Background

[PubMed]

Hypoxia in malignant tumors can affect the outcome of anti-cancer treatments. Malignant tumors are relatively resistant to chemotherapy and irradiative therapy because of their lack of oxygen, a potent radiosensitizer. This is the reason for the multitude of efforts to develop methods and imaging techniques for measuring oxygen in tissues. Radiolabeled 2-nitroimidazole compounds have been developed to specifically detect tumor hypoxia, offering a less invasive and less technically demanding alternative to the Eppendorf (oxygen) electrode method. The imaging technique also avoids sampling bias.

[¹⁸F]Fluoromisonidazole ([¹⁸F]FMISO) was proposed as a tracer for determining tumor hypoxia in vivo with clinical positron emission tomography (PET) in 1984. Since then, it has become the most widely used nitroimidazole derivate for this technique (1, 2). [¹⁸F]FMISO has been shown to selectively bind to hypoxic cells both in vitro and in vivo. It is used to quantitatively assess tumor hypoxia in lung, brain, and head-and-neck cancer patients (3, 4) and in the hearts of patients with myocardial ischemia (5, 6).

[¹⁸F]FMISO is reduced and binds selectively to macromolecules within hypoxic cells. [¹⁸F]FMISO is relatively hydrophilic and diffuses across cell membranes, showing a passive distribution in normal tissues (4). Because of the slow reaction mechanisms and the absence of active transport of the tracer molecules, the identification and quantification of hypoxic tumor areas necessitate long examination protocols. Also, in severe conditions (pO₂ ≤ 2-3 mmHg), hypoxia is underestimated by [¹⁸F]FMISO uptake measurements, possibly because of the existence of a threshold below which the bioreduction and cell uptake of [¹⁸F]FMISO does not increase any further (7).

Synthesis

[PubMed]

Two main strategies for synthesizing [¹⁸F]FMISO are reported in the literature: a nucleophilic substitution on a protected precursor (8, 9) or epoxide ring opening; and the production of the intermediate [¹⁸F]epifluorohydrin, followed by coupling to 2-nitroimidazole (10). Grierson et al. (11) proposed a two-step synthesis producing [¹⁸F]FMISO with a high yield (40% end of bombardment (EOB)), high purity (>99%), and a specific activity of 37 TBq/mmol. Through this method, the fluoroalkylating agent [¹⁸F]epifluorohydrin is first obtained by displacing (2R)-(−)glycidyltosylate (GOTS). [¹⁸F]FMISO is then obtained by reaction of [¹⁸F]epifluorohydrin with 2-nitroimidazole and further purification through high-performance liquid chromatography (HPLC).

One of the most promising methods seems to be a nucleophilic substitution of the tolysate-leaving group by [¹⁸F]fluoride on the tetrahydropyranyl-protected precursor 1-(2′-nitro-1′-imidazolyl)-2-O-tetrahydropyranyl-3-O-toluenesulfonylpropanediol (NITTP), with hydrolysis of the protecting group. An automated synthesis of [¹⁸F]FMISO by this method has been reported in the literature, using either HPLC or Sep-Paks for the purification of the radiotracer (1, 8). The chemical yield obtained when using NITTP was found to be ≤40% (and reproducible), with a radiochemical purity ≥97%, and a specific activity of about 34 TBq/mmol (8).

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

[¹⁸F]FMISO uptake was reported in various studies using tumor cell lines such as V-79, EMT-6, RIF-1, and canine osteosarcoma, incubated under hypoxic conditions (7). Those experiments showed in vitro good selectivity of [¹⁸F]FMISO for hypoxic cells. Reported uptake ratios between hypoxic and normoxic cultures ranged from 12 to 27, depending on the tumor type. In other in vitro studies using V-79, EMT-6(UW), RIF-1, and CaOs-1 cell lines, it was shown that the decreased uptake in hypoxic cells was directly related to increased oxygen concentrations, and that an O₂ level between 720 and 2300 ppm reduced [¹⁸F]FMISO binding by 50%, relative to binding under anoxic conditions (12). Studies using Chinese hamster V79-171b spheroids exposed to 50 mm [³H]FMISO for 1 to 6 h under aerobic (5% CO₂ in air), hypoxic (5% CO₂, 5% O₂, in N₂), or anoxic (5% CO₂ in N₂) conditions showed that the uptake in anoxic spheroids was similar to that in anoxic cell monolayers. In experiments performed by Casciari et al. (13), [¹⁸F]FMISO-PET showed a tumor hypoxic fraction of 15%, a value consistent with those obtained from radiation survival assays (17%) and measurements of oxygen consumption (22%).

Human Studies

[PubMed]

[¹⁸F]FMISO has been used in humans to assess hypoxia and perfusion in brain tumors. Data obtained by Bruehlmeier et al. (4) using [¹⁸F]FMISO-PET and [¹⁵O]H₂O on patients with glioblastoma showed a positive correlation between [¹⁸F]FMISO tumor uptake at 0-5 min after injection and [¹⁵O]H₂O, with r values between 0.42 and 0.86. No relation between [¹⁸F]FMISO uptake and perfusion was found at 150-170 after injection. Despite the nature of the covalent binding of [¹⁸F]FMISO metabolites (19), a degree of reversibility of [¹⁸F]FMISO tumor binding was reported by Bruehlmeier et al., who suggested this might be attributable to a limited “back diffusing” effect from the cells.

[¹⁸F]FMISO-PET human studies, including comparative studies using both [¹⁸F]FMISO and FDG (20), showed that [¹⁸F]FMISO uptake varied greatly, depending on the tumor types and the tumor sites in the same patient (21, 22). Generally, increased [¹⁸F]FMISO tumor uptake was found in tumor margins rather than in the centers (4).

[¹⁸F]FMISO-PET has also been used as a noninvasive method to assess tumor hypoxia in humans with head-and-neck cancer (HNC) and non-small cell lung cancer (NSCLC). In a reported study (3) involving 40 patients with advanced HNC (n = 26) or NSCLC (n = 14) before curative radiotherapy, dynamic (0-15 min) and static PET scans were performed over a 4-h time period after injection. SUVs and ratios-to-reference tissues (mediastinum or muscle) were calculated. Results for HNC showed that patients with local recurrence could be separated from disease-free patients by SUV, 4 h after injection (SUV > 2 for all recurrences). On the other hand, for NSCLC, no similar correlation was observed. The tumor-to-muscle ratios (T/Mu) and tumor-to-mediastinum ratios (T/Me) at 4 h after injection correlated with the risk of relapse in both tumor entities: all patients with a T/Me > 2.0 (NSCLC, n = 5) or with a T/Mu > 1.6 (HNC, n = 5) had tumor recurrence, whereas only 3 of the remaining 11 patients experienced a recurrence (27%).

A variety of compartment models using human [¹⁸F]FMISO-PET data have been proposed to quantify tumor hypoxia. In 2005, Thorwarth et al. (23) developed a model based on dynamic HNC patient data. This model takes into account the variations of tumor vasculature obtained by immunohistochemical studies and the slow uptake of severely hypoxic and necrotic tissues overlooked by previous models based on SUVs (24).

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