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Tumor Hypoxia: Chicken, Egg, or a Piece of the Farm?

Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD

THE ARTICLE BY Fyles et al¹ in this issue of the Journal of Clinical Oncology is a well-done and welcome study on the clinical importance of tumor hypoxia for patients with carcinoma of the cervix. The impact of hypoxia in increasing radiation resistance to a variety of biologic end points has been known for almost 70 years. It was demonstrated 35 years ago that hypoxia limited the radiocurability of murine tumors.² That hypoxia exists in human tumors was demonstrated 45 years ago by Thomlinson,² who observed that necrosis occurred in tumor cells greater that 150 µmol/L from blood vessels, which is the approximate diffusion distance of oxygen. This diffusion-limited hypoxia was felt to be an important reason for the inability of radiation therapy to achieve local tumor control, and consequently, an extensive laboratory and clinical effort was undertaken to overcome this effect starting with the use of hyperbaric oxygen in the mid-1960s. This effort has included and continues to include increasing the delivery of oxygen, oxygen-mimetic radiation sensitizers, hypoxic cytotoxic agents, agents that alter hemoglobin affinity for oxygen, agents that alter blood flow, and deposition of heat to alter blood flow and take advantage of the relative sensitivity of hypoperfused cells.^2-5 In essence, the fields of radiation oncology and radiation biology have been engaged in well-conceived translational laboratory-to-the-clinic-and-back research for many decades.

In a meta-analysis by Overgaard and Horsman,⁶ there was a statistically significant benefit to combining radiation therapy with antihypoxia therapies compared with radiation alone. Nonetheless, the observation that antihypoxia therapies with radiation did not improve local control rates by a substantial percentage has resulted in several conclusions, all of which are partly correct. Among them are (a) hypoxia may not exist in human tumors; (b) hypoxia is not important in treatment outcome; (c) the antihypoxia treatments evaluated were not effective due to insufficient potency, concentration, or timing of administration; (d) tumor reoxygenation occurs during treatment so that initial tumor hypoxia resolves during a course of radiation treatment;² (e) hypoxia is but one example of the complex phenotype within a tumor owing to the abnormal tumor physiology;^3,4,7,8 and (f) hypoxia per se is not the problem, it is simply an indicator of a "bad" cancer.

So, with the problem being a "bad outcome" in terms of local tumor control and survival for the patient, is hypoxia the cause of the problem (the Chicken)? Is hypoxia the result of the problem (the Egg)? Or is the constellation of hypoxia, which includes both the reason for and the consequence of its presence, an important component of cancer progression, treatment resistance, and, therefore, a critical therapeutic target (the Farm)? This metaphor lends itself to the inclusion of other agrarian terms, but we will leave those to the reader’s imagination.

The role of hypoxia in clinical oncology can be addressed with the following questions, focusing this discussion on the outcome with radiation therapy, although the general conclusions apply to other therapies as well:

1. Is hypoxia present in human tumors?

2. What happens to hypoxia during a course of treatment?

3. What is the impact of hypoxia on tumor progression?

4. What is the impact of hypoxia on therapy?

5. What tools are present or under development to image and otherwise detect hypoxia?

6. What are the therapeutic approaches against hypoxia?

A first general point to make is that although this discussion relates to an abnormal concentration of oxygen within the tumor, other molecules are also distributed abnormally within the tumor when compared with normal tissues. The second general point is that there are two types of hypoxia: (1) chronic or diffusion-limited hypoxia, in which tumor cells are too far from a functioning blood vessel, and (2) acute/intermittent or perfusion-limited hypoxia, in which the abnormal periodic flow within a tumor vessel renders some cells underperfused for a period of time.^3,4,8 This latter mechanism may be somewhat analogous to ischemia-reperfusion. The third general point is that hypoxia relates to molecular biology, biochemistry, physiology, angiogenesis, and imaging. This editorial cannot cover these issues in the detail available for each of the various components of these fields.

