Nuclear Oncology, 1 Ed.

CHAPTER 35

RADIONUCLIDE IMAGING OF TUMOR HYPOXIA AND ITS CLINICAL IMPLICATIONS

Gang Niu • Xiaoyuan Chen

INTRODUCTION

Hypoxia is a pathologic condition in which tissues lack oxygen required for normal cell metabolism. In tumors, hypoxia occurs when the size of a tumor results in an imbalance between oxygen supply and consumption. In locally advanced solid tumors, the O2 consumption rate of neoplastic (as well as stromal) cells may outweigh a restricted oxygen supply and result in the development of tissue areas with very low O2 levels. After several decades of preclinical and clinical investigations, there is now a consensus that the prevalence of hypoxic tissue areas (i.e., areas with O2 tensions [pO2 values] ≤ 2.5 mm Hg) is a characteristic of locally advanced solid tumors and a relevant pathophysiologic feature. Accumulating evidence has shown that up to 50% to 60% of locally advanced solid tumors may exhibit hypoxic and/or anoxic tissue areas that are heterogeneously distributed within the tumor mass.1Variability in oxygenation among tumor sites is greater than intratumor variability. Local recurrences have a higher hypoxic fraction than the primary tumor. However, there is no clear-cut difference in the oxygenation status between primary and metastatic malignancies.1

Hypoxia in solid tumors is mainly caused by rapid proliferation and severe structural and functional vascular abnormalities.2 It has been found that intratumoral vessels have significant structural abnormalities. They are dilated, saccular, tortuous, and heterogeneous in their spatial distribution3 which contributes to perfusion-limited O2 delivery. This type of hypoxia is also called “acute hypoxia” because it is usually transient. Hypoxia in tumors can also be caused by an increase in diffusion distances which is termed “diffusion-limited hypoxia,” also known as “chronic hypoxia.”1 In addition to enlarged diffusion distances, adverse diffusion geometry can also cause hypoxia. Tumor-associated or therapy-induced anemia can contribute to the development of hypoxia, known as “anemic hypoxia.” This type of hypoxia is particularly relevant in tumors or tumor areas exhibiting low perfusion rates.4

Cells exposed to hypoxia respond by reducing their overall protein synthesis which in turn leads to restrained proliferation and subsequent cell death. Sustained hypoxia can also change the cell cycle distribution and the relative number of quiescent cells leading to alterations in response to radiation and many drugs. Hypoxia initiates both p53-dependent and p53-independent apoptosis pathways including those involving genes of the BCL-2 family. At the same time, hypoxia-induced proteome and/or genome changes may promote tumor progression via mechanisms enabling cells to overcome nutritive deprivation, to escape from the hostile environment, and to favor unrestricted growth. Sustained hypoxia may also lead to cellular changes resulting in a more clinically aggressive phenotype.5

In 1953, Gray et al.6 first demonstrated that the presence of hypoxic cells in solid tumors was associated with treatment failure following radiotherapy. Later on, it has been demonstrated that hypoxic cell radioresistance is a result of lack of oxygen in the radiochemical process by which ionizing radiation is known to interact with cells.7 The magnitude of this effect is well described by the oxygen enhancement ratio (OER), which is typically in the order of 2.7 to 3. The phenomenon is most clearly seen after large single doses of radiation but also exists in fractionated radiotherapy. It is often observed in solid tumors, whereas normal tissues tend to have sufficient oxygen to sustain radiation sensitivity. The level of hypoxia that results in radioresistance is 5 mm Hg or less, which is at the more extreme end of the hypoxic scale, whereas the influence of other hypoxic-related biologic phenomena may happen at hypoxia levels that are less severe and in the range of 5 to 20 mm Hg.8

Hypoxia-induced expression of certain genes may also result in a drug-resistant phenotype by expansion of populations of cells with altered biochemical pathways.9 For example, hypoxia is selective for cells that have lost sensitivity to p53-mediated apoptosis and for cells that are deficient in DNA mismatch repair, which may, in turn, be resistant to platinum-based chemotherapeutic agents.10,11 Transient hypoxia has been reported to cause amplification and increased expression of the genes encoding P-glycoprotein and dihydrofolate reductase, which induce drug resistance to substrates of P-glycoprotein and to folate antagonists, respectively.12 Transient hypoxia associated with glucose deprivation can also disrupt protein folding in the endoplasmic reticulum, which may confer resistance to topo-isomerase II–targeted drugs and enhance P-glycoprotein expression and multidrug resistance.13In the presence of oxygen, many anticancer drugs generate free radicals that damage DNA. These drugs accept electrons from biologic sources and then transfer the electrons to oxygen.14 For example, doxorubicin undergoes chemical reduction to a semiquinone radical, which in turn reduces oxygen to a superoxide that may contribute to cytotoxicity.15 Thus, at low oxygen concentrations, the cytotoxicity of drugs whose activity is mediated by free radicals is decreased.16

Consequently, hypoxia represents a compelling therapeutic target.17 The currently developed small molecule drugs to kill hypoxic cells include bioreductive prodrugs that are activated selectively under hypoxic conditions and drugs that inhibit molecular targets in hypoxic cells. The former category mainly is comprised of five different chemical moieties, including nitro groups, quinones, aromatic N-oxides, aliphatic N-oxides, and transition metals. The compounds have the potential to be metabolized by enzymatic reduction under hypoxic conditions, and thus provide the basis for the design of bioreductive prodrugs to exploit tumor hypoxia.18 On the other hand, the identification of molecular mechanisms that mediate cellular responses to hypoxia has stimulated interest in targets that might compromise the survival of hypoxic cells if inhibited. The two main oxygen-responsive signaling pathways that mediate adaptation to hypoxia are centered on the hypoxia-inducible factor (HIF) family of transcription factors19 and the unfolded protein response (UPR).20

In view of the major role hypoxia plays in tumor development and resistance to therapy, the ability to detect hypoxia within tumors has significant implications for cancer management and therapy. Currently, the gold standard for in vivo measurement of tumor oxygenation is the Eppendorf needle electrode system, which allows for direct measurement of pO2 in tumors. However, it is an invasive procedure that often requires ultrasound-guided placement of the electrode. Its use, therefore, is limited to easily accessible tumors. Thus, evaluation of hypoxia in the clinic is shifting to monitoring endogenous markers, especially the transcriptional targets of the HIFs, and exogenous 2-nitroimidazole probes, such as pimonidazole, that bind covalently to SH-containing molecules (thiols) in hypoxic tissue.21,22 These differences in oxygen concentration have important implications for hypoxic cell targeting as well as differences in the spatial distribution, duration of hypoxia, and the genetic and environmental context in which hypoxia occurs. In particular, these factors will dictate the choice of hypoxia-targeted therapy that best complements existing agents used to treat the nonhypoxic tumor cell population.17 However, the understanding of tumor hypoxia and physiologic hypoxia in some normal tissues is far from complete. This chapter summarizes the noninvasive imaging of hypoxia and the potential clinical applications.

