Pilot study of PET imaging of ¹²⁴I-iodoazomycin galactopyranoside (IAZGP), a putative hypoxia imaging agent, in patients with colorectal cancer and head and neck cancer

There is substantial evidence that tumor hypoxia is an important indicator of clinical prognosis for human cancer. The hypoxic tumor phenotype is complex; its various aspects include reduced sensitivity to low LET radiation, differential patterns of gene expression, enhanced angiogenesis, and an increased propensity to metastatic dissemination [1–8]. Although a full understanding of all the implications of hypoxia has yet to be achieved, relatively simple polarographic probe measurements in accessible tumors typically show a difference in survival between patients with hypoxic tumors and those with non-hypoxic tumors [9–11]. It follows that a non-invasive imaging method, even if it could provide only the same information as polarographic probes, could potentially help predict tumor behavior and enable individualized courses of treatment to be designed on a rational basis.

Positron emission tomography (PET) using hypoxia-selective radiotracers has been proposed as a non-invasive method of visualizing hypoxia within solid tumors. However, the optimal radiotracer has yet to be identified. Typical candidate radiotracers for hypoxia imaging are radiolabeled small molecules that freely diffuse into (and out of) cells but are differentially metabolized in regions of low pO₂. In hypoxic cells, the molecules are irreversibly reduced and their radiolabeled metabolites bind to macromolecules, resulting in intracellular trapping. Conversely, in non-hypoxic cells, the molecules remain operationally intact and diffuse back out again. Achieving a hypoxia imaging differential requires that the radiolabeled tracer accumulates in sufficient quantity in hypoxic regions while clearing relatively quickly from normoxic regions.

However, hypoxia in tumors is, at least partly, a consequence of a dysregulated and chaotic vascular network leading to an imbalance between oxygen supply and demand. It is likely that this dysfunctional vasculature will also impose some restriction on the delivery of molecular tracers for hypoxia imaging. In contrast, the supply of such tracers to better-vascularized non-hypoxic tumor regions is likely to be greater. Accordingly, the intratumoral distribution of a hypoxia tracer would be expected to undergo a kinetic evolution from an initial state, where it is dominated by vascular supply (higher tracer concentrations in non-hypoxic regions), toward a final state, where it is dominated by the accumulation of trapped metabolites (higher concentrations in hypoxic regions). The hypoxia selectivity of such tracers would thus be expected to increase with time.

There are, however, practical limits to the length of time that is available between tracer administration and imaging. The most commonly used 2-nitroimidazole-based hypoxia radiotracers (¹⁸F-fluoromisonidazole [12, 13] (¹⁸F-FMISO), ¹⁸F-fluoro-azomycin-arabinoside [14, 15] (¹⁸F-FAZA), ¹⁸F-2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide [16, 17] (¹⁸F-EF5), ¹⁸F-fluoro-erythronitroimidazole [18, 19] (¹⁸F-FETNIM)) are all labeled with the positron-emitting radionuclide fluorine-18 (¹⁸F), and its relatively short (110 min) half-life limits the time for PET image acquisition to a maximum of approximately 3 h post-injection. This may be insufficient to produce a static image that reflects only tumor hypoxia. In principle, the longer (12.6 h) half-life of ⁶⁴Cu allows later imaging times for the candidate hypoxia radiotracer ⁶⁴copper-diacetyl-bis(N4)-methylthiosemicarbazone (⁶⁴Cu-ATSM). However, current clinical imaging studies of ⁶⁴Cu-ATSM typically involve early (<1 h post-injection) imaging [20, 21], partly due to historical experience with positron-emitting isotopes of Cu with shorter half-lives (e.g., ⁶⁰Cu = 24 min, ⁶²Cu = 10 min) [22, 23].

As an alternative to static imaging, kinetic analysis of dynamically acquired PET data has been proposed for assessment of tumor hypoxia [24, 25]. This enables generation of parametric images of uptake rate constants or time-activity curve slopes which may, due to the deconvolution of ‘supply’ (i.e., vascular functionality) and ‘demand’ (i.e., hypoxic bioreduction), provide a more accurate representation of tumor hypoxia. This approach, however, has problems of its own related to the long and complex imaging protocols required, image noise, and the accurate determination of arterial input function.

Based on the foregoing considerations, we decided to investigate the use of a long-lived radiolabeled 2-nitroimidazole compound, ¹²⁴I-iodoazomycin galactopyranoside (¹²⁴I-IAZGP) to enable late static imaging of tumor hypoxia. The positron-emitting radionuclide ¹²⁴I has a half-life of 4.2 days enabling, in principle, imaging at times in excess of 24 h post-injection. Additionally, a series of in vitro studies with tumor cells in culture and in vivo studies in murine xenograft models provided support for the hypothesis that ¹²⁴I-IAZGP might be a superior radiotracer for non-invasive imaging of tumor hypoxia using late PET imaging [26–28]. In order to study ¹²⁴I-IAZGP in human subjects, an investigational new drug application (IND 72,013) was submitted to the US Food and Drug Administration (FDA). Subsequently, we performed a clinical study to evaluate the safety, biodistribution, and imaging characteristics of ¹²⁴I-IAZGP in a group of patients with advanced colorectal cancer and head and neck cancer using serial PET imaging. We now present the results of this clinical study.

