Comparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients

doi:10.1016/S0360-3016(02)04503-0

International Journal of Radiation OncologyBiologyPhysics

Volume 56, Issue 2, 1 June 2003, Pages 375-383

Presented at the Radiation Research Annual Meeting, Reno, NV, April 2002.

https://doi.org/10.1016/S0360-3016(02)04503-0 Get rights and content

Abstract

Purpose

To compare the Eppendorf Po₂ histograph and the alkaline comet assay as methods of measuring tumor hypoxia in patients with head-and-neck squamous cell carcinomas.

Materials and methods

As part of a larger clinical trial, 65 patients with head-and-neck squamous cell carcinoma nodal metastasis underwent tumor oxygenation measurements with Eppendorf Po₂ histographs and comet assays, performed on fine-needle aspirates at 1 and 2 min after 5 Gy. Fifty-four patients had sufficient tumor cells for comet analysis at 1 min and 26 at both 1 and 2 min. Individual cells were examined for DNA single-strand breaks by alkaline gel electrophoresis, and the distribution of values was quantified using median tail moment (MTM). Nonirradiated tumor cells from pretreatment fine-needle aspirates received 5 Gy in vitro to establish the oxygenated response.

Results

There was a significant correlation between the 1- and 2-min MTM (slope = 0.77 ± 0.03). There was no relationship between DNA damage in tumor cells irradiated in vitro and in vivo. No correlation was found between Eppendorf Po₂ measurements and comet MTM. There was a statistically significant correlation between the treatment response in the node studied and comet MTMs, whereas no correlation was observed between treatment response and Eppendorf measurements.

Conclusion

Comet assays are reproducible, as shown by biopsies at 1 and 2 min. Intertumor variation in the MTM is not a result of intrinsic radiosensitivity but of tumor hypoxia. There was no correlation between Eppendorf Po₂ measurements and comet MTM. Comet assays were better than Eppendorf in predicting treatment response as an end point for short-term outcome. Longer follow-up is needed to determine the role of the comet assay as a predictor for locoregional tumor control and survivals.

Introduction

Tumor hypoxia has been shown to promote tumor progression through the selection of tumor cells with diminished apoptotic potential, to stimulate pro-angiogenic gene expression, and to increase metastatic potential (1). Past studies have demonstrated a strong correlation between pretreatment tumor Po₂, tumor control, and survival in patients with head-and-neck squamous cell carcinoma (HNSCC) 2, 3, 4. Studies have also indicated that hypoxia increases tumor invasiveness and dissemination in human solid tumors 5, 6, 7.

Several methods have been used to assess tumor hypoxia and to provide prognostic information for solid tumors 2, 7, 8, 9. One of the most commonly used methods is the Eppendorf Po₂ histograph, which uses a polarographic oxygen sensor to determine tumor Po₂. Numerous groups have used this instrument to make Po₂ measurements in solid tumors in patients and in xenograft models 2, 3, 10, 11, 12, 13. Early results from clinical studies suggested that this method could be used to identify hypoxic tumors and, more importantly, to predict treatment outcomes in patients treated with radiotherapy 2, 3, 4.

The comet assay, also known as single-cell gel electrophoresis assay, was first applied to the study of hypoxia in murine tumors by Olive and Durand (14). In this assay, tumors are exposed to 5–15 Gy of irradiation, and the cells are aspirated using a fine needle (≥22 gauge), lysed in an alkaline solution, subjected to electrophoresis, and viewed under a microscope. DNA broken by irradiation travels further down the gel, producing a “comet”-shaped image with a “tail” that lengthens in direct proportion to the number of DNA breaks. The basis for measuring the proportion of hypoxic cells is that ionizing radiation produces approximately 3 times fewer DNA single-strand breaks (SSBs) in anaerobic than in aerobic cells 14, 15. It is therefore possible to use the amount of DNA damage produced to estimate the degree of hypoxia at the time of irradiation.

Although both methods are advocated for measuring tumor hypoxia, the relationship between the comet assay and Po₂ microelectrode measurements is unclear. Kavanagh et al. found no correlation between the Eppendorf Po₂ histograph measurements and those of the alkaline comet assay in murine tumor models (12). However, Aquino-Parsons et al. noted a weak but significant correlation in the hypoxic fractions derived from the two approaches in a group of patients with advanced tumors (11). Specifically, both methods were able to identify patients with the most hypoxic tumors in the study. However, the type of tumors evaluated was heterogeneous; most tumors were advanced or metastatic, and there was no correlation with treatment outcomes. The present study was therefore undertaken to compare these two methods of detecting tumor hypoxia in a group of patients with a uniform diagnosis of HNSCC and to determine the ability of each approach to predict for treatment response in these patients.

