioma xenograft: a quantitative multi-parameter analysis. Int J Radiat Oncol Biol Phys. 2000;48:571–82.PubMedGoogle Scholar
  33. 33.
    Hoebers FJ, Janssen HL, Olmos AV, Sprong D, Nunn AD, Balm AJ, et al. Phase 1 study to identify tumour hypoxia in patients with head and neck cancer using technetium-99m BRU 59-21. Eur J Nucl Med Mol Imaging. 2002;29:1206–11.PubMedCrossRefGoogle Scholar
  34. 34.
    Troost EG, Bussink J, Kaanders JH, van Eerd J, Peters JP, Rijken PF, et al. Comparison of different methods of CAIX quantification in relation to hypoxia in three human head and neck tumor lines. Radiother Oncol. 2005;76:194–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Esther G. C. Troost
    • 1
    Email author
  • Peter Laverman
    • 2
  • Mariëlle E. P. Philippens
    • 1
  • Jasper Lok
    • 1
  • Albert J. van der Kogel
    • 1
  • Wim J. G. Oyen
    • 2
  • Otto C. Boerman
    • 2
  • Johannes H. A. M. Kaanders
    • 1
  • Johan Bussink
    • 1
  1. 1.Department of Radiation OncologyRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  2. 2.Department of Nuclear MedicineRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

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