Michael J. Eble

Repeatability and prognostic impact of the pretreatment pO2 histography in patients with advanced head and neck cancer

PURPOSE The purpose of this study was to evaluate the repeatability and the predictive relevance of the pretreatment pO(2) histography on the survival of patients with advanced head and neck cancer. PATIENTS AND METHODS From July 1995 to August 1998, polarographic pO(2) measurements of lymph node metastases before therapy were performed in altogether 60 patients with histologically proven squamous cell carcinoma of the head and neck using the Eppendorf histograph. Forty-one of 60 patients were treated with an accelerated-hyperfractionated radiotherapy regimen with or without simultaneous chemotherapy as part of a multicenter phase III study. In 23 of 60 patients, two repeated independent measurements of the same tumor were performed with a time interval of approximately 24 h between the two measurements. RESULTS The multivariate analysis revealed the fraction of pO(2) values </=2.5 mmHg as the only significant prognostic factor for the survival (P=0.05) in the 41 study patients. No correlation was found between tumour oxygenation and the volume of the measured lymph node metastases or the haemoglobin concentration. The coefficient of variation of the repeated measurements representing the assay variability was 57-68% of the total variation. CONCLUSION Our data support the concept of the relevance of the pretreatment tumour hypoxia for the prognosis of patients with head and neck cancer after fractionated radiotherapy. Because of the relative poor repeatability of the pO(2) histography and the small patient number, further studies are required to confirm this finding and to evaluate the most relevant oxygenation parameter for clinical endpoints.

Oxygenation of advanced head and neck cancer: Prognostic marker for the response to primary radiochemotherapy

Recent clinical studies suggest that the degree of tumor oxygenation may be predictive of the response of radiation therapy for cancer. In an exploratory investigation of cervical lymph node metastases in 27 patients with advanced squamous cell carcinoma of the oropharynx and hypopharynx, this relationship was investigated by means of oxygen measurements with an Eppendorf PO (2) histograph. The measurements were made before the start of radiation therapy and after the first week of therapy. Clinical response was evaluated 6 weeks after the completion of therapy. Before therapy, marked hypoxia was observed in the lymph node metastases, with a mean PO (2) value of 16.1 +/- 8.2 mm Hg and a hypoxic fraction (PO (2) < 10 mm Hg) of 56.4% +/- 20.0%. After the first week of radiation (9 Gy) there was a general reoxygenation (DeltaPO (2) = 5.0 +/- 10.1 mm Hg, P < 0.05; Deltahypoxic fraction = -11.3% +/- 31.3%, P = 0.11). A relationship between the degree of reoxygenation and tumor response was not observed. Patients without at least partial lymph node response (n = 8) showed poorer pretherapeutic oxygenation (PO (2) mean = 11.1 +/- 2.9 mm Hg) than those who responded to the therapy (n = 19, PO (2) mean = 18.2 +/- 8.8 mm Hg). In this investigation of a defined set of patients with advanced carcinoma of the oropharynx and hypopharynx, we found that pretherapeutic oxygenation data are predictive for the therapeutic response to radiation therapy or radiochemotherapy.

Additive effects of cisplatin and radiation in human tumor cells under oxic conditions

The interaction of cisplatin and irradiation was studied in vitro in four human cell lines. Additive effects were observed for the combination given either simultaneously or sequentially. No influence on recovery was seen in split-dose experiments. It is concluded that radiosensitization cannot be presumed in every clinical setting of combined treatment with radiation and cisplatin.

Measurement of Hypoxia Using the Comet Assay Correlates with Preirradiation Microelectrode pO2 Histography in R3327-AT Rodent Tumors