INVASIVE MEASUREMENT OF HYPOXIA

A variety of techniques are being proposed to assess hypoxia and the apparent extent of hypoxia in human tumors. These methods can be broadly categorized into direct measurements and indirect measurements according to different principles and ability to quantify tissue oxygenation. Direct measurements, including polarographic needle electrode, phosphorescence imaging, near-infrared spectroscopy (NIRS), blood oxygen level dependent (BOLD) and 19F magnetic resonance imaging (MRI), and electron paramagnetic resonance (EPR) imaging can detect oxygen partial pressure (pO2), oxygen concentration or oxygen percentage.23 Indirect measurements, including measuring exogenous and endogenous hypoxia markers, can provide parameters related to tissue or tumor oxygenation.24

The invasive polarographic electrodes have been extensively used to measure pO2 in human tumors and animal studies since the 1990s.25,26 Regarded as the gold standard, these electrodes have been applied to more easily accessible tumors such as head and neck cancer, cervical cancer, soft tissue sarcomas of the extremities, astrocytic brain tumors, lung cancer, pancreatic cancer, prostate cancer, and lymph node metastases. The typical pretreatment median pO2 is 11.2 mm Hg (range: 0.4 to 60 mm Hg)25 and values obtained by such measurements can predict treatment response27 and the metastatic potential of tumors.28 Currently, the polarographic electrode can be used under CT or ultrasound guidance to assay the pO2 of a tumor in deep-seated organs and obtain overall tumor oxygen status.29 However, insertion of an electrode into the tumor disrupts tissues, making it difficult to distinguish necrotic areas to discern the patterns of hypoxia. Moreover, the technique requires great expertise and has a large interobserver variability.

Indirect methods utilize exogenous probes to measure molecular reporters of oxygen. These reporter agents usually form stable adducts with intracellular macromolecules when the oxygen pressure is less than 10 mm Hg.30 2-nitroimidazole derivatives such as misonidazole (1-(α-methoxymethylethanol)-2-nitroimidazole),31 pimonidazole (1-(2-nitro-1-imidazolyl)-3-N-piperidino-2-propanolol),32 and EF5 (nitroimidazole[2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentaflouropropyl) acetamide)33 have been used. Immunohistochemical (IHC) staining with specific antibodies against hypoxia marker adducts in situ can provide quantitative information on the relative oxygenation at cellular resolution.30,34 For example, in one animal study, 120 mg/kg of pimonidazole was intravenously administrated followed by antibody staining against pimonidazole. The result showed that pimonidazole staining was decreased when pO2 returned to normal by carbogen reoxygenation.35 IHC methods are particularly useful for in vitro studies, including assays of human biopsy specimens. Many studies have confirmed that these exogenous markers for areas of chronic hypoxia are more sensitive under severely hypoxic conditions than the polarographic needle electrode.33,36

NONINVASIVE IMAGING OF HYPOXIA

Although the polarographic electrode and IHC staining can provide relatively accurate measurement of tumor oxygenation, these methods have a selection bias and only identify partial, rather than complete information of the entire tumor region.37 Therefore, there has been growing interest in using noninvasive functional and molecular imaging techniques which can yield a plethora of high quality experimental data per protocol by increasing the number of times that quantitative data can be collected.38 To date, several imaging techniques have been developed using either direct measurement or indirect measurement of tumor oxygenation. Most of these techniques are still immature for clinical application. For example, electron paramagnetic resonance (EPR) spectroscopy uses the spin of unpaired electron species to obtain images and spectra. It is now being evaluated in animals to provide a quantitative measure of oxygenation in tissues.39 Although this method has a lot of potential to be developed as a tumor oximeter, particularly to monitor changes after tumor oxygenation,40 development of a suitable paramagnetic marker that has low toxicity for human is currently unavailable. The lack of appropriate EPR instrumentation in the clinic also prevents widespread implementation of this otherwise promising technique.41

Tumor vascular pO2 can also be measured by phosphorescence imaging following the injection of an albumin bound metal– porphyrin complex (Oxyphor) into the vasculature. For instance, Lebedev et al.42 described a general approach to construct phosphorescent nanosensors with tunable spectral characteristics, variable degrees of quenching, and a high selectivity for oxygen. The performance of the probes was demonstrated in measuring vascular pO2 in the rat brain with in vivo microscopy. NIRScan also be used to analyze tumor oxygenation in vivo through recording the spectral changes by hemoglobin in the vasculature. An animal study by Kim and Liu43 showed good efficacy for NIRS compared to electrode measurements in assessing tumor hypoxia. However, low spatial resolution, light scattering, limited path length, low sensitivity, and being easily affected by the environment limit clinical translation of both phosphorescence imaging and NIRS.

Photoacoustic Imaging

Photoacoustic tomography (PAT), also referred to as optoacoustic tomography or thermoacoustic tomography, is a hybrid noninvasive imaging modality that combines high optical contrast and high ultrasonic resolution.44,45 PAT involves optical irradiation, ultrasonic detection, and image formation. The tissue is usually irradiated by a short-pulsed laser beam to produce thermal and acoustic impulse responses. Locally absorbed light is converted into heat, which is further converted to a pressure rise via thermoelastic expansion of the tissue. The initial pressure rise, determined by the local optical energy deposition and other thermal and mechanical properties, propagates in the tissue as an ultrasonic wave, which is referred to as a photoacoustic wave. The photoacoustic wave is detected by ultrasonic transducers placed outside the tissue, producing electric signals. The electric signals are then amplified, digitized, and transferred to a computer to form an image.46

This technique can assess relative changes in concentrations of both oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (Hb).47 Wang et al.48 implemented PAT to image cerebral blood oxygenation of rats in vivo and the results showed that PAT can image the change of the alternation from hyperoxia to hypoxia. However, at this time, there is no report related to clinical application, primarily because of the depth limitation of this technique.