In the report in this issue of the Journal of Clinical Oncology, Fyles et al¹ measured tumor hypoxia with an invasive technique using the Eppendorf electrode. Using the percentage of readings of oxygen less than 5 mmHg (hypoxic proportion [HP₅]), an oxygen concentration below which cells are relatively radioresistant in tissue culture (Fig 6.5 in Hall²), the patients with poorly oxygenated tumors had a statistically inferior progression-free survival (PFS) rate. Poor tumor oxygenation status was correlated with positive nodal status, as assessed by imaging with all of the potential limitations of imaging acknowledged by the authors. HP₅ was an independent predictor of PFS in the node-negative patients but not in the node-positive patients. This suggests that once the nodes are involved, the impact of hypoxia on PFS is not important. Using our farm metaphor, the horse is already out of the barn (the Barn Door Effect). For node-negative patients, the patients with hypoxic tumors had an inferior PFS. The authors report that their analysis did not demonstrate that the hypoxic tumors had an inferior pelvic control rate. Data were not specifically reported by nodal status, although the report indicates that pelvic control was not impacted by HP₅ in either the node-positive or node-negative group. The effect of lower HP₅ on PFS but not on pelvic control for the node-negative group suggests that hypoxia is an indicator of a Barn Door Effect in that one can control the pelvis in the face of the lower HP₅, but there is a higher incidence of metastases. Thus the barn door is open (increase in metastases and decrease PFS) in the face of hypoxia. Analogous results have been obtained by Brizel et al⁹ for soft tissue sarcomas in which hypoxic tumors had an increased risk of distant metastases.

Returning to the questions:

IS HYPOXIA PRESENT IN HUMAN TUMORS?

Yes. It has been noted to be present before initiation of treatment in cervix, head and neck, pancreas, brain, and breast tumors and sarcomas.^3,10

WHAT HAPPENS TO HYPOXIA DURING A COURSE OF TREATMENT?

There is only a limited amount of information in this regard. Cooper et al¹¹ studied changes in oxygenation before and after 40 to 45 Gy of radiotherapy for patients with carcinoma of the cervix. Mean tumor partial pressure of oxygen (_PO₂) increased and HP₅ decreased in most but not all of the patients. However, even those with improvement still had substantial HP₅ readings after treatment. In patients with head and neck cancer, a decrease in median _PO₂ and an increase in HP₅ were observed at the end of radiation therapy.¹² A small trial with positron emission tomography imaging demonstrated a decrease in hypoxia.¹³ A trial in cervical cancer using radiation plus biologic radiation modifiers indicated that an increase in oxygenation during treatment correlated with an improved outcome.¹⁴ For patients with head and neck cancer, repeat measurements after 10 to 15 Gy of irradiation were similar to baseline values.¹⁵ More information is clearly needed as to the changes in hypoxia and also the best measure of hypoxia that predicts response. For example, is it median _PO₂, HP₅, a certain imaging procedure, or another measure?

WHAT IS THE IMPACT OF HYPOXIA ON TUMOR PROGRESSION?

In the last few years, there has been remarkable progress in understanding the changes in cellular function as a result of tissue hypoxia. The hypoxia-inducible factors (HIF-1 and HIF-2)¹⁶ dimerize with the constitutively expressed HIF-1ß subunit to bind to a hypoxia-responsive element^17,18 to activate a wide array of genes, including those involved in anaerobic metabolism, cell cycle arrest, differentiation, stress adaptation, angiogenesis, and others.^4,8,19,20 These can result in profound alterations on tumor and cellular phenotype, including an obvious role in angiogenesis by upregulation of angiogenic factors such as vascular endothelial growth factor.^21-25 The expression of HIF is dependent on protein degradation. Binding of the von Hippel-Lindau (VHL) protein (pVHL) to HIF targets it for ubiquitylation and proteosomal degradation.^26,27 The mechanism of the protein-protein interaction between pVHL and HIF has recently been described.^28,29 Abnormalities in HIF biochemistry, such as constitutive presence of HIF or a defect in pVHL, will allow HIF-directed gene expression that in turn will allow for the expression of the hypoxic phenotype in nonhypoxic cells, as demonstrated by the faulty vascular proliferation seen in patients with VHL disease.³⁰ Overexpression of HIF is found in common human tumors,³¹ HIF is a positive factor in solid tumor growth,³² and HIF is a negative prognostic factor.^33,34 Therefore, HIF and its effector genes seem to be excellent targets for innovative molecular therapeutics.^35,36

