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Originally published as JCO Early Release 10.1200/JCO.2005.02.7474 on January 17 2006 © 2006 American Society of Clinical Oncology. Endogenous Markers of Two Separate Hypoxia Response Pathways (hypoxia inducible factor 2 alpha and carbonic anhydrase 9) Are Associated With Radiotherapy Failure in Head and Neck Cancer Patients Recruited in the CHART Randomized TrialFrom the Departments of Radiotherapy/Oncology and Pathology, Democritus University of Thrace, Alexandroupolis, Greece; Gray Cancer Institute and the Cancer Centre, Mount Vernon Hospital; University College London; Cancer Research UK Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom; and the Barbara Ann Karmanos Cancer Institute, Detroit, MI Address reprint requests to Adrian L. Harris, MD, Cancer Research UK, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom, OX3 7LJ; e-mail: aharris.lab{at}cancer.org.uk
PURPOSE: Randomized controlled trials have generally shown a benefit from accelerated radiotherapy in head and neck squamous cell carcinoma (HNSCC). However, the large randomized United Kingdom trial CHART (Continuous Hyperfractionated Accelerated Radiotherapy) failed to show a benefit of strongly accelerated over standard radiotherapy (RT) in 918 patients with HNSCC. In this study, we investigated the impact of tumor hypoxia on the outcome of HNSCC patients in the CHART trial. There are two distinct hypoxia inducible factors (HIFs) that control different gene response pathways and we assessed them both with endogenous markers of hypoxia, hypoxia inducible factor HIF-2 alpha (HIF-2) and carbonic anhydrase CA9, an indicator of HIF-1 alpha (HIF-1) function. METHODS: Tissue from pre-RT biopsies performed in 198 of 918 patients recruited was analyzed for the immunohistochemical expression of HIF-2 and CA9. RESULTS: A significant association of high HIF2 and of high CA9 reactivity with poor locoregional control (P < .0001 and P = .0002, respectively) and poor survival (P = .0004 and 0.002, respectively) was noted. In multivariate analysis, HIF-2 and CA9 maintained their independent prognostic significance. Coexpression of both pathways had an additive effect, supporting their independent role. The uni-directional hypothesis, that a benefit from randomization to CHART should be seen in the nonhypoxic tumors, was supported by the data (one-tailed P = .04). CONCLUSION: Expression of endogenous markers of hypoxia for the HIF-1 and HIF-2 pathway is strongly associated with radiotherapy failure. Using immunohistochemical methods it is possible to identify subgroups of HNSCC patients who are highly curable with radiotherapy, or who are excellent candidates for clinical trials on hypoxia-targeting drugs in two distinct pathways.
Radiotherapy is an important curative treatment modality for head and neck squamous cell carcinomas (HNSCC), with cure rates exceeding 80% in early stages of the disease. However, in locally advanced tumors, locoregional control remains unsatisfactory, with 3-year cure rates rarely exceeding 50% to 60%.1 Because protraction of overall treatment time reduces the effectiveness of radiotherapy,2 it has been suggested that accelerated radiotherapy, defined as radiotherapy schedules employing a dose-intensity exceeding 10 Gy per week,3 may significantly improve tumor control in HNSCC, provided that the total dose is large enough. During the last two decades, a number of randomized controlled trials have provided evidence supporting this hypothesis,4,5,6 and variety of such accelerated radiotherapy schedules have been devised and been tested in phase III clinical trials. Perhaps the most biologically informative of these has been the CHART (Continuous Hyperfractionated Accelerated Radiotherapy) head and neck trial.7 There are several reasons for this. First, CHART delivered a total dose of 54 Gy in 36 fractions of 1.5 Gy administered three times daily over 12 consecutive days. This is the strongest accelerated schedule tried in a phase III study with a rate of dose accumulation exceeding 30 Gy per week. This is the only schedule tested so far that completed radiotherapy in less than 3 weeks. Second, it is one of the largest trials, randomly assigning 918 patients to CHART versus conventional fractionated radiotherapy, 66 Gy in 33 fractions over 6 to 7 weeks. The trial showed no significant difference in locoregional tumor control or in survival between the two trial arms. This contrasts the expected gain from CHART if the benefit of acceleration after 6 to 7 weeks of radiotherapy is back-extrapolated down to a 12-day schedule.8 Two main hypotheses have been put forward to explain the outcome of CHART. One