Combinatorial Treatment with mTOR Inhibitors and Streptozotocin Leads to Synergistic In Vitro and In Vivo Antitumor Effects in Insulinoma Cells.

Streptozotocin-based chemotherapy is the first-line chemotherapy recommended for advanced pancreatic neuroendocrine tumors (pNETs), whereas targeted therapies, including mTOR inhibitors, are available in second-line treatment. Unfortunately, objective response rates to both treatments are limited. Because mTOR pathway activation, commonly observed in pNETs, has been reported as one of the major mechanisms accounting for chemoresistance, we investigated the potential benefit of mTOR inhibition combined with streptozotocin treatment in a subset of pNETs, namely insulinomas. To evaluate the potential of mTOR inhibition in combination with streptozotocin, we selected four different inhibitors acting at various levels of the pathway (everolimus: inhibition of mTORC1; MK-2206: inhibition of AKT; BKM120: inhibition of PI3K, mTORC1, and mTORC2; and BEZ235: inhibition of mTORC1 and mTORC2). Effects on cell viability and apoptosis were assessed in insulinoma cell lines INS-1E (rat) and MIN6 (mouse) in vitro and were confirmed in vivo by using a mouse model of hepatic tumor dissemination after intrasplenic xenograft. In vitro, all four combinations display synergistic effects. These combinations lead to heterogeneous mTOR pathway inhibition, in agreement with their respective target, and increased apoptosis. In vivo, tumor growth in the liver was significantly inhibited by combining streptozotocin with everolimus (P = 0.0014), BKM120 (P = 0.0092), or BEZ235 (P = 0.008) as compared to each agent alone. These results suggest that targeting the mTOR pathway in combination with streptozotocin could be of potential benefit for insulinomas and pNET patients and thus support further clinical investigations. Mol Cancer Ther; 17(1); 60-72. ©2017 AACR.

Prediction of response to everolimus in neuroendocrine tumors: evaluation of clinical, biological and histological factors.

Objectives Several targeted therapies are available for metastatic neuroendocrine tumours (NETs) but no predictive factor of response to these treatments has been identified yet. Our aim was to identify and evaluate clinical, biological, histological and functional markers of response to everolimus. Methods We retrospectively reviewed 53 patients with NETs treated with everolimus (68 % in clinical trials). Clinical, biological and histological data were analyzed. The functional marker p-p70S6K, a main effector of the mTOR pathway, was studied by immunohistochemistry in 43 cases. Prognostic factors of progression-free survival (PFS) were studied by Kaplan Meier analysis. Results All patients had metastatic and progressive disease before everolimus treatment. Objective response was 9 % and median PFS was 8.1 (4.7-11.5) months. Hypercholesterolemia (HR = 0.13, p < 0.0001) was associated with longer PFS, whereas presence of bone metastases (HR = 3.1, p < 0.001) and overexpression of p-p70S6K by tumor cells (HR = 2.5, p = 0.01) were associated with shorter PFS under everolimus at multivariate analysis. Conclusion Clinical markers are not useful to predict response to everolimus. However, occurrence of hypercholesterolemia under treatment may be an early marker of response. Prospective studies are required to confirm these results and to assess whether p-p70S6K immunostaining is a prognostic or predictive marker of no-response to everolimus.

Antitumor effect of everolimus in preclinical models of high-grade gastroenteropancreatic neuroendocrine carcinomas.

BACKGROUND/AIMS: While the range of therapeutic options for well-differentiated gastroenteropancreatic neuroendocrine tumors has recently increased with the emergence of targeted therapies, such as mTOR inhibitors, there is no recent progress in the treatment of poorly differentiated neuroendocrine carcinomas (PDNECs). Since PDNECs have been shown to strongly express mTOR pathway components, the aim of the present study was to assess the antitumor effect of the mTOR inhibitor everolimus in preclinical models of PDNECs. METHODS: The expression of mTOR pathway components and their response to everolimus were assessed in two neuroendocrine cell lines: STC-1 and GluTag. A xenograft model of intrahepatic dissemination in the nude mouse, based on the intrasplenic injection of either STC-1 and GluTag tumor cells, was used. Animals were started on everolimus treatment 3 days after injection. The effects of treatment on tumor growth, proliferative capacities, apoptosis and in situ expression of mTOR pathway components were assessed. RESULTS: The expression of mTOR pathway components was comparable in STC-1 and GluTag cells and in human PDNECs and could be inhibited in vitro by everolimus. In vivo, the tumor volume of STC-1 and GluTag xenografts was significantly reduced in treated animals (6.05 ± 1.84% as compared to 21.76 ± 3.88% in controls). Everolimus treatment also induced a significant decrease in Ki67 index and in the phosphorylation levels of the two major effectors of mTOR, p70S6K and 4E-BP1. CONCLUSION: Our experimental data suggest that mTOR inhibition could be considered a therapeutic option for high-grade gastroenteropancreatic neuroendocrine tumors.

Contrast-enhanced ultrasound after devascularisation of neuroendocrine liver metastases: functional and morphological evaluation.

OBJECTIVE: To evaluate morphological and perfusion changes in liver metastases of neuroendocrine tumours by contrast-enhanced ultrasound (CEUS) after transarterial embolisation with bead block (TAE) or trans-arterial chemoembolisation with doxorubicin-eluting beads (DEB-TACE). METHODS: In this retrospective study, seven patients underwent TAE, and ten underwent DEB-TACE using beads of the same size. At 1 day before embolisation, 2 days, 1 month and 3 months after the procedure, a destruction-replenishment study using CEUS was performed with a microbubble-enhancing contrast material on a reference tumour. Relative blood flow (rBF) and relative blood volume (rBV) were obtained from the ratio of values obtained in the tumour and in adjacent liver parenchyma. Morphological parameters such as the tumour's major diameter and the viable tumour's major diameter were also measured. A parameter combining functional and morphological data, the tumour vitality index (TVI), was studied. The Wilcoxon rank-sum test and Fisher's test were used to compare treatment groups. RESULTS: At 3 months rBF, rBV and TVI were significantly lower (P = 0.005, P = 0.04 and P = 0.03) for the group with doxorubicin. No difference in morphological parameters was found throughout the follow-up. CONCLUSIONS: One parameter, TVI, could evaluate the morphological and functional response to treatments.