.

      Advantages and Disadvantages of PET-Based Hypoxia Imaging

Besides being a time-consuming procedure with hours between administration and imaging and a poor contrast between hypoxic and normoxic tissue, PET-based hypoxia imaging has the major disadvantage of a low resolution, resulting in problematic detection of small but potentially relevant volumes of hypoxic tissue [23]. A more general challenge of hypoxia imaging is its dynamic nature; it remains questionable whether a single PET can provide enough reliable information to make the treatment dependent on this biomarker [41–43]. The advantage of imaging biomarkers for hypoxia is that they provide 3D information about the tumor tissue, in a noninvasive manner. This enables longitudinal monitoring and facilitates further research in this field.

      MRI-Based Hypoxia Imaging

      Besides PET, hypoxia and perfusion may also be visualized with MRI. Different MRI protocols have been suggested for this, for instance dynamic contrast enhanced (DCE) MRI in which gadolinium is typically used as a paramagnetic contrast agent. With DCE MRI, different parameters can be retrieved (e.g., k^trans/k_ep, v_e) for the visualization of tissue perfusion.

Reduced perfusion might indicate tumor hypoxia, and therefore DCE MRI can be used as a surrogate for hypoxia imaging. However, increased vascular permeability leads to higher intratumoral contrast agent levels, while it may be associated with increased hypoxia [37, 44].

Other examples of interesting sequences/protocols, although not (yet) with a proven predictive value, are blood-oxygen level-dependent (BOLD) MRI [45], which visualizes the ratio between paramagnetic deoxyhemoglobin and diamagnetic oxyhemoglobin, and the less mature tissue oxygen level dependent (TOLD) MRI, and mapping of oxygen by imaging lipid relaxation enhancement (MOBILE). These could be used for the visualization of tissue oxygenation in water and in lipids, respectively [44].

      In MRI, the acquisition itself can be performed with many different settings, and many different parameters can be retrieved from the acquired data. Without denying the value of this field of research, in depth discussion about the advantages and disadvantages of these different techniques and applications is not included here.

      Imaging of Proliferation

      Proliferation and Radioresistance

In cancer tissue, a typical misbalance between the rate of cell death and the rate of cell proliferation occurs, resulting in tumor growth. Generally, within tumor categories the tumors with the highest proliferation rate have (without treatment) the worst prognosis [12], although conflicting study results have been published [46]. Generally, a higher proliferation rate before treatment and early during treatment may be related to a worse response to radiotherapy [8, 16], and especially accelerated proliferation during the course of radiotherapy is related to a poorer outcome.

      Causes of Accelerated Proliferation

Irradiation results in the killing of well-oxygenated tumor cells, and therefore the tumor volume reduces. For the remaining malignant cells this leads to improved supply of oxygen and nutrition, a process referred to as “reoxygenation.” Before therapy, these cells had to compete with many more tumor cells, which apparently would have led to spontaneous cell loss. Instead, due to radiotherapy, these cells are enabled for further proliferation. Proliferation itself is also stimulated in response to ionizing radiation [47]. This mechanism of proliferation of tumor cells, stimulated or enabled by radiotherapy, is called accelerated repopulation [48].

      Counteracting Radioresistance due to Proliferation

      Especially in fast-responding tumors, accelerated repopulation should be considered in the treatment design. Practically, specifically for head-and-neck squamous cell, and non-small-cell and small-cell lung cancer, this risk of repopulation during the treatment course is known [48]. In case of a longer treatment time, in these cancers accelerated repopulation plays a more prominent role and reduces the effectiveness of the radiotherapy. In this situation, tumor cell kill due to radiotherapy is attenuated by accelerated repopulation. In order to increase the treatment response by limiting the effect of accelerated repopulation, different approaches, such as accelerated radiotherapy or radiotherapy combined with another therapy, e.g., cetuximab, have been suggested. Since these are all at the cost of increased side effects [16], careful patient selection for this treatment modification is crucial. Imaging of proliferation, or accelerated repopulation, has the potency to select those patients or patient groups that require an intensified treatment, and could thereby contribute to an improved treatment outcome.

      PET-Based Proliferation Imaging

One way to measure proliferation, or the effect of proliferation, is to measure the anatomical size of a tumor. Besides the delay in the visibility of the size-effect, even after weeks, size does not provide information on the tissue viability and is therefore an impaired biomarker. This makes ¹⁸F-fluorothymidine (¹⁸F-FLT) PET imaging, which visualizes cell proliferation, an attractive alternative. As it is not (yet) routinely used in the clinical practice of radiotherapy, it is an often-used imaging biomarker in research. Dose escalation based on ¹⁸F-FLT PET has been suggested and was shown to be technically feasible [49].

      Mechanism of ¹⁸F-FLT Uptake and Accumulation

¹⁸F-FLT is a radiolabeled variant of the nucleoside thymidine. After intravenous administration, ¹⁸F-FLT gets transported into the cell by nucleoside transporters. The enzyme thymidine kinase 1 (TK1) leads to monophosphorylation of ¹⁸F-FLT and thereby to trapping of ¹⁸F-FLT in the cell [9, 31]. During the S-phase of the cell cycle (DNA synthesis) TK1 is increased about 10-fold [49], which makes ¹⁸F-FLT visualize cell proliferation. This is confirmed by comparison of the ¹⁸F-FLT uptake with the cellular proliferation biomarker Ki-67. Ki-67 is exclusively present in the active phases of the cell cycle, and therefore strictly associated with cell proliferation [50]. ¹⁸F-FLT uptake and immunohistological analysis of Ki-67 expression showed a positive correlation [3], with the advantage of ¹⁸F-FLT being noninvasive and providing information of the entire imaged volume (whole body is possible), at the cost of a much lower resolution.

      Imaging of ¹⁸F-FLT

¹⁸F-FLT PET provides reproducible quantitative imaging results [