18F-FAZA is, like 18F-FMISO, a 2-nitroimidazole compound, but sugar-coupled (Figure 1). As already mentioned, 2-nitroimidazole compounds undergo reduction under hypoxic conditions, forming highly reactive oxygen radicals. Subsequently, they bind to macromolecules in the intracellular compartment and trapped inside hypoxic cells30. When 18F-FAZA is labeled with the radioisotope 18F, it can be detected by a PET scanner.

Kumar et al31 were the first that reported on the synthesis of 18F-FAZA by fluorination of 1-α-D-(2,3-di-O-acetylarabinofuranosyl)-2-nitroimidazole with DAST followed by deprotection. Their main objective was to develop a PET imaging compound that was similar to the SPECT compound 123I-IAZA, but that was less lipophilic than 18F-FMISO. Theoretically, a less lipophilic compound may produce higher perfusion and faster clearance from blood resulting in a better hypoxia-background-ratio. In a rat model, they showed that 3H-FAZA had similar biodistribution, tumor uptake, and pharmacokinetics as 123I-IAZA, but was indeed less lipophilic than 18F-FMISO.

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Figure 1. Molecular structure of 18F-FAZA

IN VITRO

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F-FAZA EXPERIMENTS AND

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F-FAZA-PET XENOGRAFT MODELS

As all new radiopharmaceuticals, 18F-FAZA is also intensively investigated in both in vitro and in vivo experiments. The detection of hypoxia-dependent radiopharmaceutical accumulation is fundamental, just like exact characterization of pharmacokinetic features.

In the first in vitro study by Busk et al.32, oxygenation-dependent 18F-FAZA retention was compared to that of 18F-FDG in different carcinoma lines. 18F-FAZA accumulation was measured after radiopharmaceutical incubation in different oxygenation conditions. Significant 18F-FAZA retention was observed after 3 hours of anoxia and no binding in nonhypoxic cells. Furthermore, it showed superior in vivo hypoxia specificity compared with 18F-FDG. This study concluded that 18F-FAZA has excellent in vitro characteristics for hypoxia imaging. Another study33 compared the hypoxia-selective uptake of 18F-FAZA to that of 18F-FMISO, as the mostly used hypoxia radiopharmaceutical, and found no differences in the vitro experiments with tumor cell lines, but in the in vivo animal PET study the elimination of

18F-FAZA was found to be faster. This feature seems to be an advantage in imaging, but the concentration of 18F-FAZA was lower, which suggests a lower sensitivity of 18F-FAZA. In a later animal PET study, obvious superior biokinetics for 18F-FAZA were found as compared to 18F-FMISO, for example, significantly higher T/M and tumor-to-blood ratios34.

Beck et al35 evaluated the predictive value of 18F-FAZA in a hypoxic xenograft tumor model and found that delayed tumor growth by radiotherapy can be further enhanced by adding the hypoxic sensitizer tirapazamine to radiotherapy. This effect has only been observed in hypoxic tumors, but not in normoxic ones. High

18F-FAZA uptake was found to be an independent negative prognostic factor for tumor progression. Furthermore, 18F-FAZA-PET was able to predict the success of the hypoxia sensitizing treatment of tirapazamine and radiotherapy.

The distribution of 18F-FAZA in xenograft tumors was examined by Busk et al.36. The 18F-FAZA-PET images were compared to the spatial distribution of 18F-FAZA using autoradiography, to polarographic hypoxia mapping using Eppendorf electrode measurements, and to pimonidazole fluorescence imaging. The consistency of

18F-FAZA distribution with hypoxia was verified using these various methods. The spatial link between 18F-FAZA and pimonidazole was further justified by the same research group37. Interestingly, the same paper describes substantial spatial overlap but also areas of mismatch of 18F-FAZA and the endogenous hypoxia marker GLUT-1. The authors explained these conflicting data by regional blood flow changes in solid tumors and the latency of endogenous hypoxia markers. Namely, acute decrease in tumor perfusion can result in 18F-FAZA retention without accumulation of GLUT-1, or on the contrary, reoxygenated regions with the presence of GLUT-1 have no 18F-FAZA accumulation. There is of course one more explanation, that is, the possible unreliable features of endogenous hypoxia markers, which is already described earlier38.

The same research group published their data on dynamic 18F-FAZA-PET imaging in three squamous cell carcinoma xenograft models39. In this study, 3 hours after

18F-FAZA administration, high tumor-to-reference tissue ratios were found. At the same time, using pimonidazole and 18F-FAZA autoradiography, a strong correlation was observed between hypoxic cell density and 18F-FAZA concentration. These results confirmed that late 18F-FAZA imaging provides a reliable measure of hypoxia, but the time-activity curves were more dependent on tumor type than on the extent of hypoxia. These conflicting data are explained to be the result of the generic 2-compartment model applied in the study, which assumes homogeneous tumors; however, intratumoral hypoxia is known to be heterogeneous.

