(18)F-FDG Is a Surrogate Marker of Therapy Response and Tumor Recovery after Drug Withdrawal during Treatment with a Dual PI3K/mTOR Inhibitor in a Preclinical Model of Cisplatin-Resistant Ovarian Cancer

Abstract

Aim: Targeting the phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway is a potential means of overcoming chemoresistance in ovarian cancer. We investigated the capability of (18)F-fluororodeoxyglucose ((18)F-FDG) small-animal positron emission tomography (SA-PET) to predict the effects of a dual PI3K/mTOR inhibitor (BEZ-235) in a cisplatin-resistant ovarian cancer model.

Methods: In a first experiment, nude rats bearing subcutaneous SKOV3 tumors received BEZ-235 for 3 days given alone or after paclitaxel and were compared to controls (either untreated or that were given the excipients of paclitaxel and BEZ-235). SA-PET was performed at baseline, on day 3, and day 7. In a second experiment aiming at further exploring the kinetics of (18)F-FDG tumor uptake during the first 48 hours following drug cessation, untreated controls were compared to rats receiving BEZ-235, which were imaged at baseline, on day 3, on day 4, and on day 5. SA-PET results were compared to cell proliferation assessment (Ki-67), PI3K/mTOR downstream target expression studies (pAKT and phospho-eukaryotic translation initiation factor 4E-binding protein 1), and apoptosis evaluation (cleaved caspase-3).

Results: In the first experiment, BEZ-235, compared to untreated controls, induced a marked decrease in (18)F-FDG uptake on day 3, which was correlated to a significant decrease in cell proliferation and to a significant PI3K/mTOR pathway inhibition. No tumor necrosis or apoptosis occurred. Four days following treatment cessation, tumor recovery (in terms of PI3K/mTOR inhibition and cell proliferation) occurred and was identified by (18)F-FDG SA-PET. Paclitaxel plus BEZ-235 showed results similar to BEZ-235 alone. In the second experiment, PI3K/mTOR pathways exhibited partial recovery as early as 24 hours following treatment cessation, but both (18)F-FDG SA-PET and cell proliferation remained unchanged.

Conclusions: (18)F-FDG SA-PET is a surrogate marker of target inhibition during treatment with BEZ-235 and predicts tumor recovery 4 days after drug withdrawal, but not during the first 48 hours following drug cessation, when a lag between PI3K/mTOR pathway recovery and metabolic recovery is observed. (18)F-FDG SA-PET could be used for therapy monitoring of PI3K/mTOR inhibitors, but our results also raise questions regarding the potential impact of the delay between PET imaging and the last drug intake on the accuracy of FDG imaging.

Figure 1

Impact of BEZ-235 on tumor growth. Mean ± SD value in each group is shown.

Figure 2

¹⁸F-FDG SA-PET response to BEZ-235 and paclitaxel plus BEZ-235 compared to control groups. (A) Percentage variation (mean ± SD) in SUV_mean compared to baseline is shown on day 3 and day 7. The differences among different groups were tested with the Kruskal-Wallis test, and post hoc test was performed with the Dunn test for multiple comparisons; *, **, and *** indicate two-tailed P < .05, P < .01, and P < .001, respectively. (B) Representative transverse slices at the level of the subcutaneous pelvic or thoracic tumors are shown for the different treatment and control groups.

Figure 3

BEZ-235 inhibits the PI3K/mTOR pathways. (A) Immunohistochemistry studies of representative tumors. The phosphorylation of 4E-BP1 is markedly reduced on day 3 for the BEZ-235 conditions within all the tumor sections. Staining is similar to the control groups on day 7. (B) Western blot of representative tumors: The administration of BEZ-235 inhibits the phosphorylation of AKT and 4E-BP1 on day 3, and this effect is no longer observed on day 7.

Figure 4

BEZ-235 inhibits tumor proliferation. (A) Quantification of Ki-67 staining (percentage of positive cells). The differences among different groups were tested with the Kruskal-Wallis test, and post hoc test was performed with the Dunn test for multiple comparisons; *, **, and *** indicate two-tailed P < .05, P < .01, and P < .001, respectively. (B) Immunohistochemistry studies of representative tumors. Cell proliferation, as assessed by Ki-67 immunostaining, is markedly reduced on day 3 in the BEZ-235 and paclitaxel plus BEZ-235 groups but is similar to control groups on day 7.

Figure 5

No evidence of cell death (apoptosis, necrosis) is observed after 3 days of treatment with BEZ-235. Immunohistochemistry and hematoxylin eosin safran (HES) studies of representative tumors are shown. No necrosis or cleaved caspase-3 is observed in any of the treatment conditions.

Figure 6

Further analysis on ¹⁸F-FDG, PI3K/mTOR pathways, and cell proliferation kinetics following BEZ-235 withdrawal. (A) Percentage variation (mean ± SD) in SUV_mean compared to baseline is shown on day 3, day 4, and day 5. The differences among different groups were tested with the Mann-Whitney test. (B) Immunohistochemistry studies of representative tumors. Cell proliferation, as assessed by Ki-67 immunostaining, is markedly reduced from day 3 to day 5 in the BEZ-235 group compared to the control group. In contrast, p4E-BP1 is markedly decreased on day 3 in the BEZ-235 group compared to the control group, but staining is almost similar to the control groups on days 4 and 5. (C) Quantification of Ki-67 staining (percentage of positive cells). The differences among different groups were tested with the Mann-Whitney test. (D) Western blot of representative tumors: The administration of BEZ-235 inhibits the phosphorylation of AKT and 4E-BP1 on day 3. pAKT increased in a time-dependent manner on days 4 and 5, in contrast with p4E-BP1, whose phosphorylation is more clearly visible on day 5.