Patterns and Levels of Hypoxia in Head and Neck Squamous Cell Carcinomas and Their Relationship to Patient Outcome

Purpose

EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels, and prognostic significance of hypoxia in primary head and neck squamous cell tumors.

Methods and Materials

Twenty-two patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analyses of EF5 immunofluorescence was carried out, and these data were compared with patient outcome.

Results

EF5 immunostaining showed substantial intra- and intertumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria based on the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5-binding levels with event-free and overall survival irrespective of the pattern of cellular binding or treatment regimen. Patients with tumors containing EF5-binding regions corresponding to severe hypoxia (≤0.1% oxygen) had a shorter event-free survival time than patients with pO₂ values greater than 0.1% (p = 0.032). Nodal status was also predictive for outcome.

Conclusions

These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO₂ and provide support for the development of noninvasive hypoxia positron emission tomographic studies with fluorine 18–labeled EF5.

Introduction

Squamous cell carcinoma of the head and neck (HNSCC) is a common and debilitating disease, with 31,000 new cases and 7,500 deaths in the United States in 2006 (1). Treatment with curative intent involves the combination of surgery, radiation therapy, and/or chemotherapy. Nonetheless, these cancers are biologically aggressive, with 80% of recurrences developing within 2 years (2). More aggressive combined-modality treatment has resulted in 5-year survival rates as high as 40–60% 3, 4, 5, 6, 7. Because of the very poor prognosis for patients with recurrent disease and the morbidity of subsequent aggressive therapies, it is critical to identify patients early in their treatment regimens who might benefit from such therapy. This would also spare patients with responsive tumors from the toxicity of intensive treatment. The key to initiating this paradigm is to identify resistance factors in individual patient tumors. Adjuvant therapy that modulates hypoxia, for example, could then be prescribed. Studies have shown that in addition to causing radiation resistance 8, 9, hypoxic tumors are more resistant to chemotherapy (10) and more susceptible to genetic instability (11). The latter contributes to biologically aggressive tumors with increased invasive and metastatic potential. Hypoxia is highly heterogeneous between HNSCC tumors and is not measurable using such standard characteristics as tumor grade or size. To individualize treatment, tumor pO₂ must be measured in individual patient tumors. Several clinical approaches were used to accomplish this (for review, see [12]). Two clinically relevant “invasive” method that have been used are needle electrode techniques and 2-nitroimidazole–binding studies, but the former can seldom be used in primary tumors of the head and neck. For the latter, 2-nitroimidazole adduct formation in hypoxic cells can be assayed using immunohistochemistry (IHC), flow cytometry, and/or positron emission tomographic (PET) imaging (for example, 13, 14, 15, 16, 17).

An early study of in vivo hypoxia was performed using polarographic needle electrodes in enlarged squamous cell carcinoma–containing neck lymph nodes (18). This work showed that hypoxia could be measured in a minimally invasive manner and the resulting values correlated with short-term treatment response (19). Subsequent studies using the Eppendorf pO₂ Histograph (Eppendorf, Hamburg, Germany) indicated that hypoxia was a predictive factor for patient outcome in HNSCC (20), as well as cervical cancers (21), soft tissue sarcomas (22), and prostate tumors (7). The majority of electrode measurements in head-and-neck sites were in enlarged lymph nodes, rather than primary tumors, because of the difficulty accessing many tumors and/or making measurements safely near bone or large vessels. For patients with tumors in difficult sites who are going to surgery, a 2-nitroimidazole hypoxia-measuring agent can be administered. These drugs bind to cells at a rate inversely proportional to cellular pO₂. For IHC analyses, surgical biopsies made 12–48 hours after drug administration can be stained for adducts that form intracellularly.

We studied binding of the 2-nitroimidazole agent EF5 in several human tumor sites to improve our understanding of in vivo hypoxia 23, 24, 25, 26. Using this detection system, patterns of hypoxia can be described and the pO₂ in individual cells and/or tumor regions can be quantified. This method also allows examination of the spatial relationship between hypoxia and blood vessels, proliferating cells, apoptotic cells (27), and other endpoints. We have previously reported that EF5 binding correlated with the level of tumor aggressiveness in supratentorial glial neoplasms (25) and with outcome in patients with extremity soft tissue sarcoma (24).

In the present study, we investigated EF5 binding in previously untreated HNSCC tumors of the oral cavity, oropharynx, and larynx. After excisional surgery, patients received varying treatments based on the physician's recommendation and patient's preferences. Despite the heterogeneity of this population, high EF5 binding was significantly associated with poor outcome. Thus, we established a proof of principle for the use of this and/or noninvasive EF5-based hypoxia imaging assays to identify patients who require aggressive therapy. More extensive studies of the additional impact of patient demographics and treatment regimen are planned, as are noninvasive studies using fluorine 18–labeled EF5 (¹⁸F-EF5) PET imaging.