An interesting study by Meier et al.40 tried to answer the question whether different breathing protocols affect oxygenation status of xenograft tumors models. The eventual hypoxic areas have been visualized using 18F-FAZA-PET in vivo and by in vitro pimonidazole immunohistochemical staining. Beside the

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visualization of tumor hypoxia, this study also focused on the reversibility of

18F-FAZA binding in normoxic muscle applying three different breathing protocols.

The uptake and clearance of 18F-FAZA has found to be oxygen supply dependent in both examined carcinomas, but it remained constant in normal muscle tissue.

18F-FAZA accumulation showed correlation with the hypoxia immunostaining.

These results confirm that 18F-FAZA is a reliable hypoxia biomarker.

Recently, a Danish group compared the prognostic value of 18F-FAZA-PET scanning and Eppendorf needle sampling in an animal model41. Oxygenation status of subcutaneously grown tumors in mice has been defined with both methods before single dose radiation. Both 18F-FAZA-PET and Eppendorf pO2 histography have found to be prognostic of therapy response.

The change of hypoxia during radiotherapy remains an interesting issue. Busk et al.42 monitored tumor hypoxia change in xenograft models before and after fractionated radiotherapy using 18F-FAZA-PET. Although during treatment, overall radiopharmaceutical accumulation changed in the experimental animals there was no signs of re-oxygenation. These results prove the high reproducibility of hypoxia PET scanning with 18F-FAZA but contradict the results of other human studies, where significant changes were observed during therapy22.

Based on these in vitro and xenograft model experiments 18F-FAZA-PET seems to be a reliable and reproducible imaging technique to visualize hypoxia.

HUMAN

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F-FAZA-PET STUDIES

To the best of our knowledge, only eight studies have been published evaluating

18F-FAZA-PET in humans16,43-49. The main results of these studies are summarized in Table 1. All 11 head and neck cancer patients included in the publication by Souvatzoglou et al.43 were also analyzed in the publication by Grosu et al.16, but the latter study included an additional seven patients. Therefore, both publications are considered as one study.

In the study of Grosu et al.16, 18F-FAZA-PET was performed before radiotherapy in patients with head and neck cancer. Hypoxic areas were identified in the primary tumor in 15 of 18 patients. In four of these patients, the hypoxic area was diffusely dispersed throughout the primary tumor. Therefore, no single area of hypoxia could be identified in these patients. Postema et al.44 found large differences in the amounts of hypoxic areas across different tumor sites. In their study, hypoxic areas

were seen in all patients with high-grade gliomas. For the other tumor sites there were significantly less hypoxic areas, especially in patients with lymphomas; only 14% of these subjects had increased 18F-FAZA uptake. Unfortunately, the reasons for these large differences in hypoxia between tumor sites are not discussed. In the study by Schuetz et al.45, 18F-FAZA-PET scans were performed before, during and after external beam radiotherapy in patients with cervical cancer. Five of the 15 patients showed increased 18F-FAZA uptake with inhomogeneous patterns.

Due to the small number of patients, 18F-FAZA-PET could not be used to predict treatment outcome. In the study by Shi et al.46, five patients with head and neck cancer were scanned by dynamic PET using 18F-FAZA and 15O-H2O. The results of this study show that different kinetic models provide different estimates of tumor hypoxia, and different models may even rank patients differently. Accordingly, although pharmacokinetic analysis may be superior to static scans, additional studies are required in order to assess the true value of kinetic analysis in this respect. In the recently published study using 18F-FAZA-PET by Garcia-Parra et al.47, 14 patients with prostate cancer were scanned before a prostatectomy. They found no existing clinically relevant hypoxic area’s in localized prostate tumors that could be possibly detected by 18F-FAZA-PET, meaning the limited value of 18F-FAZA in this specific tumor type. A recent human 18F-FAZA-PET study48 investigated the hypoxia using 18F-FAZA-PET in forty patients with head and neck cancer treated by (chemo)radiotherapy. Tumors were located at different primary sites and tumor stage varied between patients. Scans were made before and during therapy. There were 25/40 hypoxic tumors before and 6/13 during treatment. Significantly poorer prognosis was observed of patients with hypoxic tumors (disease-free survival, 60%), compared to non-hypoxic counterparts (disease-free survival, 93%). Despite the relatively low patient population and the diversity of the tumors sites that were investigated, this study further strength the idea that 18F-FAZA-PET scan is a reliable method for hypoxia imaging with prognostic potential. The most recent human

18F-FAZA-PET study compared 18F-FAZA-PET with 18F-FDG-PET scans in 11 patients with stage III-IV non-small cell lung cancer just before chemoradiotherapy49. They concluded that 18F-FAZA-PET imaging is able to detect heterogeneous distributions of hypoxic subvolumes within homogeneous 18F-FDG background. Furthermore, no correlation between 18F-FAZA and 18F-FDG uptake was found. The authors of this study claim that 18F-FAZA provides additional information on tumor hypoxia to18 F-FDG and might be used to develop a tool for guiding individualization of treatment of advanced non-small cell lung cancer.

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In document University of Groningen Discovery of prognostic markers in laryngeal cancer treated with radiotherapy Bruine de Bruin, Leonie (Page 33-38)