Section snippets

Patient protocol

Between 7/1996 and 7/2001, 65 patients with newly diagnosed node-positive Stage III–IV HNSCC were enrolled in an NCI-sponsored protocol (Trial NCI-T94–01190). All patients signed an institutional review board–approved informed consent for all procedures and treatments in the study. Patients with Stage III tumors were randomized to radiation alone or radiation with tirapazamine (TPZ), whereas those with Stage IV tumors were randomized to cisplatin-based induction chemotherapy followed by

Patient characteristics and Eppendorf Po₂ results

Table 1 shows detailed characteristics of the patients in this study. Three had Stage III, and 62 had Stage IV tumors. The median age was 58 years (range: 39–80 years), and 8/65 were female. One patient with a Stage III tumor had RT alone, and 2 had RT and TPZ; 29 patients with Stage IV tumors had chemoradiation alone, and 33 received TPZ in addition to chemoradiation.

The median number of total and acceptable oxygen readings obtained in the tumor was 88 (range: 34–180) and 78 (range: 43–153),

Discussion

To qualify for the study, patients needed to have documented tumor metastatic to the largest palpable neck node by a fine-needle aspiration. This node was then used as the index node for oxygen measurement by both the Eppendorf Po₂ histograph and the comet assay. Except for lymph node <2 cm, which required CT guidance for Eppendorf Po₂ measurement, all tumors were felt to be clinically accessible by either the electrode or a fine needle without image guidance. Although the number of Po₂

References (26)

D.M Brizel et al.
Oxygenation of head and neck cancerChanges during radiotherapy and impact on treatment outcome
Radiother Oncol
(1999)
V Rudat et al.
Repeatability and prognostic impact of the pretreatment pO(2) histography in patients with advanced head and neck cancer
Radiother Oncol
(2000)
M Nordsmark et al.
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)
A.W Fyles et al.
Oxygenation predicts radiation response and survival in patients with cervix cancer
Radiother Oncol
(1998)
C Aquino-Parsons et al.
Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia
Radiother Oncol
(1999)
M.C Kavanagh et al.
A comparison in individual murine tumors of techniques for measuring oxygen levels
Int J Radiat Oncol Biol Phys
(1999)
M.J Dorie et al.
DNA damage measured by the comet assay in head and neck cancer patients treated with tirapazamine
Neoplasia
(1999)
M Nordsmark et al.
Pretreatment oxygenation predicts radiation response in advanced squamous cell carcinoma of the head and neck
Radiother Oncol
(1996)
M Nordsmark et al.
Invasive oxygen measurements and pimonidazole labeling in human cervix carcinoma
Int J Radiat Oncol Biol Phys
(2001)
W.T Jenkins et al.
Hypoxia and necrosis in rat 9L glioma and Morris 7777 hepatoma tumorsComparative measurements using EF5 binding and the Eppendorf needle electrode
Int J Radiat Oncol Biol Phys
(2000)

J.M Brown et al.

The unique physiology of solid tumorsOpportunities (and problems) for cancer therapy

Cancer Res

(1998)

D.M Brizel et al.

Tumor oxygenation predicts for likelihood of distant metastasis in human soft tissue sarcoma

Cancer Res

(1996)

M Hockel et al.

Hypoxic cervical cancers with low apoptotic index are highly aggressive

Cancer Res

(1999)

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^☆: Supported by the American Society of Therapeutic Radiology Young Investigator Grant (Q.T.L.) and PHS Grant CA 67166, awarded by the National Cancer Institute, DHHS (Q.T.L., J.M.B.)

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Clinical investigation: head and neckComparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients☆

Abstract

Purpose

Materials and methods

Results

Conclusion

Introduction

Section snippets

Patient protocol

Patient characteristics and Eppendorf Po2 results

Discussion

Radiother Oncol

Radiother Oncol

Radiother Oncol

Radiother Oncol

Radiother Oncol

Int J Radiat Oncol Biol Phys

Neoplasia

Radiother Oncol

Int J Radiat Oncol Biol Phys

Int J Radiat Oncol Biol Phys

The unique physiology of solid tumorsOpportunities (and problems) for cancer therapy

Cancer Res

Tumor oxygenation predicts for likelihood of distant metastasis in human soft tissue sarcoma

Cancer Res

Hypoxic cervical cancers with low apoptotic index are highly aggressive

Cancer Res

Clinical investigation: head and neck
Comparison of the comet assay and the oxygen microelectrode for measuring tumor oxygenation in head-and-neck cancer patients☆

Patient characteristics and Eppendorf Po₂ results