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Statistics from the American Cancer Society suggest that over 1.5 million new cases of cancer will be diagnosed in 2010, and close to 570,000 deaths will occur this year (American cancer society cancer facts 2010_) Over two thirds of diagnosed patients will receive radiation therapy.  Solid tumors comprise 90% of all human cancers. These cancers, which develop from a single mutated normal cell, and either infiltrate surrounding normal tissue, such as occurs in brain tumors, or for most cancers, metastasize to vital organs throughout the body, leading to substantial morbidity and mortality. The inherently unorganized nature of the tumor microenvironment can lead to a phenomena known as tumor hypoxia, or low oxygen levels within tumor cell populations. These low oxygen values within both primary and secondary malignancies can confer resistance to both radiotherapy and chemotherapy, and represent a pharmacological hurdle in the successful treatment of cancer patients. Elucidation of the pathogenesis and cellular responses of hypoxia could result in identification of new therapeutic targets, which might transform the problem of tumor hypoxia into a targetable parameter, potentially resulting in better prognosis and increased long term survival rates.
1.1. Characterization of Tumor Hypoxia
The presence of low oxygen concentrations- known as hypoxia- in tumor cell populations was first postulated by Tomlinson and Gray in 1955, based on their histological observations in human lung cancer.  Convincing evidence has since been presented that tumor hypoxia is a characteristic of almost every solid tumor, including malignancies of the breast, uterine cervix, head and neck, rectum and pancreas, brain tumors, soft tissue carcinomas, and malignant melanomas. There also appears to be no difference on the oxygen concentrations of primary tumors and metastatic malignancies. [312,371,1000] Up to 50-60% of these solid tumors may exhibit areas classified as hypoxic. Hypoxic conditions have been shown to exist irrespective of tumor size, stage, nodal status, or other characteristics. (May want to use blood flow illustration, Paper 371, pg 200, or paper261,pg 60, in this part of paper)
While most normal tissues exhibit a median oxygen partial pressure (pO2) of between 40 and 60 mmHg, some areas of hypoxia in solid tumors may have a median pO2 of only 10 mmHg.(356) Severely hypoxic areas of ï‚£ 2.5 mmHg may also be distributed within a solid tumor mass, significantly reducing the response to radiation and chemotherapy. Vaupel and others showed a significant difference in pO2 values collected from locally advanced squamous cell carcinomas of the uterine cervix compared with normal tissue. Measurements from normal tissue indicated a median of 42 mmHg and a mean of 40 mmHg, in stark contrast to the respective values of 10 mmHg and 16 mmHg for measurements collected in patients with cervical cancer. (Figâ€¦. (227 Source
The hypoxia levels in tumors of the cervix were also found to be independent of age, parity, menopausal status, or smoking habits of the patients.
1.2. Pathogenesis of Tumor Hypoxia
Tumor hypoxia results when the supply of oxygen to the tumor cells cannot keep up with O2 consumption. Both abnormal microvasculature of the tumor, as well as increasing diffusion distances between tumor cells and vessels are significant factors responsible for the pathogenesis of hypoxia. Additionally, anemia, whether tumor-associated or therapy-induced, can also result in poor tumor oxygenation. (227- the oncologist)
While tumor hypoxia can be classified either as acute or chronic, it is important to note that definite classification can be rather arbitrary. A mixture of both types could potentially manifest within a solid tumor, and regions exhibiting an intermediate state of hypoxia can also exist.(50)
"Perfusion-limited", or acute hypoxia, is caused by inadequate blood flow throughout tumor tissues. Abnormalities of tumor microvessels such as an unorganized vascular network, dilations, lack of blood flow regulation and incomplete endothelial lining contribute to pO2 deficiencies (Fig2). Perfusion related hypoxia leads to transient ischemia. (227)
Diffusion or chronic hypoxia can be caused by diffusion distances of >70 ïm from the vessel, with necrosis occurring at 180ïm. The uncontrolled proliferation of malignant cells causes the tumor to outgrow its blood supply, thus restricting nutrient and O2 delivery. (70, 319)
Anemia is a common condition in cancer patients. Its presence can be treatment related, or as a result of disease. Reduced. O2 transport capacity of the blood in anemia leads to anemic hypoxia. Hypoxia is intensified when hemoglobin levels drop below 10-12 g/dl. This is especially true with the concurrent presence of a low perfusion rate.