This study was designed to evaluate the safety, biodistribution, and imaging characteristics of ¹²⁴I-IAZGP in human patients. In particular, the primary imaging function was to assess the time-dependent biodistribution of ¹²⁴I-IAZGP throughout the whole body, rather than its intratumoral distribution, and image acquisition parameters were, necessarily, suboptimal for this latter role. It is also important to note that, like other tumor hypoxia PET tracers, ¹²⁴I-IAZGP is not a tumor imaging agent per se, and its potential clinical role would be to characterize tumors that had been pre-identified by other means. Nevertheless, the fact that no differential uptake of ¹²⁴I-IAZGP was observed in any disease site must be considered disappointing. It is unlikely that this finding is due to the absence of tumor hypoxia in the study patients. For example, Figure 1 shows large volume FDG-avid, centrally cold liver metastases suggestive of significant central necrosis and thus likely to contain perinecrotic hypoxic regions.

There is a major discrepancy between the previous pre-clinical studies of ¹²⁴I-IAZGP, which demonstrated its ability to detect hypoxia in tumor cells both in vitro and in small animal models [27, 28, 33, 34], and these clinical findings. One factor in this discrepancy may be related to the dilution effect when transitioning from a small animal to a human subject (in terms of both mg/kg and MBq/kg). Our pre-clinical studies used activities of approximately 16.7 MBq in 25-g mice [27] or approximately 18.5 MBq in 200-g rats [28], corresponding to approximately 100 to 700 MBq/kg. In contrast, patients in this study were injected with approximately 148 MBq corresponding to approximately 2 MBq/kg, a factor of 50 to 350 times less than the pre-clinical studies.

We hypothesized that the concentration of radioactivity in tumors might just be too low to be detected with the clinical PET scan parameters used. In order to address this, after the ten-patient imaging study was complete, we included one additional patient who was pre-scheduled for surgical resection of a sigmoid tumor. For this patient, approximately 300 MBq (8 mCi) ¹²⁴I-IAZGP was administered the day before surgery, clinical PET/CT images were acquired at 6 and 21 h post-administration, and following surgery, the entire surgical specimen was imaged on a microPET scanner. In addition, tumor tissue sections cut from the surgical specimen were exposed to a phosphor imaging plate for up to 7 days for digital autoradiography (DAR). However, not only were the clinical PET scans negative as before, but the ex vivo evaluation of the surgical specimen also returned negative results. (Additional file 1: Figure S1 shows the DAR results).

The biodistribution of ¹²⁴I-IAZGP was characterized by rapid clearance from the blood and whole body primarily via the urinary and, to a lesser extent, GI tracts, with no significant uptake in the parenchymal tissues apart from the salivary glands, presumably reflecting dissociated iodine. In terms of radiation exposure, 148 MBq (4 mCi) of ¹²⁴I-IAZGP produced absorbed doses to normal parenchymal tissues less than or equal to those produced by typical clinical activities (370 to 444 MBq; 10 to 12 mCi) of ¹⁸F-FDG. However, the absorbed doses to the large intestinal and urinary bladder walls were significantly greater for ¹²⁴I-IAZGP than ¹⁸F-FDG. As absorbed doses to the urinary bladder wall depend on the frequency of voiding, study patients were encouraged to hydrate and to void frequently in order to minimize this.

There is no doubt that the use of long-lived positron-emitting nuclides for imaging has intrinsic dosimetric disadvantages compared to short-lived radionuclides, where the balance between image quality and patient dose is usually more favorable. However, the use of a long half-life radionuclide is unavoidable if it takes a long time for sufficient biological contrast to develop; our hypothesis was that PET imaging of tumor hypoxia was such a case. An additional disadvantage associated with ¹²⁴I is its relatively low positron yield (approximately 24%) coupled with the presence of multiple high-energy cascade gamma rays which act to further decrease the ratio of PET image quality to patient radiation dose.

We found that PET imaging of ¹²⁴I-IAZGP in 2D mode was possible up to, at least, 6 to 8 h post-administration and possibly 24 h, although with an enhanced likelihood of low-count reconstruction artifacts. However, imaging at 48 h was generally not feasible with the activities (approximately 148 MBq) and emission times (typically 6 to 8 min per bed position) used in this study. This contrasts to the case where ¹²⁴I is used to label antibodies with prolonged biological retention, for which clinically useful images may be acquired at times of up to 1 week post-administration [35, 36]. This deficiency may be alleviated to some extent by the use of 3D reconstruction methods as long as appropriate corrections are included for cascade coincidences.

However, even with foreseeable improvements in image acquisition and reconstruction methods, our data indicate the uptake of ¹²⁴I-IAZGP in tumors is insufficient to suggest any clinical role in the management of patients with colorectal or head and neck cancer.

The study reported here is the first trial describing the toxicity and biodistribution of ¹²⁴I-IAZGP in human subjects. We found that the administration of ¹²⁴I-IAZGP was safe and that it was rapidly cleared from the body. PET imaging failed to show any differential uptake of ¹²⁴I-IAZGP in any disease site. While we continue to believe that characterization of tumor hypoxia will be important for individualizing patient treatments, ¹²⁴I-IAZGP PET in its current form is unlikely to provide any clinical utility for assessing hypoxia in colorectal cancer and head and neck cancer. Despite these negative results with ¹²⁴I-IAZGP, there is still a strong theoretical rationale for the concept that late PET imaging should provide the most specific indication of tumor hypoxia. It is hoped that new hypoxia agents will emerge, perhaps featuring alternative long-lived positron-emitting radionuclides such as ⁶⁴Cu, ⁸⁹Zr, or ⁷⁶Br, which can provide a convenient way to perform single time-point imaging of tumor hypoxia.