is that the putative biologic trigger for accelerated tumor repopulation (whether this is linked to an accelerated proliferation or decreased apoptosis rate) is activated only some 3 to 4 weeks after the start of radiotherapy. The other hypothesis is related to the well-recognized role of hypoxia in failure of radiotherapy.9-12 The CHART schedule may be too short to allow maximum tumor cell reoxygenation to take place before most of the dose is delivered. Tumors able to undergo reoxygenation during standard fractionation may not do so during CHART, and this could partly offset a gain from accelerated radiation.13 The two hypotheses might be linked; animal experiments on a xenograft tumors supported the idea that reoxygenation may be the trigger for accelerated tumor cell repopulation.14 The motivation for this study was, therefore, to establish diagnostic tests able to predict the causes of therapeutic failure, which would in future allow the individualization of radiosensitization policies with specific hypoxia targeting or antiangiogenic agents. A major effect of hypoxia, apart from its adverse effect on oxygen potentiation of radiation, is the induction of gene expression via hypoxia inducible factor 1 alpha (HIF-1), a transcription factor stabilized under hypoxia that induces the angiogenic factor vascular endothelial growth factor (VEGF). Many other pathways are induced, including glycolysis, invasion, and growth factors. HIF-1 expression has been shown to predict poor prognosis after radiation in several tumor types.15-17 A homologous member of the family, HIF-2 alpha (HIF-2) is regulated in a similar way, and in transfection experiments can regulate similar genes, but recently was shown to be tethered in the cytoplasm in embryonic fibroblasts.18 One pathway regulating pH is carbonic anhydrase 9 (CA9),19 a hypoxia inducible transmembrane enzyme, which has been shown to correlate with direct measurement of oxygen tension in cervical cancer.20 We have shown by RNA interference that the two factors regulate different pathways and the hypoxia inducible CA9 is regulated by HIF-1, not HIF-2.21 Hu et al22 also showed that HIF-1 and HIF-2 regulate different pathways, with HIF-1, but not HIF-2, regulating glycolysis. In this study, we wanted to assess whether either pathway was related to outcome and whether activation of both pathways (HIF-2 and CA9) had additional impact. Thus we investigated the impact of tumor hypoxia, as assessed with endogenous markers of two pathways of gene regulation by hypoxia, on the outcome of accelerated and of standard radiotherapy for head and neck cancer.
Trial From 1990 to 1995, 918 patients with HNSCC (laryngeal, pharyngeal, nasopharyngeal and oral cavity tumors) were randomly assigned between CHART and conventional fractionation. The allocation ratio was 3:2 in favor of CHART.7 Radiotherapy was administered with radical intent without surgery or concurrent chemotherapy. To investigate molecular features that define the differential response of tumors to conventional versus accelerated radiotherapy, in September 1995, the CHART Steering Committee consulted and granted permission for the retrospective collection of histologic material from patients entered onto the trial for the purpose of immunohistochemical analysis of factors that may influence the outcome of either treatment. The histologic material was anonymized in the laboratory by trial number, and the clinical data were held in a separate database controlled by a biostatistician (S.M.B.). Presence of tumor was verified in each specimen, of which 198 were available. Histologic staining and assessment were carried out without knowledge of patient information, and statistical analysis was performed without involvement in the histologic study. Out of these 198 patients, 116 were treated with CHART (1.5 Gy x 3 per day, for 12 days: 54 Gy total dose) and 82 with conventional radiotherapy (2 Gy per day, 33 fractions: 66 Gy total dose). Table 1 shows the distribution of patient and disease characteristics in the analysis group and in all randomly assigned patients. There was no significant difference between the distribution of clinical factors in the patients who were included in the analysis group and in those who were not. The only exception was histologic differentiation (P = .002); the proportion of tumors without specified differentiation was lower in the analysis group than in all trial patients. This appears to reflect a higher proportion of histopathologic grading performed in the departments contributing material to the biologic study. Thus, the analysis group appears to be clinically representative of the population in the trial.
