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4113 Background: Predictive biomarkers of response to antiangiogenics currently remain elusive, with several molecules discovered but not fully validated nor clinically applied. TALENT trial is a multicenter prospective phase II study of lenvatinib, a VEGFR1-3 and FGFR1-4 multikinase inhibitor (MKI), in advanced G1/G2 neuroendocrine tumors (NETs) from pancreatic (panNETs) and gastrointestinal (giNETs) origins, which has reported the highest overall response rate (ORR) by central radiology assessment with a MKI in this setting. Here we report the plasma biomarker study. Methods: Proangiogenic profiling of plasma samples from patients included in the trial were analyzed by multiplex ELISA (custom made Quantibody Array, RayBiotech). Quantitative determinations of VEGF-A, FGF2, FGF4, Ang2, IL8, PlGF, VEGF-C, VEGF-D and VEGFR2 were obtained from 85 samples of sufficient quality (out of 111 patients included in the study). Association and prediction of response were evaluated for each biomarker and correlated with PFS, OS and ORR in all patients and the different subgroups included in the trial. Results: While none of the factors were able to discriminate PFS or OS in the whole population, a significant association of high-Ang2 and low-FGF2 to ORR was observed. Subgroup analysis confirmed this association in the two cohorts of patients, giNETs (p = 0.003) and panNETs (p = 0.024). In the panNET cohort, prior targeted therapy was mandatory. Prior sunitinib exposure was observed in 8 patients. Of the factors studied, Ang2 and VEGFR2 were significantly decreased in plasma from sunitinib-pretreated patients (-73% p = 0.022 and -62% p = 0.042 respectively). Furthermore, in these sunitinib-pretreated patients Ang-2 and VEGFR2 levels associated to ORR (p = 0.029 and p = 0.029 respectively) and were able to discriminate PFS (log rank p = 0.027 and 0.007 respectively). ROC curve analysis defined Ang2 and VEGFR2 plasma levels with optimal predictive power to be 415pg/ml and 1770pg/ml respectively (p = 0.021). Conclusions: Plasma proangiogenic profiling of TALENT patient samples unraveled high-Ang2 and low-FGF2 as predictive biomarkers of response to lenvatinib in both cohorts. In antiangiogenic-pretreated patients, Ang2 and VEGFR2 levels significantly predict response to treatment in panNETs. Importantly, current studies with MKIs should confirm the value of these markers for advanced NETs not only to predict response, but also to stablish sequential treatment options for these patients.

551 Background: Anti-angiogenic (AA) drugs are the cornerstone of first-line (FL) treatment in renal cell carcinoma (RCC). While several mechanisms of resistance to AA have been described, second line (SL) therapies are disappointing with short PFS reported. A recent strategy makes use of individual patient-derived tumor models in animals for drug testing simultaneously with that patient’s FL and SL treatments (AVATARs). The predictive value of each specific AVATAR for that same original patient will be key in order to predict SL treatment response in the clinic trying to help in decision making in this setting. Methods: We generated a unique panel of patient-derived mouse models of RCC based on the orthotopic implantation of primary renal tumor biopsies or metastasis directly obtained from patients. Sunitinib FL treatment followed by different SL treatments are evaluated in each mouse model. Response to treatments in these models and molecular profiling in search of predictive factors of response/resistance will be performed. Results: In this subset of 12 advanced RCC patients, tumor take rate in mice to develop AVATARs was 75% at 5 months after implantation. Previous data demonstrated a positive association of grafting capacity with tumor stage and Fuhrman grade (p < 0.0001). AVATAR models faithfully reproduced each patient’s histological characteristics and metastatic capacity. Time to tumor growth in AVATAR models significantly correlated to clinical outcome in each patient (spearman correlation, p < 0.0001). These advanced AVATAR models are treated FL with AA drugs and, at the moment of emergence of resistance to therapy, treatment is switched to known and experimental SL treatments in different animal cohorts. Updated results on AVATAR treatment and prediction of patient’s responses will be presented. Conclusions: Thorough characterization of these AVATAR models together with their response to AA treatments we will be able to predict response/resistance to new SL treatments for our RCC patients as a personalized cancer therapy approach.

e15627 Background: Anti-angiogenic (AA) drugs are the cornerstone of fist-line (FL) treatment in renal cell carcinoma (RCC). While several mechanisms of resistance to AA have been described, second line (SL) therapies are disappointing with short PFS reported. A recent strategy makes use of individual patient-derived tumor models in animals for drug testing simultaneously with that patient’s FL and SL treatments (AVATAR). The predictive value of each specific AVATAR for that same original patient will be key in order to predict SL treatment response in the clinic trying to help in decision making in this setting. Methods: We generated a unique panel of patient-derived RCC mouse models based on orthotopic implantation of primary renal tumor biopsies or metastasis directly obtained from patients. Sunitinib FL treatment followed by different SL treatments are evaluated in each mouse model. Response to treatments in these models and molecular profiling in search of predictive factors of response/resistance wi...