Clinical trials indicate a significant correlation between low levels of hemoglobin and poor response to radiation and chemotherapeutic treatment. Normally the tissue can compensate for mild to moderate anemia by increasing the perfusion rate. However, in tumor masses, the abnormal vasculature can prohibit compensation. Therapies aimed at increasing oxygen delivery to hypoxic tumor fractions may convey enhanced sensitivity to radiation treatment, as well as chemotherapy. Specific treatment modalities for the correction of anemia will be discussed further in Chapter 4. [50 (31)]
In vitro and in vivo studies have investigated the relationship between hypoxia, anemia and treatment outcome. The work of Kelleher et al. showed that "tumor anemia resulted in substantial worsening of tumor oxygenation measured with polarographic needle electrodes". 
In normal tissue, hemoglobin levels above 8 g/dl can provide adequate oxygenation. Even hemoglobin levels between 8 g/dl and 4 g/dl, while reduced, can oxygenate normal tissue. This is a result of compensation due to increased perfusion, as discussed previously. Since locally advanced tumors cannot counteract oxygen supply reduction, fractions of the tumor exhibiting hypoxia are inevitable. 
May want to use Becker et al study: Demonstrates relationship between falling hemoglobin level and decreasing tumor oxygenation (227 ref 15) 307
In solid tumors, hypoxia occurs as a result of inadequate oxygen supply due to cell proliferation and inefficient vascular supply.  In normal tissues, the oxygen supply can adequately match the metabolic consumption of the cells. However, the metabolic demands in the microenvironment of a solid tumor can increase substantially as tumor cells exponentially divide. An imbalance in oxygen supply and demand inevitably leads to the development of regions of hypoxia. Development of hypoxia in solid tumors can be attributed to three major mechanisms, including abnormal structure of tumor vasculature (perfusion limitations), lengthy oxygen diffusion distances (diffusion-limited hypoxia) or reduced oxygen carrying capacity of the blood (anemia related hypoxia (Fig 2)
Figure 3. Differences in vasculature between normal tissues (upper panels) and malignant tumors (lower panels.)
1.3. Methods of Detection
Determination of the oxygenation status of solid tumors is a critical factor in structuring successful treatment protocols for human malignancies. Several factors must be taken into consideration when determining which method to utilize for an individual patient. These include the invasiveness of the procedure, the degree of resolution required, the size and location of the tumor, as well as potential financial considerations. [241, 273] An optimal method for assessing tumor hypoxia would: differentiate between normoxic, hypoxic, and anoxic cells within intertumor and intratumor regions; distinguish between perfusion-related (acute) and diffusion-related (chronic) hypoxia; be accessible to any tumor site; reflect cellular rather than vascular pO2 ; be capable of measuring pO2 on a spatial scale similar to the O2-diffusion distance; be non-invasive, non-toxic to both patient and administrator, reliable, and allow for repeated measurements. Over the past two decades, researchers and clinicians have developed several methods to quantify the levels of hypoxia within a tumor mass. While a thorough discussion of each oxygen measurement technique is beyond the scope of this paper, attention will be focused on methods that are most clinically relevant.
1.3.1. Polarographic Needle Electrode
In 1963, Kolstad and colleagues used glass-sealed polarographic oxygen electrodes to quantify levels of hypoxia in human cervical carcinomas. In doing so, they demonstrated that the presence of hypoxia was associated with poorer treatment response. One specific drawback to the glass electrodes in use during that time was poor penetration into the tumor tissue-on the order of a few millimeters. [Vaupel book pg 19]
The issue of limited penetration of earlier systems was addressed with the introduction of the Eppendorf polarographic needle electrode in the late 1980's. By covering the sensor in a steel jacket, measurement of pO2 values deep inside the tumor provided a better picture of the spatial distribution of hypoxia.[Vaupel book 20] In subsequent years, both animal and human studies have relied heavily on this technique for measurement of oxygen levels within a tumor. An advantage of this technique is the ability to directly measure pO2 values at different locations within the tumor in a timely manner. However, the inability to differentiate between necrotic areas and areas where viable tumor cell populations exist, the invasiveness of the procedure, and the limitation of measurement to readily accessible tumors has led to recent developments of other methods of hypoxia assessment.