BOLD Magnetic Resonance Imaging

HbO2 is diamagnetic and Hb is paramagnetic.49 Microscopic field gradients in the vicinity of red blood cells and vessels are modulated by changes in Hb concentration. Paramagnetic Hb can increase the transverse relaxation rates of the surrounding protons whereas HbO2 does not.50 Such magnetic field perturbations within a voxel (volume element) cause a loss of phase coherence and therefore lead to signal attenuation in gradient echo or T2* (apparent spin–spin relaxation time)-weighted sequences. This phenomenon has been called BOLD contrast.51 BOLD-MRI uses endogenous signals coming from Hb as image contrast to reflect the changes in the blood oxygenation, and has been shown to have potential to diagnose tumor hypoxia.52 The relationship between BOLD-MRI signal and vascular oxygenation allows investigators to directly estimate pO2. Many studies have been undertaken to investigate the effects of carbogen breathing in mice and oxygenation in tumor models by BOLD-MRI.53,54 A recently reported preclinical study demonstrated that under short-term generalized hypoxia induced by inhalation of 8% oxygen, BOLD contrast was measured as high as 25% from vessels at 9.4 T using a simple gradient echo (GRE) pulse sequence.54 The major disadvantage of BOLD-MRI, like in the case of phosphorescence and near-infrared fluorescence imaging, is that the data provide the change of oxygen tension in vasculature but not in the tissue. Furthermore, BOLD-MRI is not a quantitative method and can be easily affected by many factors including flow, hematocrit concentration, pH, and temperature.52,55

FIGURE 35.1. Structures of hypoxia positron emission tomography (PET) imaging agents.

19F Magnetic Resonance Imaging

19F has 100% natural abundance, a spin of 1/2, and a gyromagnetic ratio of 40.08 MHz/T, which is slightly lower than that of 1H (42.58 MHz/T), resulting in 83% of the sensitivity of 1H.56 Additionally, the chemical shift of 19F is sensitive to the molecular environment of its nucleus because of the seven outer-shell electrons of 19F atom as compared to only one electron of 1H. Finally, in contrast to the prominent 1H signal from mobile water in biologic tissue, only a trace amount of 19F (<1 μM) is present in tissue. Moreover, these 19F atoms are immobilized in a solid phase in the teeth and bones, resulting in virtually zero background 19F MR signal in vivo.57

19F MRI uses mainly perfluorocarbons (PFCs) or fluorinated nitroimidazoles as the contrast agents for hypoxia imaging. PFCs are highly hydrophobic but offer exceptional oxygen solubility.58 The 19F spin lattice relaxation rate of PFCs varies linearly with the dissolved oxygen concentration. Thus, the 19F-based oximetry allows measurements of vascular oxygenation in vivo.59 To date several PFCs have been tried, for example, hexafluorobenzene (HFB)60and perfluoro-15-crown-5-ether (PF15C5),61,62 which can be injected either intravenously or intratumorally. The use of 19F MR is progressing rapidly toward detecting changes in tumor oxygenation in response to radio-sensitizing and oxygen-augmenting treatments.63 The disadvantages of 19F MRI are that the measurements are subject to flow artifacts and the oxygen sensitivity of some 19F MR imaging compounds can be easily affected by temperature, dilution, pH, common proteins, and blood.64 After intravenous injection, majority of the PFC contrast agent is extensively ingested by the reticuloendothelial system (RES), and the slow clearance can have adverse effects. Intratumoral injection can also be risky as PFC emulsion could be accidentally injected into tumoral vein, leading to embolism.65 Another type of probes that had been tried for detecting tumor oxygenation by 19F MRI are fluorinated-2-nitroimidazole compounds including hexafluoromisonidazole (CCI-103F),66 EF5,67 and SR-4554.68 SR-4554 has shown good result at detecting hypoxia in a phase I study by Lee et al.68 and it can be further developed for the detection of tumor hypoxia in certain patients.

PET Imaging

Detection of tumor hypoxia with radionuclides was first demonstrated in 1981 by autoradiography with 14C-misonidazole, which selectively bound to metabolizing hypoxic cells within the tumor.69 So far, the intensively investigated hypoxia imaging markers include 2-nitroimidazoles, nucleoside conjugates, and Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu(II)-ATSM).7072 The structures of most popular hypoxia imaging probes are shown in Figure 35.1.

2-Nitroimidazoles

The mechanism by which 2-nitroimidazoles are reduced and retained in hypoxic tissues has been summarized previously.41 In brief, 2-nitroimidazole tracers are readily diffusible through cell membranes and undergo reversible reduction by intracellular reductases to yield nitroimidazole radical anions, which are then rapidly reoxidized to neutral molecules that diffuse out of the cell. If the concentration of oxygen in tumor tissue is low, oxidation competes with the irreversible binding of the radical anion to other substrates which forms irreversible compounds with cellular macromolecular components.73 Therefore, with decreasing intracellular concentration of oxygen, the tracer accumulates within the hypoxic tissue. There are several features that make 2-nitroimidazoles the most dominant hypoxia imaging agents. First, the reduction of the nitro group on the imidazole ring is accomplished by tissue nitroreductases that are plentiful and do not represent a rate-limiting factor.74 Second the ideal octanol–water partition coefficient75 and low nonspecific protein-binding guarantee that their biodistribution is not complicated by reduced blood flow.76 Furthermore, the pO2 where nitroimidazoles are retained in cultured cells is in the same range as that where the OER is observed. These features enable 2-nitroimidazoles to be trapped at a level proportional to the intracellular demand for O2 and is not limited by blood flow, meeting the design requirements for a hypoxia imaging agent.41

Imaging Tumor Hypoxia with 18-FMISO

18F-labeled misonidazole (18F-FMISO) is the first-generation nitroimidazole marker and the most commonly used hypoxia PET imaging agent.77 18F-FMISO is only sensitive to the presence of hypoxia in viable cells. Studies have shown that significant 18F-FMISO uptake requires a hypoxic level of less than 10 mm Hg.78 With an image-guided robotic system, the oxygen tension (pO2) in rodent tumor xenografts was measured using interstitial probes guided by 18F-FMISO PET images. The 18F-FMISO image intensities were inversely correlated with the measured pO2.79 However, another comparison study showed no correlation between data from 18F-FMISO PET and polarographic oxygen-sensitive electrodes.80

18F-FMISO has been found to reflect hypoxia in glioma, head and neck cancer, renal tumor, and non–small-cell lung cancer.78,81,82 In a clinical study by Rajendran et al.83 in glioblastoma multiforme (GBM) patients, both posttherapy FMISO and FDG images showed increased uptake and retention, suggesting that reoxygenation did not occur. Koh et al.84 found that treated non–small-cell lung carcinomas exhibited increased oxygen and decreased hypoxia distribution, as indicated by sequential FMISO imaging.