Hypoxia, with or without reoxygenation, is a mediator of malignant progression, with mechanisms that include selection pressure, genomic instability, genomic heterogeneity, decreasing apoptotic potential, increasing angiogenesis, and a chaotic microcirculation.^4,8,37,38 Lal et al¹⁹ have recently described a series of hypoxia-overexpressed genes (HOGs), an abbreviation fitting our metaphor. These are under regulation by HIF and the expression of which corresponded to hypoxia regions in tumors as defined by the pimonidazole, hypoxic-cell marker technique.^39-41 Hypoxia also regulates expression of other genes, for example, those under the control of the iron-responsive element.^42,43

WHAT IS THE IMPACT OF HYPOXIA ON THERAPY?

Radioresistance of hypoxic cells is well established.² Clinically, hypoxic tumors tend to do worse in terms of both local recurrence and distant metastases, as discussed by Fyles et al.¹ This has been reported after surgical resection as well as after radiation therapy.^3,8,44-47 Hypoxia may also adversely effect chemotherapy response.^8,48

WHAT TOOLS ARE PRESENT OR UNDER DEVELOPMENT TO IMAGE AND OTHERWISE DETECT HYPOXIA?

Both invasive and noninvasive methods are under evaluation to assess tumor hypoxia.⁸ Invasive techniques include the Eppendorf electrode as used by Fyles et al¹ and a luminescent fiber-optic sensor.⁴⁹ Noninvasive imaging techniques include blood oxygen level–dependent magnetic resonance imaging (MRI) ⁵⁰ and dynamic contrast-enhanced MRI.⁵¹ A nuclear medicine technique under development exploits the bioreductive metabolism and binding of nitroimidazole compounds using single-photon emission computed tomography or positron emission tomography.^52,53 A novel approach using electron paramagnetic resonance imaging and Overhauser MRI may allow for real-time serial assessment of both blood flow and oxygenation.⁵⁴

The fact that 2-nitroimidazole compounds are metabolized and form adducts to cellular proteins under hypoxic conditions has led to the development of immunohistochemical techniques using pimonidazole^40,41 and EF5.⁵⁵ It seems that these techniques are best suited for the identification of chronically hypoxic cells, because sufficient time is required for reduction and binding of the parent compound. In general, the measurement of the proportion of hypoxic cells between the Eppendorf electrode and the 2-nitroimidazole binding technique is similar. However, there is not a direct correlation within the subregions of a tumor, due in part to the lack of drug diffusion into necrotic regions and to the potential for both chronic and intermittent hypoxia. Knowledge of the molecular biology of hypoxia has led to the investigation of hypoxia-induced proteins as intrinsic markers of hypoxia. These might be analyzed in the serum, such as plasminogen activator inhibitor-1⁵⁶ or in tumor biopsy specimens, such as carbonic anhydrase 9.^57-60

WHAT ARE THE THERAPEUTIC APPROACHES AGAINST HYPOXIA?

One can target the hypoxic cell itself, hypoxia-inducible genes, the hypoxia-related gene and protein expression, and the underlying abnormal vasculature. The hypoxic cell has been targeted by the use of means to increase tumor oxygenation,³ including carbogen, erythropoietin, blood transfusion,⁶¹ hyperbaric oxygen, and agents that alter hemoglobin affinity for oxygen, such as RSR13,⁶² and oxygen mimetic hypoxic cell sensitizers, such as nimorazole and etanidazole,³ which enhance radiation cell killing when administered simultaneously with radiation. Taking advantage of the reductive metabolism and drug activation under hypoxia are the hypoxic cytotoxic agents such as tirapazamine and mitomycin analogs and related drugs.^3,4 Using hypoxia-activated genes for gene therapy is another approach under development.⁴ Part of the efficacy of hyperthermic treatment may be due to improved oxygenation.⁵ Targeting the normal stromal components include therapies that are antiangiogenic and antivascular. The discussion of these therapies is an enormous topic in itself. However, a few recent observations are of interest to this editorial. Angiogenesis can occur very early in tumor development, before the presence of hypoxia,^63,64 and antiangiogenic therapy might reduce hypoxia by normalizing existing tumor vasculature.⁶⁵ Antiangiogenic⁶⁶ and antivascular therapies that target the neovasculature are under development for use with radiation therapy.⁶⁷