1.3.2. BOLD MRI
Blood Oxygenation Level Dependent (BOLD) MRI is a prominent method of in vivo assessment of humans based on its ease of administration, non-invasiveness, validity, and capability for repeated measurements, and general availability. BOLD MRI does not measure pO2 directly. The contrast of magnetic resonance signals hinges on the balance of oxyhemoglobin and paramagnetic deoxyhemoglobin. Because the presence of deoxyhemoglobin increases the relaxation rate of water in the blood and perfused tissues, BOLD-MRI can offer sensitivity to pO2 within the vessels as well as the surrounding tissue. Since transient occlusion of vessels occurs in acute (perfusion-related) hypoxia, these areas are more sensitive to imaging, due to their close proximity to blood vessels within tumor tissue. Chronic (diffusion-related) hypoxia is less likely to be reflected because red blood cells are farther removed from areas of hypoxia (643). With BOLD-MRI there is no need to administer exogenous radioactive contrast material, and measurements can be obtained repeatedly with minimal patient discomfort. However, certain limitations to this method of evaluation include a low signal to noise ratio, invalid functional parameter estimates due to potential motion of the patient (643), and the influence of pH, hematocrit and blood flow on the signal  Possibly include study on high sensitivity but low specificity by Hoskin et al.
Positron emission tomography (PET) is a molecular imaging technique that has moved to the forefront of oncological assessment in the last decade. A radioactive tracer is administered to the patient via injection. Approximately 2 hours post injection a PET scan is performed, reflecting the distribution of the tracer within tissues and identifying areas of hypoxia (722). 18F-fluoromisonidazole (18F-MISO), 18F-fluoroazomycin-arabinofuranoside (18F-FAZA), and 64Cu- diacetyl-bis N4-methylthiosemicarbazone (64Cu-ATSM) are all radioactive markers which have demonstrated success in measuring hypoxia in solid tumors.
Rasey et al first reported on the use of 18F-MISO in hypoxia imaging studies using PET . Once injected, 18F-MISO freely diffuses homogenously into normal tissue, and reaches the tumor regardless of perfusion limitations. The contrast between hypoxic and aerated areas within a tumor arises from the selective entrapment of the tracer within hypoxic cells. This mechanism of retention involves reduction of the NO2 substituent in viable hypoxic and normoxic tissue where electron transport is occurring. Once reduced within normoxic tissue, rapid transfer of the electron back to oxygen reforms the original tracer molecule, which than then diffuse freely out of the cell. Under hypoxic conditions however, less oxygen is available to accept electrons from the nitro group and as a result stays in the reduced form, bonding with peptides and other macromolecules in a covalent manner. It is important to note that the concentration of tracer retained is inversely proportional pO2 within the cell. Consequently, the tracer can help scientists discern the oxygenation topography within the tumor by successfully marking hypoxic fractions that can be visualized upon PET scanning. Unlike the polarographic needle electrode technique, 18F-MISO PET samples only from viable hypoxic tumor tissue instead of a mixture of hypoxic and necrotic cells, the latter of which would be lacking a functional electron transport system required for reduction of 18F-MISO . One problem that has limited the clinical application of this tracer is the slow accumulation of 18F-MISO in hypoxic cells, as well as slow clearance from well oxygenated tissues. This can be attributed to the relatively high lipophilicity of the molecule  As a result, a newer, less lipophilic tracer was introduced by Kumar and others; 18F-FAZA [722 source 22].
CHAPTER 2. TUMOR HYPOXIA AND TREATMENT RESISTANCE
Chapter 3. Cellular Responses and Adaptations to Hypoxia
The initiation of a host of molecular pathways that enables tumor cells to adapt to conditions of hypoxia is beginning to be studied extensively. One key molecule responsible for the up-regulation of genes that enable adaptation by both normal and malignant cells to occur is Hypoxia Inducible Factor 1 (HIF-1), a heterodimeric transcription factor.