484 Background: Everolimus treatment is currently approved for a few tumor types and currently evaluated in several other malignancies, including NETs. Nevertheless, there is an urgent need for histological or molecular factors of prediction of response, as these would help discriminate subgroups of patients more likely to respond. Here we report on a biological sub-study on histological markers to unravel the relationship between the activation of the IGFR-mTOR-S6 signal transduction pathway with treatment response to everolimus plus octreotide combined therapy in patients with GI NETs. Methods: IGFR-1 (Abcam #54274), phospho-mTOR (Ser-2448, Cell Signaling #2976) and phospho-S6 (Ser-235/236, Cell Signaling #4857) were evaluated by immunohistochemistry (according to Choe G. et al, Cancer Research. 2003) from paraffin embedded blocks from patients with progressive locally advanced or metastatic non-functioning GI NETs, included in a national phase II clinical trial (EVERLAR, NCT01567488). Study endpoint was PFS, and 50% of the patients had progressed at the time of analysis. Univariant (Chi square) and multi-variant (COX) statistical analysis were performed. Results: 38 patients were evaluated with a median age of 62.7 years, balanced on gender (57 % vs. 42%), of which 76.3% were metastatic, mostly to liver. IGF1R positivity showed a nonsignificant trend to association to treatment response (50% progression vs. 21% progression, p=0.15). Nevertheless, mTOR activation (phospho-mTOR positivity) was associated with progression (median PFS of 100 days vs. 225 days, p=0.019). Multivariant COX regression analysis showed a Hazard Ratio of 2.96 [0.8-10.2] (p=0.087) for phospho-mTOR activation. Conclusions: Contrarily to expected results, mTOR activation status determined by phospho-mTOR immunohistological positivity seems to be associated to worse outcome in terms of progression to everolimus+somatostatin analogs in GI NETs patients. This could be indicative of a refractoriness or primary resistance to treatment based on a threshold of mTOR activation, possibly due to activation feedback-loops.

e22057 Background: Metronomic chemotherapy has been suggested as a maintenance administration strategy after the Maximum Tolerated Dose (MTD) treatment in a multi-targeted chemo-switch schedule (C-S). We report the effectiveness of this chemo-switch schedule in mice models of human pancreatic and ovarian cancer. Methods: In pancreas cancer models, Gemcitabine (G) was administered on four different schedules: metronomic (METG), Maximum Tolerated Dose (MTDG), chemo-switch schedule (C-SG, MTDG dosing followed by the METG) and anti-angiogenic (MTDG followed by the administration of monoclonal anti-VEGF receptor antibody DC101). In ovarian cancer model, animals were treated following a Cyclophosphamide chemo-switch schedule (C-S CTX). Results: In all these models C-S schedule had the most favorable effect, reaching at least 80% of tumor growth inhibition in absence of increased toxicity. Moreover, in the pancreas cancer model while peritoneal metastases were observed in control and MTD groups, no dissemination was observed in the MET and C-S groups. C-S treatment caused a decrease in angiogenesis and its effect on tumor growth was similar to obtained by the MTD followed by anti-angiogenic DC101 treatment. At the molecular level, C-S treatment combined an increase in thrombospondin-1 expression with a decrease in number of CD133+ cancer cells and triple positive CD133+/CD44+/CD24+ cancer stem cells. Conclusions: These findings provide confirmation that the chemo-switch schedule is a challenging clinical strategy with demonstrated inhibitory effects on tumor dissemination, angiogenesis and cancer stem cells.

4139 Background: NETs are a heterogeneous group of rare neoplasms. Preclinical studies have shown that IGFR overestimulation can lead to constitutional activation of mTOR pathway. The main objective of this study is to describe the activation status of IGF1R-mTOR pathway by immunohistochemistry in a series of NETs and to assess the association with IGF1R expression. Methods: We studied 69 paraffin tumour blocks: 28 pancreatic NETs (pNETs) (2 gastrinomas, 1 glucagonoma, 4 insulinomas and 21 non-functioning (nf) pNETs), 32 GI NETs (4 stomach, 8 colorectum, 18 ileum and 2 dudodenum) and 9 NETs from other origins (2 anterior mediastinum, 2 ovary, 3 bile duct and 2 kidney). The expression of IGF1R, phosphorylated (p) mTOR and (p)S6 has been determined by immunohistochemistry using anti-IGF1Rβ (sc-713), anti-Phospho-mTOR (S2448) (49F9, Cell Signaling) and anti-Phospho-S6 Ribosomal Protein (S235/336)(91B2, Cell Signaling). Results: Expression of IGF1R has been observed in 46 of 69 (66%) samples with the highest expression in 2/4 (50%) insulinomas, 5/21 nf pNETs (23%) and 3/18 ileum (17%). We have observed activation of the mTOR pathway by immunodetection of (p)mTOR in 14 of 69 samples (20%): 2/21 nf pNETs (9.5%), 1/8 colorectum (12.5%), 8/18 ileum (44%), 1/2 anterior mediastinum, 1/2 ovary, 1/3 biliar tract. We haven’t detected any (p)S6 activation in these samples. Consistent IGF1R-mTOR pathway activation was detected in 11/14 (78%) samples with mTOR positivity that also showed expression of IGF1R. The most relevant finding is that all of the 8 samples from ileum with activation of mTOR showed some degree of expression of IGF1R. Conclusions: 2/3 of NETs show varying levels of expression of IGF1R, but only 16% demonstrate activation of the IGF1R-mTOR pathway. While the positivity of (p)mTOR in pNETs is lower than expected, we have identified a subgroup of ileal NETs with consistent activation of both IGF1R and (p)mTOR which could help stratify patients in clinical trials involving modulation of this pathway. In contrast, none of the 69 samples studied was positive for (p)S6 which suggests this marker is not valid to be used in the clinic. Further validation studies are required to help clinical stratification for therapies against IGF1R mTOR pathways.