Because tumor hypoxia is closely related to radioresistance, pretreatment or serial 18F-FMISO PET imaging has been performed to determine prognosis or dose planning of radiation therapy.85,86 Pretreatment FMISO uptake and retention correlated with survival data, allowing clinicians to better predict the failure of tumor response to treatment.83,87 In one study, pretreatment 18F-FMISO PET was performed on 17 patients with untreated head and neck squamous cell carcinoma (HNSCC). Local control rates with radiotherapy were significantly lower in the tumor group with high uptake of 18F-FMISO compared to the tumor group with low uptake of 18F-FMISO using either SUVmax or tumor-to-muscle ratio (TMR) as the hypoxic indicator.88 Combined 18F-FMISO PET imaging and intensity-modulated radiotherapy (IMRT) planning permitted delivery of higher doses to hypoxic regions, increasing the predicted TCP (mean 17%) without increasing expected complications.89 Increased equivalent uniform dose (EUD) of the hypoxic volumes has also been confirmed.90 However, the changes in spatial distribution of tumor hypoxia, as detected in serial FMISO PET imaging, compromised the coverage of hypoxic tumor volumes achievable by dose-painting IMRT.

In addition, 18F-FMISO PET imaging has been applied to monitor tumor response to various antiangiogenesis therapeutics.9194 Treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid, Vadimezan), an antivascular compound, resulted in a marked reduction in the 18F-FMISO mean standardized uptake value (SUVmean) in approximately half of the treated tumors. The reduction in SUVmean correlated with a decrease in the fraction of tumor area staining positive for both EF5 and pimonidazole. Compared with untreated controls, tumors with decreased SUVmean had significantly fewer perfused microvessels. Thus, a reduction in 18F-FMISO SUVmean after DMXAA treatment was indicative of reduced perfusion and therefore delivery of 18F-FMISO, rather than a reduction in tumor hypoxia.91 A total of 13 patients with locally advanced HNSCC underwent 18F-FMISO PET scans before and after neoadjuvant chemotherapy (NAC). All PET indexes of 18F-FMISO significantly decreased after NAC. Although hypoxic volume (HV) in primary tumor and a few indices before NAC in responders was lower than that in nonresponders, none of the change in indices were statistically significant.93 In another preclinical study, two doses of 12 mg/kg VEGF121/rGel, administered intraperitoneally, resulted in initial delay of tumor growth but the growth resumed 4 days after tumor treatment was stopped. 18F-FMISO uptake was increased in the treated tumors at day 1 and day 3, compared to the control group. At days 7 and 14, 18F-FMISO uptake restored to the baseline level (Fig. 35.2).94

Inconsistent results and controversies still exist in 18F-FMISO PET imaging. The uptake of 18F-FMISO has a wide variation among patients and different types of tumors. In many cases, no correlation was found between patient diagnosis and the degree of reduction in FMISO uptake and retention. For example, in a prospective study, neither the presence nor the absence of hypoxia, defined by positive 18F-FMISO findings on the midtreatment PET scan, correlated with patient outcome.95 The variability in spatial uptake has been confirmed with two 18F-FMISO PET scans 3 days apart in patients with head and neck cancer by a voxel-by-voxel analysis.90

To overcome this problem, serial images performed during the course of treatment, rather than baseline volumes, would be most helpful in planning to boost radiation therapy to persistent hypoxic subvolumes. In addition, the time frame is also very important for 18F-FMISO PET imaging because it is a very dynamic process for the uptake, retention, and clearance of the tracer upon injection. Clinical studies on static 18F-FMISO PET for head and neck cancer patients revealed that 18F-FMISO image 90 to 140 minutes after injection led to an operational definition of tumor hypoxia corresponding to a tumor-to-blood activity concentration ratio of greater than 1.2 to 1.6.35,70,83 However, the physiologic clearance of 18F-FMISO from highly perfused normal tissue may result in the same tumor-to-blood ratio (T/B), which is comparable to hypoxic tumor at the time of patient imaging. At 90 to 180 minutes after injection, the activity concentration in normal tissue continually decreases as a function of time, whereas in hypoxic tumor it increases as a function of time. The possibility of a cross-over point between the decline in activity from a well-perfused tissue region and the increasing activity from a predominantly hypoxic one can result in an ambiguity in the interpretation of single time-point imaging. With dynamic contrast-enhanced (DCE) MRI using Magnevist (Gd-DTPA) and dynamic 18F-FMISO PET, it has been demonstrated that tumor vasculature is a major determinant of early 18F-FMISO uptake.96 Consequently, some investigators proposed that data acquisition of 18F-FMISO should be done 4 hours post injection to gather the optimal contrast, preferably allowing further analysis, for example, hypoxic sub volume definition for therapy planning.97

FIGURE 35.2. A: Representative decay-corrected whole-body coronal micro-PET images of mice bearing MDA-MB-435 breast cancer at 2.5 hours after intravenous injection of 18F-FMISO (1.11 MBq/mouse) on days 0, 1, 3, 7 and 14 after the treatment was initiated. The tumors are indicated by arrows. Increased tumor uptake of 18F-FMISO was observed on day 1 and day 3 but was restored to the baseline level on day 7. Upper panel: Control; Lower panel: VEGF121/rGel treatment. B: Quantitative micro-PET region of interest analysis of tumor uptake for 18F-FMISO (*p < 0.05, **p < 0.01). (Reproduced with permission from Yang M, Gao H, Sun X, et al. Multiplexed PET probes for imaging breast cancer early response to VEGF/rGel treatment. Mol Pharm. 2011;8:621–628. Copyright 2011. American Chemical Society.)

