Measurements of hypoxia using pimonidazole and polarographic oxygen-sensitive electrodes in human cervix carcinomas

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Abstract

Background and purpose: The measurement of tumour oxygenation using Eppendorf oxygen-sensitive needle electrodes can provide prognostic information but the method is limited to accessible tumours that are suitable for electrode insertion. In this paper the aim was to study the relationship between such physiological measurements of tumour hypoxia and the labelling of tumours with the hypoxia-specific marker pimonidazole.

Materials and methods: Assessment of tumour oxygen partial pressure (pO2) using an Eppendorf pO2 histograph and immunohistochemical pimonidazole labelling was carried out in 86 patients with primary cervix carcinomas. Pimonidazole was given as a single injection (0.5 g/m2 i.v.) and 10–24 h later pO2 measurements were made and biopsies taken. Tumour oxygenation status was evaluated as the median tumour pO2 and the fraction of pO2 values ≤10 mmHg (HP10), ≤5 mmHg (HP5) and ≤2.5 mmHg (HP2.5). Hypoxia was detected by immunohistochemistry using monoclonal antibodies directed against reductively activated pimonidazole. Pimonidazole binding was scored using a light microscope. Each tumour was evaluated by the relative area pimonidazole at highest score and the accumulated area of pimonidazole labelling from score 1 to 4. Necrosis was measured in HE stained sections.

Results and conclusions: The degree of hypoxia assessed by either pimonidazole binding or invasive electrode measurements varied significantly between tumours. There was a trend that the most hypoxic tumours measured by oxygen electrodes had the highest score of necrosis, and no or little pimonidazole binding. However, this observation was not consistent and there was no correlation between pimonidazole staining expressed in this way and oxygen electrode measurements of hypoxia.

Introduction

The prognostic value of tumour oxygenation measured by the Eppendorf oxygen partial pressure (pO2) histograph has been demonstrated in a number of studies on a variety of tumour types treated with either primary radiation, radiotherapy combined with surgery or surgery alone [6], [10], [12], [16], [18], [19]. However, the method is limited to clinically accessible tumours suitable for needle electrode insertion. Alternative methods with potential for wider clinical application are therefore needed [21]. A method relying on injection of a hypoxia-specific probe, biopsy and immunohistochemistry is particularly attractive provided that the method is suitable for a routine service laboratory and not too expensive.

The feasibility and usefulness of measuring the bioreductive binding of 2-nitroimidazoles was originally established using tritiated misonidazole [31]. That work led to the development of immunochemical methods using monoclonal antibodies raised to protein adducts of reductively activated 2-nitroimidazoles [24]. Both pimonidazole [15], [20] and EF5 [9] binding have now been used clinically to measure tumour hypoxia. Studies in experimental tumours showed a good relationship between pimonidazole binding and oxygen electrode measurements of hypoxia in a single tumour model where the level of oxygenation was manipulated artificially [25] whereas the relationship between Eppendorf pO2 measurements and EF5 binding in individual murine tumours was not significant [14]. While the prognostic significance of Eppendorf measurements is well documented, clinical outcome results to assess the prognostic value of pimonidazole binding are still awaited.

The aim of the current study was to examine the relationship between two biologically different measures of hypoxia using hypoxia marker labelling and Eppendorf pO2 data in uterine cervix carcinomas. By calibrating pimonidazole results against the Eppendorf measurements this might give an indication of which parameters to use as a prognostic candidate in the future. We have previously reported the preliminary analysis of a multi-centre collaborative study examining the relationship between invasive oxygen measurements and pimonidazole labelling in 28 human cervix carcinomas [20]. The final analysis of this collaborative study, incorporating estimates of micro-regional necrosis, is reported here.

Section snippets

Patients

Dual assessment of hypoxia using invasive oxygen sensitive needle electrodes and immunohistochemical pimonidazole labelling was carried out on 86 patients with primary carcinoma of the uterine cervix. Forty-three cases were included between February 1999 and August 2000 from the Christie Hospital NHS Trust, Manchester, UK. Twenty-four patients were included between February 1998 and October 2000 at the Department of Oncology, Aarhus University Hospital and 19 patients were included between

Pimonidazole labelling

Data obtained from both assays were available for further analysis in all 86 patients. The number of tumour biopsies taken for analysis of pimonidazole labelling ranged from one to six with a median of three and a median of 74 microscopic fields (range 4–299). Each field area was 0.06 mm2 and the total area analysed per tumour is listed in Table 2. Large variability in pimonidazole scores was observed within tumours. An example from a squamous cell carcinoma is presented in Fig. 1. There was

Discussion

As the work reported here was a multi-centre collaborative study, an examination was made of inter-institutional variability of pimonidazole labelling and Eppendorf pO2 data. No significant difference in tumour oxygenation was found in the data obtained at the three centres. Although one of the centres used anaesthesia, the overall median pO2 was equally low (3 mmHg) in all three centres. This level is in agreement with results reported by Fyles et al. (overall median pO2=5 mmHg, range 0–94

Acknowledgments

Financial support was provided by The Danish Cancer Society, NIH grant 74069-02, NIH grant CA37879, The BIOMED II Concerted Action MBH4-98-3006 and Cancer Research UK. The study was prepared during the Flims Workshop ‘Methods in Clinical Cancer Research’ 1999 arranged by FECS, ASCO and AACR. The authors would like to thank Dr Michael R. Horsman for constructive discussions, Ms Debra Novotny for specialist pathology advice and Ms Inger Marie Horsman, Ms K. Hillebrandt and Mr M.J. Johansen for

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