Early detection of peritoneal dissemination is essential for adequate treatment planning in the initial staging and follow-up of patients with gastric cancer. The diminishing role of surgical reassessment in treated patients has increased the reliance on cross-sectional imaging.
Accurate detection of peritoneal carcinomatosis by non-invasive means is a challenge in studies on both animals and humans. The peritoneum of the abdomen and pelvis provides a large surface area for microscopic seeding [
10]. Tumour cells may grow on free peritoneal surfaces or invaginate the peritoneal folds over the mesentery or omentum, making some tumours extremely difficult to detect.
A number of imaging modalities for small animals evolved over the last decade and were successfully applied in numerous studies [
8]. These modalities included also preclinical methods such as BLI. Functional imaging using PET was also introduced and scanners constructed for small animal use. These modalities, which differ significantly in temporal and spatial resolution, have inherently different technical requirements. To the best of our knowledge, however, few data comparing PET and BLI have yet been obtained in a single animal study. Our aim, therefore, was to evaluate the sensitivity and overall accuracy of these methods in detecting peritoneal carcinomatosis in a mouse model.
PET
PET using
18 F-FDG was more sensitive than BLI in our study. In general, PET detected lesions independently of the site but with a clear correlation to the size of the lesions. As shown in Table
1, lesions on the peritoneal surface were detected in 60 % of cases (6/10), whereas only one out of seven lesions (14 %) was identified on the diaphragm. This low detection rate might be explained by the size of the nodules, since all undetected lesions measured less than 1 mm. This agrees with the results of Kondo et al. [
11], who reported a limited sensitivity of
18 F-FDG PET for lesions smaller than 4 mm in their animal model.
We observed seven false-positive sites using PET, all of which were found in views of the bowel mesentery. These sites can probably be attributed to focal uptake of 18 F-FDG in the bowel, which is non-specific in nature and not related to tumour tissue. 18 F-FDG uses the same initial pathway as glucose and the uptake is therefore highly dependent on the presence of glucose transporters in the cell membrane. These transporters are present in tumour cells but also in many other tissues. 18 F-FDG is also taken up by inflammatory cells, which may explain false-positive findings in a clinical setting.
The lack of anatomical landmarks, relatively high costs and false-positive results due to focal uptake in non-malignant tissue might be regarded as potential limitations of PET. However, 18 F-FDG is still the most common tracer used in oncology for both clinical purposes and experimental setups. More specific tracers such as 18 F-FLT still need to be validated.
Bioluminescence imaging
Bioluminescence imaging is a modality that perfectly suits the needs of small animal imaging. It is quick, quite easy to handle, and relatively inexpensive [
12]. Bioluminescence imaging has been shown to be sensitive in various animal studies and has also been used to monitor tumour progression and relapse [
13].
In our study, however, BLI was relatively insensitive compared with PET. This finding is, most likely, attributed mainly to light scattering and absorption (amount of tissue between cancer cells and CCD camera). When tumours are growing as a well-defined focal lesion close to the surface e.g. after flank injection, a good correlation between BLI signal and actual tumour mass is usually observed. This correlation is reduced in setups in which multiple lesions grow relatively dispersed in the abdomen, as it is the case in our intraperitoneal model [
14]. The correlation between BLI light signal and tumour volume post mortem (“in vivo”) was not significant. BLI negative lesions resulted not only in a decrease in sensitivity but obviously have also substantial contribution to non-optimal correlation of light signal and tumour size (Fig.
4).
As might be expected, the sensitivity in the abdominal cavity depended on the site of the lesion. As shown in Table
1, all nodules attached to the peritoneum (
n = 10) were detected by BLI. This high detection rate is probably explained by the superficial position of the lesions. Nodules shielded by dense structures or organs such as the liver frequently remained undetected. This might explain why none of the nodules on the diaphragm and only one lesion on the liver (1/13) could be seen.
It is important to notice, however, that the sensitivity of BLI is potentially dependent on various other factors, such as luciferase expression levels, transfection stability, oxygenation and tumour viability, as well as the performance and the setting of the camera. Some uncertainty may also remain with respect to a perfectly homogeneous distribution of the implanted tumour cells and the substrate D-luciferin in peritoneal cavity.
In principle, the substrate D-luciferin can be administered to animals using intraperitoneal (i.p.), subcutaneous (s.c.) and intravenous (i.v.) injections. After i.p. injection D-luciferin is absorbed by the peritoneum and reaches luciferase-expressing tissues mainly via bloodstream. In fact, variations in absorption rate through the peritoneum may distort the reproducibility of signal quantification [
15]. A biodistribution study of radiolabeled D-luciferin demonstrated higher uptake in the gastrointestinal organs (pancreas and spleen) after i.p. injection than after i.v. injection. In addition, direct diffusion, other than the delivery via systemic circulation, may cause preferential distribution of D-luciferin in superficial intraperitoneal tumours close to the injection site [
16]. However, the contribution of direct diffusion of the substrate to the overall signal is probably low owing to poor membrane permeability of D-luciferin [
17]. Therefore, we think that i.p. administration may generally lead to overestimation of luciferase activity of IP tumours relative to extraabdominal tumours rather than causing “false” negative results in intraperitoneal lesions. However, s.c. injection of D-luciferin may be an alternative to i.p. injection for BLI of xenografts in nude mice particularly for tumours with weaker signals and when greater precision is required e.g. in signal validation studies [
18].
Quantification of the BLI light signal may also be distorted due to technical reasons associated with CCD imaging. Larger tumours may show increased optical density and thus increased quenching of the signal as well as reduced availability of substrate to the tumour core compared to smaller tumours. This implies that tumour load may be underestimated when using BLI in vivo. The duration of light exposure to the camera is another potential factor influencing the sensitivity of the system. The exposure time is characterised by a trade-off between imaging depth and overexposure of superficial lesions. The parameter is therefore specific for the experimental setup and technical equipment. The exposure time in our study was derived from experience obtained in previous studies [
8], which means that it was somewhat arbitrary in nature.
Although the correlation between light signal and tumour volume is not significant, changes in tumour mass relative to the initial BLI signal can still be accurately quantified when a uniform position of the animals towards the camera on the different imaging days is achieved. The BLI signal is, thereby, normalised to the baseline of each individual animal during the course of intervention [
14].
Limitations
Our study does have certain limitations. First, the numbers of animals and lesions undergoing imaging were relatively small. On the other hand, the pre-test probability of the presence of peritoneal tumour was quite high, because all mice had at least one tumour on post mortem examination. In consequence, our study did not address the true “sensitivity” of the methods used for detection but derived only relative sensitivities. For this reason, we cannot determine whether our findings could be extrapolated to a non-selected patient population or to other malignancies. Furthermore, the lack of a true negative fraction did not allow the specificity to be determined. The limited application of BLI in human studies is also obvious.