Compared with static imaging, dynamic imaging followed by kinetic analysis has certain advantages. It can be used to quantitatively calculate the perfusion/clearance rates. Furthermore, dynamic imaging facilitates the separation of specific signal from nonspecific signal and thus can be applied to accurately measure the binding potential of an agent.98 Dynamic imaging with 18F-FMISO has been performed either with a mathematical phantom or head and neck cancer patients.99,100 A generic irreversible one-plasma, two-tissue compartmental model was used to analyze the pharmacokinetic parameters including a potential tumor hypoxia index (Ki). There is a positive correlation of 0.86 between the average tumor-to-background (T/B) and average hypoxia index (Ki) of the region of interest. However, the direct correlation between the T/B and hypoxia of the individual pixel is not obvious because of the statistical photon counting noise in PET and the amplification of noise in kinetic analysis.100

In patients with newly diagnosed GBM, the volumetric analysis demonstrated that the viable hypoxic tissue assessed by 18F-FMISO PET is related to the neovascularization in Gd-enhanced MRI and the tumor aggressiveness assessed by 11C-MET PET.101 Moreover, the intratumoral 18F-FMISO uptake pattern matched perfectly with endogenous markers including hypoxia-responsive element (HRE) driven HSV1-TKeGFP and CA9.102 It has also been reported that the uptake volume of FMISO was larger in GBM than in non-GBM, which may distinguish GBM from lower-grade glioma.103 Swansan et al.104 performed T1Gd, T2 MRI, and 18F-FMISO PET studies on a total of 24 patients with glioblastoma preceded surgical resection or biopsy. On 18F-FMISO PET images, the HV generally occupied a region straddling the outer edge of the T1Gd abnormality and into the T2. A significant correlation between HV and the volume of the T1Gd abnormality that relied on the existence of a large outlier was observed, indicating hypoxia may drive the peripheral growth of glioblastomas.104,105

In addition to identifying tumor hypoxia, differentiating between chronic and acute hypoxia, is a worthwhile challenge for noninvasive imaging. Wang et al.106 have developed an iterative optimization method to delineate chronic and acute hypoxia based on the 18F-FMISO PET serial images. They assume that chronic hypoxia is the same in the two scans and has a gaussian distribution, whereas acute hypoxia varies in the two scans and is best represented by Poisson distributions. The model calculation generates the amount of acute hypoxia, which differed among patients, ranging from approximately 13% to 52%, with an average of approximately 34%. However, this model was not confirmed with a follow-up study by comparing microscopic fluorescent staining results with the fractions of acute hypoxia in individual tumors assessed by the model using xenografts of two tumor types. The method needs to be further tested in both experimental and clinical studies.107

Imaging Tumor Hypoxia with 18F-EF5 and 18F-FAZA

Quite a few second-generation nitroimidazoles for tumor hypoxia imaging have been developed including 18F-FETNIM (fluoroerythronitroimidazole),108,109 18F-FETA (fluoroetanidazole),110 18F-EF3 (2-(2-nitroimidazol-1-yl)-N-(3,3,3-trifluoropropyl)acetamide),111 18F-EF5,112,113 18F-NEFA (2-fluoro-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)acetamide), 18F-NEFT (2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-fluoroacetate),114 and 18F-FRAZ (1-α-D-(2-deoxy-2-fluororibofuranosyl)-2-nitroimidazole).115 The full names of these tracers are listed in Table 35.1. In general, these new markers are more water soluble and resistant to degradation by most physiologic oxidation mechanisms. For example, both 18F-FMISO and 18F-EF3 exhibited similar pharmacokinetics and biodistribution in mice and accumulated in tumors in a hypoxia-dependent manner. However, more nonspecific activity was observed with 18F-FMISO at late time points after tracer injection in normal tissues.111 However, another study found that 18F-EF3 is not superior to 18F-FMISO for PET-based hypoxia evaluation as measured in a rat rhabdomyosarcoma tumor model.121

EF5 was reported to be the most stable 2-nitroimidazole derivatives studied to date.122 EF5 distributes evenly throughout soft tissue within minutes of injection. Its concentration in blood over the typical time frame of the study (approximately 3.5 hours) was nearly constant, consistent with a previously determined EF5 plasma half-life of approximately 13 hours. Elimination was primarily via urine and bile.123 18F-EF5 was used in several human trials to determine its feasibility as a hypoxia imaging agent (Fig. 35.3).124 18F-EF5 was found to be hypoxia-specific. The increased uptake of 18F-EF5 was related to the extent of malignancy and high risk of metastasis in cancer patients.112Thus, the technique could be useful to identify high-risk patients in clinical trials to determine whether early chemotherapy will influence the occurrence of metastasis.125 One drawback of EF5 is the complex labeling chemistry and consequently low radiochemical yield.126

TABLE 35.1

LIST OF HYPOXIA IMAGING TRACERS WITH ABBREVIATIONS

Among the second generation of nitroimidazoles, 18F-FAZA has shown promise to assess tumor hypoxia. It has faster diffusion into cells and faster clearance from the normal tissues than 18F-FMISO.127 Two-dimensional spatial distribution of 18F-FAZA, demonstrated by autoradiography, showed a highly significant colocalization with fluorescence images from pimonidazole.128,129 18F-FAZA PET imaging in seven patients with high-grade gliomas showed very high tracer uptake in all patients, indicating that 18F-FAZA is a promising agent for assessing the hypoxic fraction of this tumor type.130

In patients with head and neck cancer, the T/M ratio generally decreased within the first 60 minutes of the dynamic sequence whereas generally increasing at later time points. The mean T/M ratio at 2 hours p.i. was 2 ± 0.3. However, the tumor volume displaying a T/M ratio above 1.5 was highly variable.72 In another study, there was substantial uptake in the primary tumor and/or the lymph nodes in the neck in six out of nine patients with HNSCC, primary lung tumors in 7 out of the 13 lung cancer patients. No side effects of the administration of 18F-FAZA were observed.130 In CT26 colon carcinoma models, it was found that the crucial 18F-FAZA uptake phase is during the first hour after 18F-FAZA injection and was not affected by pure oxygen breathing.131

Using 18F-FAZA to determine the predictive value for success of radiotherapy in combination with tirapazamine, a specific cytotoxin for hypoxic cells, has been evaluated in EMT6 tumor-bearing nude mice. An additive beneficial therapeutic effect of tirapazamine to RT was observed only in hypoxic tumors but not in normoxic tumors.117 In a clinical study, 18 patients with advanced squamous cell head and neck cancer were imaged with 18F-FAZA PET and CT. The results demonstrated the feasibility of using 18F-FAZA PET to guide hypoxia-directed intensity-modulated radiotherapy in head and neck cancer.132 Moreover, 18F-FAZA PET analysis showed that pretreatment tumor hypoxia was prognostic of a satisfactory radiation response. Both 18F-FAZA PET and pO2 electrode showed that the more hypoxic tumors had significantly less tumor control.133 Another clinical study confirmed that PET imaging with 18F-FAZA is feasible in patients with cervical cancer. However, because of the limited number of patients, the predictive and prognostic value of 18F-FAZA remains to be clarified.134

FIGURE 35.3. PET and CT images of one patient with 18F-EF5, 18F-FDG, and 15O-H2O. 18F-EF5 images are made at 3 hours after injection. Arrow indicates the primary tumor, and arrowhead indicates the metastatic lesion. (Reprinted with permission from SNMMI from Komar G, Seppanen M, Eskola O, et al. 18F-EF5: a new PET tracer for imaging hypoxia in head and neck cancer. J Nucl Med. 2008;49:1944–1951.)