Assessment of Hypoxia-Regulated Proteins HIF-2 protein was detected with the antibody EP190b (immunoglobulin G1 [IgG1] monoclonal antibody [MAb]; University of Oxford, United Kingdom),23 and CA9 with M75 (provided by J. Pastorek, MD, University of Bratislava, Bratislava, Slovak Republic).19 A modified streptavidin technique was used for immunohistochemistry, as previously reported.15,24 Briefly, microwaving for antigen retrieval was used (three 5-minute exposures). The EP190b antibody was applied overnight, and a 90-minute incubation was performed with M75. After washing with triethanolamine-buffered saline (TBS), sections were incubated with a secondary antibody (Kwik Kit, Cat. No. 404050; Thermo Shandon, Pittsburgh, PA) for 15 minutes and washed in TBS. Kwik Streptavidin peroxidase reagent was applied for 15 minutes and sections were again washed in TBS. The color was developed by 15-minute incubation with diaminobenzidine solution and sections were weakly counterstained with hematoxylin. All optical fields were examined (three to seven fields from each patient at x200 magnification). The percentage of cancer cells with strong cytoplasmic HIF-2 expression and with nuclear HIF-2 expression were assessed separately in all optical fields. Assessment of CA9 was based on the membrane staining noted on cancer cells. The mean value of the percentages obtained from the optical fields examined per patient was used to obtain the scores for each one of the patients. Assessment was performed by two independent observers and results were assessed for interobserver variability. Disagreement was resolved using the conference microscope. We used a previously reported HIF scoring system to divide samples in two groups of low and high HIF-2 reactivity.15 Strong cytoplasmic expression in more than 50% of cancer cells or nuclear expression in more than 10% of cancer cells was considered positive. Although one may assume that nuclear HIF is the active form, clearly it is synthesized in the cytoplasm and also degraded in the cytoplasm. There may be redistribution while collecting tissues, which would be difficult to control but the overall expression indicates upregulation of the pathway selectively in cancer. In previous studies, we showed that analysis based on pure nuclear expression provides marginal statistical association with other molecular factors or prognosis, and that strong cytoplasmic HIF expression, which is a tumor-specific finding, better reflects the HIF-upregulated pathway in paraffin-embedded material.15,25-27 This suggestion is in accordance with the scoring system proposed by Zhong et al.28 This scoring system was established from our group and validated in several human carcinoma studies providing significant correlations with histopathologic and molecular tumor variables, as well as prognosis of patients.15,25-27 We specifically chose to use the independently derived divisions based on previous studies to avoid data-specific selection of cut points.
In a previous study in head and neck carcinomas treated with radiotherapy, we proposed that tissue samples with at least 10% of cancer cells reactive for CA9 should be considered positive for CA9.24 The same cutoff point (< 10% v All immunohistochemical studies were conducted at the department of Pathology, Democritus University of Thrace (Komotini, Greece), and staining was assessed independently by two observers (A.G. and E.S.), without knowledge of clinical outcome. Discrepancies were resolved using the conference microscope.
Statistical Analysis
Definition of Cutoff Points and Groups of Patients The median HIF-2 nuclear reactivity was 0% (range, 0 to 100%), whereas the median percentage of cells with strong cytoplasmic and/or nuclear reactivity was 20% (range, 0 to 100%). Eighty-two cases were negative for both cytoplasmic and nuclear staining, whereas 41 cases were positive for both. Twenty-three cases were positive for cytoplasmic staining only, and 52 for nuclear only. One hundred sixteen patients (58%; 95% CI, 52% to 65%) had high HIF-2 reactivity (scored as HIF-2 positive) and 82 (41.5%) negative or low (scored as HIF-2 negative).