Abstract Sauer, G., Weber, K. J., Peschke, P. and Eble, M. Measurement of Hypoxia Using the Comet Assay Correlates with Preirradiation Microelectrode pO2 Histography in R3327-AT Rodent Tumors. Polarographic determination of tumor oxygenation by Eppendorf histography is currently under investigation as a possible predictor of radiotherapy outcome. Alternatively, the alkaline comet assay has been proposed as a radiobiological approach for the detection of hypoxia in clinical tumor samples. Direct comparisons of these methods are scarce. One earlier study with different murine tumors could not establish a correlation, whereas a weak correlation was reported for a variety of human tumors. Considering the different end points and spatial resolution of the two methods, a direct comparison for a single tumor entity appeared desirable. Anaplastic R3327-AT Dunning prostate tumors were grown on Copenhagen rats to volumes of 1–6 cm3. Eppendorf histography (100–200 readings in 5 parallel tracks) for 8 different tumors revealed various degrees of oxygenation, with median pO2 values ranging from 1.1 to 23 mmHg. Within 5 min after an acute exposure to 8 Gy 60Co γ rays, tumors were excised from killed animals and rapidly cooled to limit repair, and a single cell suspension was prepared for use with the comet assay. The resulting comet moment distributions did not exhibit two subpopulations (one hypoxic and the other aerobic), and a hypoxic fraction could not be calculated. Instead, the average comet moment distribution was taken as a parameter of overall strand break induction. Corresponding experiments with tumor cells grown in vitro allowed us to derive the relationship between the oxygen enhancement ratio (OER) for the average comet moment and oxygen partial pressure (Howard-Flanders and Alper formula). The validity of this relationship was inferred for cells exposed in situ, and the convolution of a pO2 distribution with the formula of Howard-Flanders and Alper yielded an array of expected OER values for each tumor. The average expected OER correlated well with the average comet moment (r = 0.89, P < 0.01), and the in situ comet moment distributions could be predicted from the Eppendorf data when 50% repair was taken into account, assuming a 5-min damage half-life. The findings confirm the potential of interstitial polarography to reflect radiobiologically relevant intracellular oxygenation, but also underscore the confounding influence of differences in repair that may occur when cells are prepared from irradiated tissues for use with the comet assay.

A Modified Computer-assisted Colorimetric Microtitre Assay (MTT) to Assess in Vitro Radiosensitivity of V79, CaSki, HeLa and WiDr Cells

The non-clonogenic MTT assay, based on the reduction of a tetrazolium salt to a purple formazan precipitate by living cells, was modified and a new procedure of analysis is proposed. The colorimetric assay could be performed semi-automatically using microtitre plate reader connected to a personal computer. Data are processed and plotted with a customized program. To ensure that both control and irradiated microtitre plates contain exponentially growing cells at the time of analysis, the calculation of relative survival is based on a series of well-defined cell numbers initially seeded. To make the two endpoints of non-clonogenic and clonogenic assays comparable, i.e. counting of living cells versus counting of colonies, radiation-induced progression delay was incorporated into the calculation. Radiation-induced cell killing and progression delay could be determined in a single analysis, but in an independent way. X-ray survival curves were generated for V79, CaSki, WiDr and HeLa cells using the non-clonogenic and a standard clonogenic assay. Using the linear-quadratic formula, the resulting parameters alpha, beta and the mean inactivation dose were not significantly different. The described assay is a feasible and reproducible technique for determination of cellular survival, which may be able to incorporate progression delay. The equivalence to a clonogenic survival assay could be proven.

Rise of oxygenation in cervical lymph node metastasis during the initial course of radiochemotherapy

It has been hypothesized that during radiation treatment a reoxygenation of hypoxic tumor tissue takes place. To test this hypothesis, we have investigated whether reoxygenation in lymph node metastases could be determined by invasive Po2 measurements. Through a hypodermic needle inserted transcutaneously into tumor-Positive lymph nodes, Polarographic oxygen determinations were made in 18 patients with advanced squamous cell carcinomas of the oropharynx and hypopharynx. These measurements were performed before therapy and a week after the onset of radiotherapy or radiochemotherapy, respectively. Low Po2 values before treatment (mean value of the patients median was 12.6 mm Hg Po2) and a mean hypoxic fraction (Po2 < 5 mm Hg) of 39.6% indicated manifest tumor hypoxia. After 1 week of treatment, a significant increase in the median PO2 (mean value of shift: 7.3 mm Hg) and a reduction in the hypoxic fraction (mean value of shift: 13.4% Po2 < 5 mm Hg, P < 0.03) were observed after both radiotherapy and radiochemotherapy. Thus invasive PO2 histography fulfills the requirements for a method to confirm tumor hypoxia in head and neck tumors. The results obtained indicate that reoxygenation occurs during the initial phases of radiotherapy and radiochemotherapy, and they will form the basis for future comparative investigations on the Possible influence of hypoxic parameters on tumor responsiveness toward radiation and radiochemotherapy.