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Abstract: Background and purpose: A subset of patients in a clinical study where sequential biopsies were to be obtained during multifraction radiotherapy received pimonidazole prior to initiating treatment, allowing a unique opportunity of following hypoxic cells in situ during therapy. Material and methods: After institutional ethics review and with informed consent, women expecting to undergo radical treatment for cancer of the cervix received pimonidazole hydrochloride, with a biopsy approximately 24h later. Therapy was then started, and weekly biopsies were obtained. In the laboratory, the biopsies were reduced to single cell suspensions for flow cytometry analysis of DNA content, pimonidazole, and proliferation markers. Results: Pre-treatment pimonidazole-positive cells were largely in G0/G1. Pimonidazole-labelled cells, though expected to be radioresistant, were markedly decreased even early into treatment, and continued to disappear with a half-time of about 3 days. Concurrently, the cell cycle distribution of the previously hypoxic cells changed from predominantly quiescent to mostly proliferating. Conclusions: While a part of the rapid apparent loss of hypoxic cells was certainly due to loss of pimonidazole adducts through repair and dilution by cell division, the speed with which this occurred suggests that many labelled cells could rapidly re-enter the proliferative pool, a result consistent with many of those pimonidazole-labelled human cervix tumour cells being cyclically, rather than continuously, hypoxic. [Copyright &y& Elsevier]

Tumour hypoxia is known to be associated with aggressiveness and poor response to treatment, which has stimulated the development of several methods able to detect hypoxic tumours. To date, only one method, the oxygen microelectrode, has been used to provide pretreatment measures of tumour oxygenation that correlate with local control and disease-free survival. In an effort to validate new methods, comparisons have been made between the Eppendorf oxygen microelectrode, the comet assay, and hypoxia marker binding in tumours of patients undergoing curative treatment or palliative radiotherapy. These comparisons suggest that tumours with median oxygen tensions below 10 mmHg have relatively high hypoxic fractions as measured by the comet assay (>0.20). The fraction of cells that binds pimonidazole, detected in cells obtained by fine-needle aspiration biopsy, correlates well with the hypoxic fraction measured using the comet assay. However, in general, hypoxic fractions measured by the comet assay and pimonidazole binding correlate only poorly with Eppendorf measurements performed for the same tumour. Factors that might be responsible for these differences, and problems associated with measuring the 'relevant' hypoxic population are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Background and purpose: Hypoxia adversely affects treatment outcome in human uterine cervical cancer. Here, we present the results of a prospective international multi-centre study evaluating the prognostic value of pre-treatment tumour oxygen partial pressure (pO2) and the hypoxia marker pimonidazole (pimo). Materials and methods: One hundred and twenty-seven patients with primary cervix cancer were entered. Pre-treatment tumour pO2 measurements were obtained, and reported by the median tumour pO2, the fraction of pO2 values ⩽10mmHg (HP10), ⩽5mmHg (HP5) and ⩽2.5mmHg (HP2.5). Following intravenous pimonidazole administration, biopsies were taken, stained for pimonidazole adducts, and scored for the area of labelled tumour cells on a scale from 0 to 4. Treatment modalities were surgery (11%), radiotherapy (98%), chemotherapy (33%) and carbogen (14%). Results: None of the hypoxia descriptors were statistically significant prognostic factors for loco-regional tumour control or overall survival when analyzed as continuous variables or divided by the sample median. By univariate analysis only tumour size and nodal status were significant prognostic factors for local control. Tumour size and FIGO stage were significant for overall survival. In a multivariate analysis stratified by centre, only tumour size above 5cm and lower pre-treatment haemoglobin predicted poorer overall survival among FIGO stage, nodal involvement, tumour size, pre-treatment haemoglobin dichotomized at 12g/dl and pimo 1, pimo 4 and HP5 as continuous variables. Conclusion: Neither Eppendorf nor pimonidazole should be dismissed based on the current results. However, further investigations are needed to readdress the hypotheses of the current study having optimized statistical designs, and a population of sufficient size treated more homogenously following rigorous protocols. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Abstract: Cigarette smokers have high carboxyhaemoglobin levels which can promote tumour radioresistance. Inhalation of carbogen gas shortens the half-life of carboxyhaemoglobin, increasing tumour radiosensitivity in animal models. Breathing 2.5% carbogen for 30min results in a greater reduction in venous blood COHb levels than breathing 5% carbogen for 7min. [Copyright &y& Elsevier]