Integrin αvβ3, a marker of angiogenesis, is highly expressed on proliferating and activated endothelial cells associated with neovascularization in malignant tumors, but not in quiescent blood vessels. PET imaging with arginine-glycine-aspartic acid (RGD)-based peptides has been used to visualize and quantify integrin αvβ3 expression levels in vivo and to further evaluate the neoangiogenesis in tumors. Under room air conditions, roughly 60% of the tumor surface displayed a spatial coupling of 18F-FAZA and 125I-Gluco-RGD uptake. However, the remaining approximately 40% of the tumor surface showed discordant 18F-FAZA and 125I-Gluco-RGD uptake, indicating that hypoxia and angiogenesis are not necessarily spatially linked to each other.135

In a feasibility study to assess hypoxia kinetic models using voxel-wise cross-analysis between the uptake of the perfusion tracer 15O-H2O and the hypoxia tracer 18F-FAZA, the compartment model, Thorwarth model, Patlak plot, Logan plot, and Cho model were applied to model the process of tracer transport and accumulation under hypoxic conditions. The results demonstrated that hypoxia kinetic modeling delivered different information from static measurements. The reversible two-compartment model gave better correspondence to the initial assumptions than the other models.136 However, with three squamous cell carcinoma tumor models, late time 18F-FAZA PET images provided an accurate measure of hypoxia against which kinetic model estimates can be validated. Tumor TACs varied widely (ranging from distinctly wash-out to accumulative type) among tumor types although pimonidazole staining revealed extensive hypoxia in all models.137

Dithiosemicarbazones

Dithiosemicarbazones were initially discovered for their antioxidant properties in the 1960s that were enhanced when they were complexed with copper. Because of the simplicity of chemistry, Cu(II)-ATSM is gaining acceptance in diagnostic imaging of hypoxia.116 Cu(II)-ATSM is a simple molecule and its biochemical interaction with cells is similarly simple, mainly based upon redox chemistry. In living cells, Cu(II)-ATSM undergoes reduction and is rapidly cleared from the aerobic cells but become trapped in the hypoxic cells, thus can differentiate between dead, hypoxic, nonfunctional, and viable tissue.138

Dearling and Packard139 suggested that the trapping mechanism is biphasic. The first phase is a reduction/oxidation cycle involving thiols and molecular oxygen. This is followed by interaction with proteins in the mitochondria leading to more permanent retention of the tracer.139 Recently, with a unique cell culture model, mitochondrial xenocybrids, Donnelly et al.140 confirmed that compromised electron transport chain (ETC) function, caused by the absence of O2 as the terminal electron acceptor or dysfunction of individual components of the ETC, is an important determinant in driving the intracellular dissociation of Cu(II)-ATSM that increases cellular retention of Cu.140Consequently, the high dynamic range of Cu(II)-ATSM uptake is representative of a narrow range of low oxygen tension whose values are dependent on microenvironment acidity, whereas FMISO uptake is representative of a wide range of pO2 values that are independent of acidity.141

Both in vitro and in vivo data showed that high MDR1-expressing tumors showed lower tracer activity on 64Cu-ATSM PET images, indicating the expression of MDR1 glycoprotein may affect the retention of 64Cu-ATSM in the tumors.142 Cu(II)-ATSM has also been applied as a marker of intracellular overreduced states for disorders with mitochondrial dysfunction, such as MELAS, Parkinson’s disease, and Alzheimer’s disease.143 Moreover, inhibition of fatty acid synthesis resulted in significant increase in 64Cu-ATSM retention in prostate tumor cells in vitro under anoxia over 60 minutes. Thus, the translation of Cu-ATSM to the imaging of prostate cancer may be limited by the overexpression of fatty acid synthase associated with prostatic malignancies.144

Most clinical copper-ATSM studies have used the agent labeled with the short-lived positron-emitting radionuclide of copper,145 60Cu (half-life, 0.395 hours; β+-decay, 92.5%; electron capture, 7.5%).146 To enable copper-ATSM to be translated for use in PET centers that do not have an in-house cyclotron, copper-ATSM labeled with one of the longer-lived positron-emitting nuclides, 64Cu (half-life, 12.7 hours; β+-decay, 17.4%; β−-decay, 38.5%; electron capture, 43%) or 61Cu (half-life, 3.33 hours; β+-decay, 62%; electron capture, 38%),145,147 is required. The longer half-lives of 64Cu and 61Cu allow for production at a regional center and distribution to PET facilities in a fashion similar to that for 18F-labeled radiopharmaceuticals. The preparation of 62Cu (half-life, 0.16 hours; β+-decay, 98%; electron capture, 2%), via a 62Zn/62Cu generator system, has been reported and commercialized148,149 and offers an additional method for radiolabeling copper-ATSM for use in humans.

Studies in animal tumor models have shown good correlations between tumor uptake of 64Cu-ATSM and oxygen electrode measurement. The imaging results were also comparable with 18F-FMISO PET.150 However, 64Cu-ATSM tumor uptake was found to be less sensitive to oxygen level change whereas 18F-FMISO tumor uptake was more responsive to changing levels of hypoxia.151 The regional comparisons between 64Cu-ATSM (10 minutes) and 18F-FDG (1 hour) resulted in a very poor correlation between the regional uptake of the two agents. The comparison between 18F-FLT and 64Cu-ATSM showed a strong relationship between the two tracers.152

Tumor uptake of Cu-ATSM, as assessed by PET, has been confirmed as a clinically important biomarker of prognosis in several human cancers. Cu(II)-ATSM was first used in human study in 2000 in normal subjects and patients who had lung cancer.153 In all lung cancer patients, 62Cu-ATSM showed high uptake and a plateau can be reached within a few minutes after tracer injection. The maximum tumor-to-background ratio of 62Cu-ATSM was up to 9.33.153 60Cu-ATSM has also been applied to detect hypoxia and to assess the relationship between Cu(II)-ATSM uptake and response to therapy and/or recurrence.154,155 The toxicology and pharmacology data demonstrated that 64Cu-ATSM has an appropriate margin of safety for clinical use compared with 60Cu-ATSM. Besides, the image quality with 64Cu-ATSM was better than that with 60Cu-ATSM because of lower noise. The pattern and magnitude of tumor uptake of 60Cu-ATSM and 64Cu-ATSM on studies separated by 1 to 9 days were similar.156