This transcription factor consists of two subunits, HIF-1ï¡ and HIF-1ï¢, the latter of which is constitutively expressed in both hypoxic and well-oxygenated tissue. Under normoxic conditions, proteasomal degradation of the HIF-1ï¡ subunit occurs through interaction of the hydroxylated HIF-1ï¡ protein, with the von Hippel Lindau protein, resulting in polyubiquitination and subsequent degradation. (Fig 4A)
Under hypoxic conditions however, hydroxylation of a proline residue within the oxygen-dependent degradation domain of the ï¡-subunit does not occur. Since prolyl hydroxylase only exhibits enzymatic activity in well-oxygenated cells, the scarcity of oxygen as a co-substrate during conditions of hypoxia is primarily responsible for the intracellular accumulation of the ï¡ subunit, and subsequent hypoxia-stimulated activation of molecular responses.
Once the HIF-1ï¡ subunit translocates to the nucleus and dimerizes with the ï¢ subunit forming the functional HIF-1 transcription complex, it binds to the hypoxia response element (HRE), a specific sequence within the promoter region of a hypoxia-responsive gene (Table 1). Upon HIF-1 interaction with the HRE, the recruitment of co-activators such as p300 occurs, and transcription of the gene ensues. Such genes that are expressed in response to HIF-1 produce protein products involved in the cellular adaptive response to hypoxia, including the promotion of angiogenesis and regulation of vascular endothelial growth factor (VEGF), as well as up-regulation of the glycolytic pathway. Additionally, the HIF-1 transcription complex is involved in regulation of tumor pH, and has been shown to function as a modulator of the
apoptotic response (Figure 4)
3.1.1. Angiogenesis and VEGF
An emerging body of evidence suggests that poorly oxygenated tumor cells trigger pro-angiogenic mechanisms in order to restore oxygen supply to ischemic areas and maintain adequate blood flow to expanding tumor cell populations. "One of the most potent stimulators of angiogenesis is VEGF, which is essential for the proliferation and migration of vascular endothelial cells, thereby enabling the formation of new blood vessels" . VEGF, or vascular endothelial growth factor, is a dimeric protein that is essential for embryogenesis, physiological angiogenesis such as for wound healing, and vascularization of malignancies 
Angiogenesis is the process of new blood vessel formation. This formation occurs in two distinct phases. First, primarily during embryonic development, the vascular framework is formed from differentiation of angioblasts, precursors to the endothelial lining. This is known as vasculogenesis. After the primitive capillary network has been formed, new vessels sprout and grow into a highly complex and organized vascular system through the process of angiogenesis (411). The mechanism of angiogenesis is carried out through several specific steps, each one closely regulated by a balance of activators and inhibitors. First, vasodilatation, involving nitric oxide, and increasing vascular permeability in response to VEGF, allow leakage of plasma proteins responsible for establishing a framework outside the vessel to which migrating endothelial cells can attach.  However, excessive permeability can lead to collapse of the vessel wall, and is kept in check via angiopoietin (Ang-1), a negative regulator of this process (411). Next, the vessel becomes destabilized, allowing endothelial cells to loosen from the smooth muscle cells, thus proliferating and migrating to distant sites (414,411). Finally, pericytes migrate to the newly formed vessel and a basement membrane is produced.
Tumor growth is dependent on angiogenesis to support its increased need for vascular expansion. Initially, small tumors (1-2mm) are poorly vascularized and grow slowly (415). As the need for adequate blood supply progresses, the angiogenic balance of activators and inhibitors is disrupted, and the tumor manifests an angiogenic phenotype. This is known as the "angiogenic switch"(414). Hypoxia, nutrient deprivation, and reactive oxygen species (ROS) are major stimuli in triggering this "switch"(415).
3.1.2. Energy Metabolism and Anaerobic Glycolysis
The metabolic profile of a solid tumor is very different from surrounding tissues. Otto Warburg first characterized over seven decades ago the major metabolic changes within the tumor microenvironment . He found that the rate of anaerobic glycolysis within tumors is elevated, even in cells with oxygen concentrations sufficient to support mitochondrial electron transport. The reason behind this apparent shift from cellular respiration has been very elusive, and has sparked new studies on the metabolic profiles of solid tumors in an attempt to understand how metabolic changes can confer a growth advantage 
During normoxic conditions, the generation of ATP occurs by oxidative phosphorylation. The low levels of oxygen in hypoxic tumor cells, coupled with a high rate of glucose consumption, necessarily require switching to anaerobic glycolysis for energy production  Adaptive metabolic changes are mediated by the transcription factor HIF-1, and play a vital role in maintaining sufficient energy levels, even when utilizing the inefficient glycolytic pathway. Up-regulation of several important glycolytic enzymes, as well as increased glucose influx occurs through activation of HIF-1 .
