Development of a Hypoxia Gene Expression Classifier with Predictive Impact for Hypoxic Modification of Radiotherapy in Head and Neck Cancer

Abstract

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.

Low Cancer Stem Cell Marker Expression and Low Hypoxia Identify Good Prognosis Subgroups in HPV(−) HNSCC after Postoperative Radiochemotherapy: A Multicenter Study of the DKTK-ROG

“…To date, the potential of hypoxia gene signatures to discriminate pretreatment tumor specimen regarding their hypoxiainduced gene expression levels has not been tested in the postoperative setting (15,16,18). However, there is some evidence from an experimental study of our laboratory, which showed that pretreatment hypoxia impacts local tumor control after radiotherapy also when radiotherapy was applied under homogenous anoxic conditions (20).…”

“…Samples were then kept at 4 C for a maximum of 18 hours and subjected to the nCounter system and processed as described elsewhere (27). Raw counts were logarithmized and then normalized to the mean of the internal level of reference genes ACTR3, NDFIP1, RPL37A, B2M, GNB2L1, RPL11, POLR2A or to the reference genes of the corresponding hypoxia gene signatures (Supplementary Table S1B), respectively (15,16). cDNA generation and RT-PCR RNA was converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Life Technologies).…”

“…Because of low RNA yield, each cDNA was preamplified for genes of interest using the pooled TaqMan gene expression assays according to the manufacturer's protocol (10 cycles). TaqMan gene expression assays were used for both hypoxia classifier and reference genes (15,16) as well as for CD44. RT-PCR was performed on an ABI StepOne Plus Real-Time PCR System (Life Technologies).…”

“…In this study, tumor hypoxia was assessed using the 15-gene signature developed by Toustrup and colleagues (15) and the 26-gene signature by Eustace and colleagues (16) in parallel. Using nanoString analyses, univariate analysis showed that hypoxic tumors are significantly associated with a high risk of locoregional tumor recurrence after PORT-C using the 15-gene signature (HR, 3.55; P ¼ 0.010) and the 26-gene signature (HR, 9.37; P ¼ 0.002; Fig.…”

“…Several gene classifiers have been developed as a measure of tumor hypoxia in biopsies routinely taken for diagnosis (15)(16)(17)(18). Toustrup and colleagues developed a 15-gene classifier for patients with HNSCC who received primary radiotherapy (15).…”