The median percentage of cells with membrane CA9 reactivity was 10% (range, 0 to 100%), which coincided with the percentage prospectively proposed for grouping cases. Using this cutoff point 115 patients (58%; 95% CI, 51% to 65%) had positive (
The interobserver variability was minimal (P < .0001, r2 = 0.91 for nuclear HIF-2 expression; P < .0001, r2 = 0.95 for cytoplasmic HIF-2 expression; P < .0001, r2 = 0.92 for membrane CA9 expression).
Relationship Between HIF-2 and CA9 Status
However, 75 tumors (38%; 95% CI, 31% to 45%) had discordant HIF-2/CA9 status. The proportion of CA9-positive tumors among the HIF-2 negative tumors was 45% (95% CI, 34% to 57%), and the proportion of CA9 negative tumors among the HIF-2 positive tumors was 33% (95% CI, 24% to 41%).
Stage of Disease at Presentation and HIF-2/CA9 Status
Correlation With Outcome
Time to Distant Metastasis Twenty-eight patients developed distant metastasis during follow-up. The only covariate associated with an increased risk of distant metastasis in a Cox proportional hazards model was N stage (P = .03; data not shown). CA9 and HIF-2 status had no statistically significant association with the risk of distant metastasis but the statistical power is low as a result of the low number of events. Among patients without CA9 or HIF-2 expression, the 5-year distant metastasis rate was 9% ± 5% (plus or minus one SE of the estimate), whereas among patients who expressed one or both of the markers, the 5-year metastasis rate was 20% ± 4%. Thus, there was moderate support for the uni-directional hypothesis that CA9 or HIF-2 expression is associated with an increased risk of developing distant metastasis (one-tailed P = .06).
Multivariate Analysis
Interaction With CHART Radiotherapy One of the prior hypotheses in this study was that a benefit from CHART relative to conventional fractionation would be seen in patients with nonhypoxic tumors. Figure 4 shows the hazard ratios for locoregional relapse after conventional relative to CHART radiotherapy. Again, the statistical power was relatively low after stratification for hypoxic marker expression, as indicated by the 90% confidence limits on the estimated hazard ratios. The point estimate showed a greater effect of CHART in patients without expression of either of the hypoxic markers. Again, there was some support for the unidirectional hypothesis that the nonhypoxic tumors would show a benefit from CHART (one-tailed P = .04). The test for interaction was borderline significant (one-tailed P = .06).
