in the hypoxic cell, which enables visualization and quantification of vital tissue hypoxia by PET imaging.
Different Nitroimidazole-Based PET-Tracers
Of the different nitroimidazole-based PET-tracers, 18F-fluoromisonidazole (18F-FMISO) was the first and still remains the most extensively studied [32, 33]. Although there is only limited clinical experience with 18F-FMISO, there are some useful reviews describing the proven, assumed, or hypothesized differences between different nitroimidazole-based tracers [10, 23, 32]. In validation studies, a good correlation between 18F-FMISO, visualized by autoradiography, and immunohistochemical visualization of 2-nitroimidazole derivatives was found [34]. Furthermore, preclinical work showed that changes in hypoxia could, to a certain extent, and based on tissue architecture, be visualized and quantified with this compound [35].
18F-FMISO has the limitation of slow pharmacokinetics (about 2 h minimum between administration and imaging) and a poor hypoxic versus normoxic tissue contrast (20–40% difference in uptake). Therefore, other nitroimidazole-based tracers were developed in order to overcome these shortcomings. The different nitroimidazole-based tracers show (small) differences in uptake and imaging characteristics. In general, compounds that are more hydrophilic have a higher clearance and therefore higher tumor-to-blood ratio (T/B) [23]. Overall, theoretical benefits of other tracers, as compared to 18F-FMISO, only lead to relatively small advantages in clinical practice. 18F- fluoroazomycinarabinofuranoside (18F-FAZA) is one of the second-generation nitroimidazole compounds and, due to its reduced lipophilicity, it has a faster washout of normoxic tissue, leading to a higher tissue-to-muscle ratio (about 2.0 at 2 h postinjection) [32]. 18F-HX4 is one of the other more hydrophilic tracers with a faster clearance than 18F-FMISO. Other examples of nitroimidazole-based tracers are 18F-EF3 and 18F-EF5, which have a more complex but also more stable labeling chemistry. Unfortunately, in practice the potential advantage over 18F-FMISO turned out to be limited [32].
Today, many different nitroimidazole-based tracers are under investigation and, while they all have a slightly different profile, it is difficult to predict which one has the best overall potential to succeed 18F-FMISO as the leading hypoxia PET tracer.
In preclinical studies, tracers labeled with other positron-emitting radionuclides have also been tested. For example, 68Ga has the advantage that it can easily be produced with a 68Ge-68Ga generator, which makes the imaging independent of the availability of a cyclotron. The disadvantage of 68Ga is the shorter half-life compared to 18F (68 min vs. 110 min), while uptake of the nitroimidazole-based tracers takes hours. These differently labeled tracers have not (yet) proved to be advantageous over 18F-FMISO.
Imaging of Nitroimidazole-Based Tracers
In general, PET imaging is performed 2–4 h after the administration of the 18F-labeled nitroimidazole-based tracer [33]. This period between administration and imaging is required for sufficient uptake and accumulation of the tracer in the hypoxic tissue. With 18F having a half-life of 110 min, the signal increase due to higher levels of the tracer in hypoxic tissue is partly counteracted by the decay leading to rapid lowering of the PET signal. The detected signal is also influenced by blood perfusion, the distance of passive diffusion, and the image acquisition protocol. Hypoxic tumors are often hypoperfused, which can lead to underestimation of the hypoxia based on imaging of the tracer uptake [10], while imaging within a short interval after administration leads to overestimation from perfusion artifacts in a well-perfused tumor.
Ideally, a parameter representing the pO2 would be calculated from the acquired data. Unfortunately, this is methodologically challenging due to the abovementioned confounders. Therefore, in general the SUV, T/B, and tumor-to-muscle ratios (T/M) are applied for analysis. Alternatively, the hypoxic volume and the hypoxic fraction can be used as parameters for the quantification of hypoxia [36]. The hypoxic fraction is the hypoxic volume, defined as the tumor volume with T/B or T/M above a certain threshold (e.g., >1.2 to >1.4 for 18F-FMISO [32, 37]), divided by the total tumor volume. Some groups use more demanding pharmacokinetic modeling methods to improve quantification by maximal correction for perfusion; however, this is a procedure where the total imaging time is substantially longer, which is cumbersome for patients [38]. Apart from the SUV, these are all relative parameters and sensitive for “inter-corporal” properties (blood volume, perfusion, clearance rate), relying on the definition of representative reference volumes (blood or muscle). Also, the parameters may vary among different protocols (e.g., amount of tracer injected, reconstruction method, correction for breathing motion). Furthermore, the time after administration influences the values, since at later time points the blood will be further cleared, while the uptake in hypoxic tissue increases as long as there is supply of the tracer.
Since hypoxia is in general defined by a signal increase of only 20–40%, the signal-to-noise ratio needs to be maximal, especially for the detection of small hypoxic subvolumes. Besides taking advantage of the optimal imaging timeframe according to the clearance and decay, image noise can be reduced by increasing the acquisition time or the administered activity. The first is at the cost of time and patient discomfort, while the second is at the cost of a higher imaging-induced radiation dose. Compared to often-used 18F-FDG-PET imaging protocols, the time per bed position is much longer. While 18F-FDG-PET is frequently used as a whole body imaging method (e.g., 6 bed positions), tumor hypoxia imaging can mostly be limited to 1 or 2 bed positions only, which reduces the overall time required for imaging.
Mechanism of Cu-ATSM Uptake in Hypoxic Tissue
The other group of PET-based hypoxia tracers consists of Cu-ATSM compounds. Although the uptake mechanism of these tracers is not fully understood, it is known that the lipophilic molecule diffuses through the cell membrane, and within the cell the copper compound undergoes reduction by thiols: Cu(II)-ATSM is converted to Cu(I)-ATSM. In the case of hypoxia, this unstable complex undergoes further reduction and the resulting free Cu(I) becomes rapidly entrapped in intracellular proteins [32].
Experiences with Cu-ATSM
Experiences with Cu-ATSM vary between promising and disappointing. The advantage of Cu-ATSM compared to nitroimidazole compounds is the higher uptake in target tissue (T/M 3.0) within only 10–15 min [32]. However, there are doubts on the hypoxic selectivity since the Cu-ATSM uptake does not always correspond to the distribution of immunohistochemical markers, especially at early imaging timepoints (<16 h after injection) [39, 40]. In future it will be elucidated whether further development of Cu-ATSM tracers can overcome these doubts [10].