4140 Background: The PAZONET trial (Grande et al, ESMO 2012) performed by the GETNE group analyzed the efficacy and safety of the use of pazopanib (800 mg/qd) in patients (pts) with progressive metastatic NETs that had been previously treated with other novel targeted agents. Here we report on the relationship between clinical outcome and circulating and tumor-related biomarkers. Methods: Kaplan-Meier analysis was used to correlate progression-free survival (PFS) with: blood circulating markers at baseline and after 12 weeks of treatment (VEGF-A and sVEGFR2 circulating plasma, Circulating Tumor Cells (CTC) and Circulating Endothelial Cells (CEC)), tumor functional status, Ki67, concomitant somatostatin analogues (SSA), chromogranin A (CgA) and primary tumor location. Results: 44 pts were enrolled, 42 were evaluable for response. sVEGFR2 decreased after 12 weeks of treatment (median decrease 20%, p20% and 10% 4.1 mo). Conclusions: sVEGFR2 and baseline CTC are promising predictive biomarkers for pazopanib in NETs. The updated results confirm the efficacy of pazopanib as a sequencing treatment of pts with progressive NETs, particularly in those with pancreatic primary tumors. The combination of pazopanib and SSA seems to be synergistic. Clinical trial information: NCT01280201.

16070 Background: Germ cell tumors (GCTs) of the testis are highly curable, but patients refractory to cisplatin (CDDP) based chemotherapy have a poor prognosis. Several studies support an important role of angiogenesis in GCT, suggesting anti-angiogenic treatment as a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor with antiangiogenic and antitumoral activities. We evaluate the effect of sunitinib, CDDP or the combination of both compounds on tumoral growth using an orthotopic model of human testicular GCTs (Piulats et al., AACR 2006; abstract 2760). Methods: Mice were implanted with 4 different tumors: a yolk sac without prior exposure to CDDP; a choriocarcinoma from a CDDP-resistant patient; and finally both a choriocarcinoma without prior exposure to CDDP and its CDDP refractory variant induced in mice by a continous CDDP exposure. Mice were treated with sunitinib (40 mg/kg, daily for 15 days), CDDP (2 mg/kg three times at 5-day interval) or combination of both. Animals wer...

12031 Background: Human tumor xenografts in mice can be remarkably predictive of response in humans to cytotoxic chemotherapeutic drugs. Tumor endothelial cells are sensitive to the action of conventional cytotoxic drugs when they are regularly administrated at low doses. This concept, known as metronomic chemotherapy, has been demonstrated in preclinical studies using transplanted tumor models. We aim to investigate the potential anti-tumoral activity of Gemcitabine (G) when administered in a low-dose schedule in an ortothopic implantation model of human pancreatic carcinomas. Methods: Standard gemcitabine schedule: NP18 tumor orthotopically implanted nude mices were randomly distributed to experimental (n = 13, G100 mg/kg intraperitoneally on days 0, 3, 6 and 9 post-implantation) and control group (n = 13, saline). Animal were sacrificed after 4 weeks and we compared weigths (grams) and volume (cm3) of tumors betwen the two groups by the Mann-Whitney U test. Metronomic schedule: After a toxicological study an optimal metronomic dose of 1 mg G /kg per day was chosen. Thirty xenografted mices were randomly distributed to experimental group (n = 15, intraperitoneal G1 mg/kg) and control group (n = 15, saline) and treated for 30 days. Animal were analysed as described before. Results: Standard schedule: Tumor weight mean of treatment group was 0.01 grams ± 0.01 versus 0.54 grams ± 0.48 of the control group. Tumor volume mean in G group was 0.01 cm 3 ± 0.01 versus 0.51 cm 3 ± 0.67) in the control group.Treatment significantly inhibited NP18 tumour growth (p < 0.001). No differences in mice weight were observed between both groups. Metronomic schedule: Tumor weight mean in the treatment group was 0.04 grams ± 0.08 versus 0.53 grams ± 0.46 in control group. Tumor volume mean in G group was 011 cm 3 ± 0.19 versus 0.37 cm 3. Treatment with low-dose of G significantly inhibited NP18 tumour growth (p < 0.003). There were no differences in mice weight between the two groups. Conclusions: Our data show that G administered in a metronomic schedule is effective in inhibiting the growth of NP18 tumor orthotopically implanted in the nude mice. We now aim to study the angiogenic profile of tumors receiving the standard and metronomic schedule and to set up a new experiment to compare survival benefit in the animal model. No significant financial relationships to disclose.