3.1.3. Tumor pH and Carbonic Anhydrase 9
3.1.4. Hypoxia and Apoptosis
3.2. Role of Hypoxia in Malignant Progression
There are two methods by which tumor hypoxia can be exploited as a theraputic target. First, a drug which is cytoxic only to hypoxic cells, would selectively attack the tumor cells. Secondly, hypoxic cells are resistant to chemo and radiation therapies. By using drugs which kill hypoxic cells, the tumors resistance is lessened.
Mitomycin C is a quinone antibiotic which was first introduced into clinical use in 1958. It has exhibited efficacy toward a number of different tumors in both chemotheraputic drugs and radiation. In 1988, Marshall and Ranth[20,hypoxia specific cytotoxins] in their pioneering workwith Chinese hamster cells in vitro, showed that while mitomycin c clearly exhibits preferential toxicity under hypoxic conditions, the oxygen levels which are required to produce the maximum resistance to radiation are so low (0.02%)(~0.15mmHg) that there is little differential toxicity of mitomycin c for aerobic and hypoxic cells. As a result, when radiation and mitomycin c are combined, there will be a population of cells which are at intermediate oxygen concentrations, that are sensitive neither to radiation or mitomycin c  While mitimycin c is a standard chemotherapy agent with systemic toxicity to well oxygenated cells, it has not been convincingly demonstrated the the higher cure rates over radiotherapy alone were the result of selective killing of hypoxic cells. Nontheless, Phase --- clinical trials are underway [ check status of trials]
Need to talk about the results of at least one other study
Fig 8 : The Hypoxic Cell 
From old paper: It has been shown in preclinical studies that tirapazamine outperforms the nitroimidizole sensitizers, with radiation doses similar to those given clinically. . Tirapazamine is 40 to 300 times more toxic to cells under hypoxic conditions than under normoxic conditions. THis drug undergoes a one electron reduction (include figure in source 57) to form cytotoxic free radicals, which lead to single strand and double strand breaks in DNA, chromosome aberations, and cell death under hypoxic conditions. The main side effects of tirapazamine, as shown in phase I and II trials are nausea/vomiting and muscle cramps. These were mostly grade 1-2, with only two patients showing grade 3-4 cramps. Only two patients showed grade 3-4 nausea/vomiting  The results of earlier trials have been promising, and phase III trials are currently underway
History: In the mid-1980s, William Lee and J. Martin Brown were looking for new classes of hypoxic radiosensitizers. They were interested in those that did not include a nitro group, since they believed this might be responsible for the neurotoxicity of the 2-nitroimidazole class of radiation sensitizers. While testing a particular benzotriazine di-N-oxide, initially known as SR 4233, and later called TZP, Brown and Lee discovered that TZP killed a larger number of hypoxic cells than any previously tested radiosensitizing drug of equal concentration. Tirapazimine's differential toxicity to hypoxic cells was much larger than any other drug[343,102]
Mechanism of Action:
The selective hypoxic cytotoxicity of tirapazamine occurs by the mechanism in figureâ€¦ Once taken up into the cell, TPZ is an excellent substrate for reductase enzymes that are present within the cell. Acting upon the parent TPZ molecule, the reductase adds an electron to the drug, and after subsequent protonation, a neutral radical is formed. The highly reactive nature of this radical causes single and double-stranded breaks in DNA through abstraction of hydrogen. In normoxic conditions however, the additional electron can be removed from the TPZ radical by oxygen. This oxidizes the radical back to the nontoxic parent drug, with concomitant formation of a superoxide radical. Differential toxicity arises due to much more toxic nature of the TPZ radical as compared with the superoxide radical, which is only generated under oxygenated conditions.