60Cu-ATSM PET may be predictive of survival and, possibly, tumor response to neoadjuvant chemoradiotherapy in patients with rectal cancer. In patients with locally invasive (T2–T4) primary or node-positive rectal cancer, both overall and progression-free survivals were worse with hypoxic tumors (tumor-to-muscle activity ratio >2.6) than with nonhypoxic tumors (tumor-to-muscle activity ratio ≤2.6).157 In patients with lung cancer of pathohistologically different types, the intratumoral distribution patterns of 62Cu-ATSM and 18F-FDG were different between SCC and adenocarcinoma in lung cancers, indicating that intratumoral regions of high glucose metabolism and hypoxia could differ with the pathohistologic type of lung cancer.158 In addition, Cu(II)-ATSM PET has been shown to distinguish patients likely and unlikely to respond to conventional therapies for cancers of the lung146 and uterine cervix.159

It was found that the Auger radiation emitted by 64Cu can cause DNA damage and induce apoptosis in hypoxic cells, thus 64Cu-ATSM can serve as both an imaging and a therapy agent.160 Although Cu-ATSM may be a valid hypoxia agent for some tumor types, the efficacy varies among different tumors and may be tumor-type specific.154

Galactopyranoside

The iodinated analogs 124I-iodoazomycin galactopyranoside (IAZG) and 124I-iodoazomycin galactoside have also been developed as hypoxia radiotracers and have shown good tumor contrast in ­several xenograft models.161However, compared with 18F-labeled compounds, deiodination, long half-lives of 124I, low positron abundance, high positron energy and accompanied high-energy γ-rays resulted in varied biodistribution, poor image resolution, and higher radiation exposure for 124I-labeled imaging tracers.119,162

SPECT Imaging of Hypoxia

Single photon emission computed tomography (SPECT) uses single photon-emitting radionuclide-labeled hypoxia-specific compounds to generate a signal from hypoxic areas of tumors.63 123I was initially used for this purpose. In one study, 123I-iodoazomycin arabinoside (123I-IAZA) was used in 51 human patients with malignancies and the results demonstrated hypoxia in small-cell lung cancer and squamous cell carcinoma of head and neck.163 The radiopharmacokinetics and the radiation dosimetry of 123I-IAZA confirmed that 123I-IAZA can be used for clinical hypoxia tissue imaging.118,164 Because 123I-IAZA has inconsistent uptake in various tumors, modified iodinated azomycin arabinosides.165 IAZA, iodoazomycin pyranoside (IAZP), β-D-iodinated azomycin galactopyranoside (IAZGP), and iodoazomycin xylopyranoside (IAZXP) have been developed and evaluated as newer agents based on the azomycin-nucleoside structure.120 125I-IAZGP in FM3A mouse tumors 24 hours after administration showed high accumulation within tumors.166

99mTechnetium [99mTc]-labeled nitroimidazoles and nonnitroimidazoles identified as BMS-181,321, BRU 59–21, N2IPA, and 99mTc-HL91 have been used to study tumor hypoxia.120 BMS-181,321 was the first 99mTc-labeled 2-nitroimidazole widely studied for the detection of ischemic and hypoxic myocardium. BMS-181,321 is unstable, has a high partition coefficient, slow clearance from the blood and high background levels in normal tissues, and thus was not optimal for tumor hypoxia imaging.167 BRU 59–21 has greater stability in vitro and more rapid clearance in vivo than BMS-181,321. This compound had shown good retention in tumors compared to muscle and blood and is suitable for tumor hypoxia imaging, as confirmed by pimonidazole staining.168 Yutani et al. found that there is a very good correlation between 99mTc-HL91 retention and hypoxia, as measured by the polarographic electrode. 99mTc-HL91 accumulated to significantly higher levels in hypoxic tumor areas and 99mTc-HL91 uptake was strongly correlated with the expression of GLUT1 in the viable cancer cell area.169,170

ENDOGENOUS HYPOXIA MARKERS

Endogenous molecular markers represent proteins and genes whose expressions are associated with hypoxia. A wide range of potential markers have been studied over the last several years and have been found to be useful to monitor hypoxia.171 Because the biology of tumor hypoxia in tumors is rather complicated and no single marker characterizes prognosis in clinical practice, attempts have been made to combine various markers to create a hypoxia-prognostic profile.172

Hypoxia-Inducible Factor 1 (HIF-1)

HIF-1 is one of the most intensively studied oxygen response pathways in molecular biology. HIF-1, activated by hypoxia, regulates genes that are involved in cell metabolism, angiogenesis, invasion, metastasis, and apoptosis. HIF-1–induced cellular changes are extremely important diagnostic and therapeutic features of cancer cells, particularly in cancers refractory to therapy. The transcription factor HIF-1 is a heterodimer consisting of an oxygen-sensitive α-subunit (HIF-1α) and the constitutively active β-subunit (HIF-1β).173 Under normoxic conditions, prolyl hydroxylases (PHDs) hydroxylate proline sites in the oxygen-dependent degradation domain (ODD) of the HIF-1α protein, and further binding of the von Hippel–Lindau protein (VHL) leads to the ubiquitination and proteasomal degradation of HIF-1α. In addition, oxygen-dependent hydroxylation of three residues within HIF-1α affects the ability of this complex to activate transcription.174

Under hypoxic conditions, HIF-1α is stabilized by hypoxia upon activation of HIF-1. This leads to increased levels of HIF-1α and reductions in hydroxylation, ubiquitination, and degradation of the protein. Accumulated HIF-1 binds to the HRE and triggers the recruitment of coactivators essential for transcriptional activation, thereby promoting the transcription of numerous genes including VEGF, VEGFR2, MMP2, and the genes encoding the glucose transporters. Several cofactors are involved in the transcriptional regulation of various target genes, and the first main inhibitory pathway of HIF-1α is factor inhibiting HIF-1 (FIH-1). The hydroxylation of the C-terminal transactivation domain (CAD) of HIF-1α by FIH-1 inhibits binding of the HIF-1 complex with p300/CBP, which is a cofactor necessary for transcription. Certain receptors of the tyrosine kinase family, like insulin-like growth factor receptor (IGFR), epidermal growth factor receptor (EGFR), and HER2/neu, can activate HIF-1α and regulate the transcriptional activity through the PI3K/Akt/mTOR pathway.173 HIF-1α overexpression is present in a wide variety of malignant tumors such as clear cell carcinoma, ovarian carcinoma, gastric carcinoma, breast cancer, soft tissue sarcoma, bladder cancer, head and neck cancer, rectal, lung cancer, and cervical carcinoma.175,176 Some studies showed that high level of HIF-1α expression indicates poorer outcomes.177 Because of the widespread overexpression of HIF-1 and its influence on multiple cellular functions, HIF-1 has become a promising diagnostic and therapeutic target as an endogenous hypoxia-related marker.

