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

Yaromina, Ala; Zips, Daniel; Thames, Howard D.; Eicheler, Wolfgang; Krause, Mechthild; Rosner, Andrea; Haase, Michael; Petersen, Cordula; Raleigh, James A.; Quennet, Verena; Walenta, Stefan; Mueller-Klieser, Wolfgang; Baumann, Michael

doi:10.1016/j.radonc.2006.08.010

« Previous Next »Radiotherapy & Oncology
Volume 81, Issue 2 , Pages 122-129, November 2006

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

Ala Yaromina
- Department of Radiation Oncology
Daniel Zips
- Department of Radiation Oncology
Howard D. Thames
- Department of Biostatistics and Applied Mathematics, University of Texas M.D. Anderson Cancer Centre, Houston, TX, USA
Wolfgang Eicheler
- Department of Radiation Oncology
- Experimental Centre, and
Mechthild Krause
- Department of Radiation Oncology
Andrea Rosner
- Department of Radiation Oncology
Michael Haase
- OncoRay – Centre for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Germany
Cordula Petersen
- Department of Radiation Oncology
James A. Raleigh
- Department of Radiation Oncology, Chapel Hill, NC, USA
Verena Quennet
- Institute of Physiology and Pathophysiology, University of Mainz, Germany
Stefan Walenta
- Institute of Physiology and Pathophysiology, University of Mainz, Germany
Wolfgang Mueller-Klieser
- Institute of Physiology and Pathophysiology, University of Mainz, Germany
Michael Baumann
- Department of Radiation Oncology
- Experimental Centre, and
- OncoRay – Centre for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, University of Technology Dresden, Germany
- Corresponding author. Michael Baumann, Department of Radiation Oncology, Medical Faculty Carl Gustav Carus, Technische Universität Dresden, Fetscherstrasse 74, 01307 Dresden, Germany.

Received 18 April 2006; received in revised form 24 July 2006; accepted 11 August 2006. published online 06 September 2006.

Abstract

Objective

To investigate the influence on local control after fractionated radiotherapy of hypoxia measured in unirradiated tumours using the hypoxic marker Pimonidazole, using multivariate approaches.

Material and methods

Five human squamous cell carcinoma lines (FaDu, UT-SCC-15, UT-SCC-14, XF354, and UT-SCC-5) were transplanted subcutaneously into the right hind-leg of NMRI nude mice. Histological material was collected from 60 unirradiated tumours after injection of Pimonidazole. The relative hypoxic area within the viable tumour area (Pimonidazole hypoxic fraction, pHF) was determined in seven serial 10μm cross-sections per tumour by fluorescence microscopy and computerized image analysis. Local tumour control was evaluated in a total of 399 irradiated tumours at 120 days after 30 fractions given within 6 weeks with total doses between 30 and 115Gy.

Results

Tumour lines showed pronounced heterogeneity in both pHF and TCD₅₀. Mean pHF values varied between 5% and 37%, TCD₅₀ values between 47 and 130Gy. A Cox Proportional Hazards model of time to recurrence with two covariates, dose and pHF, yielded significant contributions of both parameters on local control (p<0.005) but violated the proportional hazards assumption, suggesting that other factors also influence tumour control. Introduction of histological grade as an example of a confounding factor into the model improved the fit significantly. Local control rates decreased with increasing pHF and this effect was more pronounced at higher doses.

Conclusions

This study confirms that tumour hypoxia measured using Pimonidazole in untreated tumours is a significant determinant of local control after fractionated irradiation. The data support the use of multivariate approaches for the evaluation of a single prognostic biomarker such as Pimonidazole, and more generally, suggest that they are required to establish accurate prognostic factors for tumour response.

Keywords: Hypoxia, Pimonidazole labelling, Heterogeneity, Human tumour xenograft, Fractionated radiation, Local tumour control