Will eradicating hypoxic cells early in the course of a tumor improve local control and/or will it decrease the risk of distant metastases? The answer is probably both, in that persistent local disease can become a source of distant metastases.⁶⁸ The presence of hypoxic cells in patients in the primary tumor in a setting without evidence of regional/or nodal disease, as with that defined by Fyles et al,¹ may define a high-risk population of patients who are excellent candidates for systemic therapies targeting metastatic disease.

It is worth emphasizing that there are other aspects of the abnormal physiology of tumors beyond hypoxia that might enhance tumor aggressiveness and might also provide novel therapeutic targets. These include increased interstitial pressure,⁶⁹ acidosis,²⁴ and lactate.^70,71 Moreover, the expression of HIF-related genes and proteins can occur in the presence or absence of hypoxia, making the HIF system an excellent novel molecular target.^35,36

CONCEPT OF MOLECULAR TARGET CREDENTIALING

Future challenges in clinical oncology will require the scientific linkage among three of the Extraordinary Scientific Opportunities as defined in the National Cancer Institute Bypass Budget: cancer imaging, cancer signatures, and molecular targets of prevention and treatment. Tumor hypoxia lends itself as a potential model for molecular target credentialing, that is, studying the interrelationship of these three areas.

CHICKEN, EGG, OR FARM?

Hypoxia is the Chicken in that it can be the cause of treatment resistance, and hypoxia-inducible changes in the cell can enhance tumor progression. Hypoxia may also be the Egg, or the consequence of the problem, as a result of the abnormal angiogenesis and increased interstitial pressure that occurs in malignant tumors. With the supposition that genes activated by HIF are a problem, this gene activation can occur under hypoxia but also potentially under nonhypoxic conditions in which HIF is misregulated. Thus the Egg, the secondary problem, may be related to abnormal HIF regulation in the hypoxic and nonhypoxic tumor. But, most importantly, hypoxia is, indeed, a fertile Farm for improved cancer treatment. The constellation of events that produce and result from hypoxia are important to tumor progression and offer a host of new targets for research and clinical intervention.

Summarizing the current crop:

• Hypoxia exists in common human tumors. Its presence is generally a poor prognostic factor, but in certain clinical settings, other features of the tumor may be more powerful in predicting outcome than hypoxia, as noted in the node-positive patients in the article by Fyles et al.¹
  
• Hypoxia can lead to treatment resistance. However, the change in hypoxic fraction during treatment may be an even stronger predictor of outcome than the initial value.
  
• Hypoxia can enhance tumor progression through hypoxia-related gene expression. Similar genes can be expressed in the face of misregulated HIF. The biochemical changes under hypoxia are related to gene transcription, protein-protein interaction (eg, pVHL with HIF), and protein degradation (eg, proteolysis of HIF).
  
• Hypoxia can be detected in tumors by noninvasive imaging techniques, invasive oxygen measurement, and molecular markers (eg, carbonic anhydrase 9). Much more research is needed to understand how to use and interrelate these various techniques.
  
• There are a variety of antihypoxia treatment strategies already in the clinic, including hypoxic cell radiosensitization and hypoxic cytotoxic therapy. A wide variety of new treatments are under development, including hypoxia-induced gene therapy, HIF-target therapy, and antiangiogenic and antivascular therapies.
  
• The presence of hypoxia and possibly the concurrent assessment of metastases genes or proteins may define a population of patients with local tumors but who are at high risk for metastatic disease and, consequently, excellent candidates for novel adjunctive therapies. These may include standard cytotoxic treatments, immunologic therapies, vaccines, and antiangiogenic treatment.
  
• Because oxygen can be imaged, can be accurately measured, is diffusible, alters gene expression and protein function, and impacts on therapy, hypoxia can serve as an excellent paradigm for the integrated study of molecular imaging, signatures, and therapy (molecular target credentialing).
  

The field of hypoxia is a fertile area for fruitful investigation that should reap a harvest of new knowledge and budding new therapies.

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