Hypoxia, a common feature of the microenvironment in solid tumors, is associated with resistance to radiotherapy, reduced therapeutic response, and a poorer clinical outcome. In head and neck squamous cell carcinomas (HNSCC), the negative effect of hypoxia on radiotherapy can be counteracted via addition of hypoxic modification to the radiotherapy. To predict which patients harbor hypoxic tumors and would therefore benefit from hypoxic modification, clinically applicable methods for pretherapeutic hypoxic evaluation and categorization are needed. In this study, we developed a hypoxia classifier based on gene expression. Through study of xenograft tumors from human squamous cell carcinoma cell lines, we verified the in vivo relevance of previously identified in vitro derived hypoxia-induced genes. We then evaluated a training set of 58 hypoxia-evaluated HNSCCs to generate a gene expression classifier containing 15 genes. This 15-gene hypoxia classifier was validated in 323 patients with HNSCC randomized for hypoxic modification or placebo in combination with radiotherapy. Tumors categorized as hypoxic on the basis of the classifier were associated with a significantly poorer clinical outcome than nonhypoxic tumors. This outcome was improved and equalized to the nonhypoxic tumors by addition of hypoxic modification. Thus, findings show that the classifier attained both prognostic and predictive impact, and its pretherapeutic use may provide a method to identify those patients who will benefit from hypoxic modification of radiotherapy. Cancer Res; 71(17); 5923-31. Ó2011 AACR.

This study emphasizes the role of FAZA PET/CT imaging as a suitable assay with prognostic potential for detection of hypoxia in HNSCC.

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The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O(2)), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.

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