This clinicopathologic study is based on tissue collected from patients recruited in the CHART trial for HNSCC. The tissue sample analyzed concerned 21.6% of the total number of patients recruited in the CHART trial. This sample, however, is considered representative of the treated population because this was not selected and because it comprised all assessable tissue blocks retrieved from the various hospitals where diagnosis had been performed. The intratumoral hypoxia was assessed by two endogenous markers of hypoxia (ie, HIF-2 and CA9). Not only are the HIFs key transcription factors regulating the expression of VEGF and proteins involved in the anaerobic metabolism, but they are also potential therapy targets.29,30 In previous uncontrolled studies in locally advanced HNSCC, we noted a strong association of HIF-2, HIF-1, and CA9 expression with poor response to radiotherapy and short relapse-free survival.15,24 A recent study in nasopharyngeal cancer showed similar results,16 whereas HIF-1 has been shown to adversely affect the efficacy of radiotherapy for oropharyngeal carcinomas.17 Recent evidence shows that HIF-1 and HIF-2 regulate different pathways,21,22 so in this analysis both were assessed for their association with prognosis. The strong association of HIF-2 and CA9 with poor locoregional control of patients with HNSCC treated with radical radiotherapy was confirmed. Although there was an association of expression of both pathways, expression of either marker alone was associated with similar poor outcome, although they regulate different pathways in vitro. The relapse-free and overall survival curves show some differences in that they split much earlier when stratified by HIF-2 versus CA9. It is possible that this reflects different functions (eg, the role of HIF-1 in glycolysis and apoptosis regulation), resulting in tumors more dependent on glycolysis and with higher apoptotic rates, versus enhanced free-radical stress response pathways in HIF-2 positive cases,21,22 although this is speculative and will need further study. Both markers did predict for local and distant relapse, although HIF-2 was slightly more powerful for distant relapse. The combined expression of both pathways conferred additive poor prognosis, again suggesting different pathways may be regulated. It is of interest that hypoxia as assessed by these markers predicted for poor outcome for either schedule of radiotherapy. Although acceleration of radiotherapy was expected to show a dramatic benefit, the CHART trial failed to substantiate a clear benefit in HNSCC patients.7 It seems that biologic factors other than rapid tumor repopulation are at least as equally important in outcome. Tumor hypoxia could underlie results of CHART in HNSCC; although the exact time point of the onset of reoxygenation during fractionated radiotherapy is unknown, presumably depending on the individual tumor response to radiation, reoxygenation has been demonstrated after the administration of several radiotherapy fractions (from 1 up to 3 weeks after the onset of radiotherapy).31-34 The degree of reoxygenation expected during the 12 days of CHART may not, however, be sufficient for restoration of cancer cell radiosensitivity. Furtheremore, small doses per fraction used in CHART or even in standard radiotherapy (1.5 to 2 Gy) may be ineffective to overcome radioresistance conferred by hypoxia, because larger radiotherapy fractions are required to exceed the shallow shoulder of the dose-response curves expected under hypoxic conditions. Further subgroup analysis was therefore carried out to explore if CHART may in fact be effective in nonhypoxic tumors. The results show that there is a significant effect in favor of CHART in this subgroup. This was the only group with a significant difference in outcome based on schedule. It is a retrospective analysis, but the cut points for HIF analysis were chosen independently of this data set, the reproducibility of HIF-2 and CA9 scoring was excellent as shown by the high correlation values between the two observers, and the pathologic analysis was blinded to results. Expression of these markers was associated with a poor outcome for both schedules of radiation therapy. Standard radiotherapy should be more effective in hypoxic tumors able to undergo reoxygenation during the 7 weeks of therapy, but such a subgroup of hypoxic tumors is currently impossible to identify and analyze. The reduced efficacy of standard radiotherapy (v CHART) to eradicate tumors with intense repopulation ability may be compensated by its superior efficacy against tumors able to undergo reoxygenation, so that the final result reflects in overlapping CHART and standard RT survival curves. Because hypoxia emerges as a potent factor counteracting the efficacy of CHART, combination with hypoxia targeting strategies should be evaluated in future studies. This rationale is supported by the results of a randomized study, where combination of CHART with mitomycin proved more efficacious than CHART alone or standard radiotherapy in patients with HNSCC.35 Combination of CHART with potent bioreductive drugs, such as tirapazamine,36 is expected to enhance the efficacy of this highly accelerated regimen. Other means of overcoming