Optical imaging methods have played an important role in evaluating hypoxia, especially in the evaluation of biopsy specimens. Several elegant methods have been developed to directly measure HIF-1 activity by the introduction of transgenes with HREs as promoter sequences coupled to reporter genes such as luciferase178,179 or green fluorescent protein (GFP).180 In these studies, a luciferase reporter gene under the regulation of an HIF1-dependent promoter has been developed. It can express a 100-fold increase of luciferase response to hypoxia and has been used to evaluate the efficacy of hypoxia-directed therapy in animals.181 Shinae et al. developed an imaging probe for HIF-1–active cells with a PTD–ODD fusion protein. Because PTD–ODD fusion proteins underlie the same ODD control as HIF-1α, they are expected to be colocalized with HIF-1α.182 The group first constructed PTD–ODD-enhanced GFP labeled with near-infrared fluorescent dye Cy5.5 for use as a model protein. The results showed that the imaging probe permeated cell membrane with high efficiency, and its stability was controlled in an oxygen concentration-dependent manner. The probe accumulated in hypoxic tumor cells with HIF-1 activity, and in contrast to the surrounding cells that were under aerobic conditions, the hypoxic tumor cells with HIF-1 activity were thus imaged with good contrast.182 Viola et al.183 also used bioluminescence imaging to noninvasively image the upregulation of HIF-1α in vivo after chemotherapy. These imaging tools are useful for studying the biology of hypoxia and mechanisms of response to experimental therapy but the heterogeneity of the gene response makes HIF-1 a complex target.

However, many studies showed only weak correlation between HIF-1α expression and oxygen electrode or PET imaging measurements.184,185 For example, the HIF-1α–positive tumor section surface was much smaller than the tumor section surface of increased 18F-FAZA uptake, suggesting that both markers are identifying distinctly different biologic processes associated with hypoxia.135 Moreover, the HIF-1α–positive tumor cell fraction was not significantly influenced by breathing conditions and covered between 0% and 35% of the total tumor section surface.

Carbonic Anhydrase IX

One downstream target of HIF-1 is carbonic anhydrase IX (CA IX). CA IX is one of the 14 members of the CA family, existing in cytosolic, membrane-associated, mitochondrial, and secreted carbonic anhydrases.186 CA IX is a membrane-associated enzyme involved in the respiratory gas exchange and acid–base balance. The presence of CA IX is limited in normal tissue in gastric mucosa, small intestine, and muscle. The overexpression of CA IX under hypoxic conditions was demonstrated in different types of cancer where a more general staining pattern is observed.187

Dubois et al. imaged CA IX with fluorescently labeled sulfonamides to distinguish hypoxic and (re)oxygenated cells in a tumor xenograft model. The in vivo imaging results confirmed previous in vitro data that CA IX binding and retention require exposure to hypoxia.188 The G250 monoclonal antibody against CA IX has been evaluated in renal-cell carcinoma xenografts and resulted in a significant inhibition of tumor growth.189 G250-based radioimmunoimaging has been used in phase II clinical trials for primary and metastatic lesion detection and guiding radioimmunotherapy after G250 was labeled with effective therapeutic radioisotopes including 177Lu, 90Y, or 186Re.190 Using phage display technology, Ahlskog et al.191 described the generation of high-affinity human monoclonal antibodies (A3 and CC7) specific to human CA IX, which may serve as broadly applicable reagents for the noninvasive imaging of hypoxia and for pharmacodelivery applications. Although the combination of CA IX and a proliferation marker can identify cells that are proliferating under hypoxic conditions,192,193 there is no correlation between the amount of CA IX and direct oxygen measurement with the needle electrode.194

Many additional hypoxia-related markers have been reported to be induced by hypoxia and expressed in hypoxic human tumors. For example, both VEGF and glucose transporters (GLUTs) were upregulated by increased HIF-1 activity under hypoxia condition.195197 Imaging strategies targeting to these proteins have also been intensively investigated to reflect tumor vasculature and proliferation. A direct relationship between pO2 measurements and protein expression, however, has not been established.198,199

CONCLUSIONS

Among the various techniques to measure tumor hypoxia, PET imaging with hypoxia-specific probes is definitely the most intensively investigated. Several imaging agents, such as 18F-FMISO, 18FAZA, and Cu(II)-ATSM, have been extensively applied in the clinic and the data so far supports the promising potential of PET imaging in hypoxia evaluation. Consequently, noninvasive measurement of hypoxia in solid tumors is predictive of drug resistance and radioresistance, dose painting, and monitoring tumor response early after the onset of the treatment. These approaches are promising although inconsistency and controversy still exist. More accurate and comprehensive evaluation of tumor hypoxia is expected with novel imaging probes, advanced or combined imaging instruments, and better alignment of microscopic observation and macroscopic evaluation.

FUTURE CONSIDERATIONS

The ideal technique for hypoxia measurement should be clinically safe, readily available, minimally invasive, with high resolution, and ease-of-use. With the number of techniques available to measure hypoxia, including polarographic needle electrode methods, IHC staining, PET, MRI, optical imaging with NIR fluorescence or bioluminescence, it is crucial to determine the pros and cons of each method before clinical application. There are many PET imaging probes available, each with its own merits and shortcomings. Thus, more preclinical animal experiments and clinical data are required to identify which method best defines hypoxia. Furthermore, a technique to differentiate acute from chronic hypoxia is needed.

Tumor tissue oxygenation is very heterogeneous, both spatially and temporally. Heterogeneity has an important impact on application of the imaging techniques to stratify patients and predict outcomes. Consequently, an effective way to assess heterogeneity has to be established. Synergistic effect of multimodality imaging and multiplexed imaging may be helpful to address the heterogeneity issue. In addition, coregistration of microscopic finding and macroscopic images will be needed at least at the preclinical level.

Reproducibility and repeatability are important aspects to be considered for quantification and comparison of hypoxia imaging data. Inconsistency and variance have been observed with most of PET imaging tracers even in the same tumor type in both preclinical and clinical studies. Thus, imaging acquisition requirements and data analysis methods need to be standardized, although it is generally agreed that more data is needed for such standardization to take place.

ACKNOWLEDGMENTS

This work was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH).

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