hypoxia have been piloted in early clinical trials, and these include CHART combined with carbogen breathing and nicotinamide37 and CHART combined with the hypoxic cell sensitizer nimorazole.38 Our study shows that endogenous markers of hypoxia (eg, HIF-2 and CA9) are easily and reliably assessed in biopsy material and provide a tool to identify subgroups of HNSCC patients who should be assessed for combinations of radiotherapy with hypoxia-targeting regimens. The rather modest effect seen in randomized studies on hypoxic radiosensitizers39,40 may be tempered if reanalysis based on immunohistochemical assessment of hypoxia markers for the two pathways could be achieved. It should be stressed, however, that endogenous markers of hypoxia do not reflect exclusively a reduced intratumoral oxygen tension, because activation of the HIF pathways may also occur through oncogenic pathways or as a result of cancer-specific metabolic demands, regardless of the extracellular oxygenation conditions. The radio-resistance herein associated with HIF- and CA9-overexpressing tumors may also be attributed to molecular pathways downstream of HIF (eg, reduced apoptosis, increased DNA repair) in addition to the traditional concept of reduced DNA radiation damage fixation in an oxygen-deficient environment. Assessment of endogenous hypoxia markers, apart from being a simpler method associated with clinically relevant hypoxia, also reflects metabolic and biochemical conditions, presumably involved in radiation resistance, impossible to predict by electrode oxygen tension measurements. We conclude that immunohistochemical assessment of the expression of endogenous markers of two transcription pathways regulated by hypoxia are strongly correlated with radiotherapy failure, and this cannot be averted by radiotherapy acceleration. By using simple immunohistochemical methods, it is possible to identify subgroups of HNSCC patients who are highly curable with radiotherapy or who are excellent candidates for clinical trials on hypoxic sensitizers or molecules blocking the HIF molecular cascade. Assessment of hypoxic tumor profile in archival material from patients recruited in large randomized studies performed with hypoxic sensitizers or chemoradiotherapy may unmask important clinical information and accelerate the quest for a highly effective individualized radiotherapy. We do not know the basis for expression of one pathway versus the other, but this may be related to genetic changes (eg, von Hippel-Lindau mutation in renal cancer selecting for HIF-2 relative to HIF-1). It will be of interest to compare gene expression analysis with the presence of these markers.
The authors indicated no potential conflicts of interest.
Bioreductive drugs: Drugs that become activated under hypoxic conditions to produce toxic metabolites. CA (carbonic anhydrase): Carbonic anhydrases (CAs) are involved in several physiological processes, including pH regulation, CO2 and HCO3 transport, and water and electrolyte balance. Eight distinct CA isozymes and additional CA-related proteins have been identified. Carbogen breathing with nicotinamide: A combination of drug and gas therapy that produces vasodilation and enhanced oxygenation of tumours. CHART (continuous hyperfractionated accelerated radiotherapy): A technique of delivering radiation dose over a short period of time, 12 days rather than 30 days.
HIF-2 Hypoxia: Oxygen concentration below normal physiological limits in a specific tissue. Reoxygenation: The effect of radiation that, by reducing in terstitial pressure in tumors, allows better oxygen delivery and collapsed blood vessels to reopen. Tirapazamine: A bioreductor drug activated under hypoxia to inhibit topoisomerase II; also binds to DNA. Tumor repopulation: The regrowth of cancer cells into a radiated area after initial therapy.
Supported by the University of Thrace (M.I.K., A.G., and E.S.), the National Health Service (S.M.B., S.D., M.I.S., and F.M.D.), the University of Oxford (A.L.H.), the Medical Research Council, the Tumor and Angiogenesis Research Group, Cancer Research UK, and European Union grant Euroxy. Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
10%) was used to define two groups of tumors with low/negative and high/positive CA9 reactivity. Thus the criteria used in this study are not study derived, but applied prospectively from previous data set. This is important if such immunohistochemical techniques are to be applied in the clinical practice of radiotherapy to predict response and guide radiotherapy and radiosensitization policies. 
2 test was used for testing relationships between categoric tumor variables. Survival and locoregional control (time to locoregional disease progression after complete tumor regression) curves were plotted using the Kaplan-Meier method, and the Mantel-Cox log-rank test was used to assess statistical differences in outcome between groups. A Cox proportional hazard model was used to analyze the effects of patient and tumor variables locoregional control and overall survival. Two-tailed P values are provided except where a unidirectional hypothesis is tested. In both cases, P values less than .05 were considered statistically significant. 
(hypoxia-inducible factor 2
