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Purpose:FGFR1 amplification (FGFR1amp) is recurrent in metastatic breast cancer (MBC) and is associated with resistance to endocrine therapy and CDK4/6 inhibitors (CDK4/6is). Multi-tyrosine kinase inhibitors (MTKIs) and selective pan-FGFR inhibitors (FGFRis) are being developed for FGFR1amp breast cancer. High-level FGFR amplification and protein expression by IHC have identified breast cancer responders to FGFRis or MTKIs, respectively.Experimental Design:Here, we used preclinical models and patient samples to identify predictive biomarkers to these drugs. We evaluated the antitumor activity of an FGFRi and an MTKI in a collection of 17 breast cancer patient–derived xenografts (PDXs) harboring amplification in FGFR1/2/3/4 and in 10 patients receiving either an FGFRi/MTKI. mRNA levels were measured on FFPE tumor samples using two commercial strategies. Proliferation and angiogenesis were evaluated by detecting Ki-67 and CD31 in viable areas by immunofluorescence.Results:High FGFR1–4 mRNA levels but not copy-number alteration (CNA) is associated with FGFRi response. Treatment with MTKIs showed higher response rates than with FGFRis (86% vs. 53%), regardless of the FGFR1–4 mRNA levels. FGFR-addicted PDXs exhibited an antiproliferative response to either FGFRis or MTKIs, and PDXs exclusively sensitive to MTKI exhibited an additional antiangiogenic response. Consistently, the clinical benefit of MTKIs was not associated with high FGFR1–4 mRNA levels and was observed in patients previously treated with antiangiogenic drugs.Conclusions:Tailored therapy with FGFRis in molecularly selected MBC based on high FGFR1–4 mRNA levels warrants prospective validation in patients with CDK4/6i-resistant luminal breast cancer and in patients with TNBC without targeted therapeutic options.

Amyotrophy lateral sclerosis is a disease that causes disability, chronicity and physical handicap. Moreover, the dependence of the patient will increase. Objectives To assess the efficacy of specific interventions designed to train non-professional caregivers of patients diagnosed with amyotrophic lateral disease. Method Descriptive cross-sectional study of an intervention designed for non-professional caregivers of patients with ALS. Pilot study. The target audience of this study are non-professional caregivers of patients with ALS. The level of dependence of the patients is measured with the Barthel index, the Revised Scale of Functional Assessment of Amyotrophic Lateral Sclerosis and Cognitive Behavioural Screening in ALS. The needs of these patients’ caregivers were evaluated according to the Kreutzer questionnaire. A multidisciplinary intervention designed to modify this situation was designed and re-evaluated. Results Finally, seven caregivers were included in the pilot study. They were women, with an average age of 56 years, partners of the patients, with parallel economic activity and with an average level of education. The patients were men with a mean age of 63 years and 1-3 years of disease duration; they presented a moderate-mild Barthel, cognitive impairment, gross motility and respiratory function. The most demanded needs were for "medical / health information" and "involvement in treatment and care". Conclusion Specific interventions to meet the needs of non-professional caregivers are highly effective. Using tools that identify them contributes to improving caregivers' care.

Sickle cell disease (SCD) is a common hemoglobinopathy, secondary to alterations in the β globin chain, resulting in an abnormal hemoglobin variant named as hemoglobin S. These disorders show a wide phenotypical spectrum, and the prevalence of these disorders has significantly changed over the time because of multiple factors such as migration. We report a case of a 17-year-old black male, born in Gambia, diagnosed with sickle cell disease, who presented an associated mutation only described in a Japanese family (Oshima et al., 1996).

The aim of this study was to estimate the incidence of diabetes mellitus in the Basque Country and the risk factors involved in the disease by reassessing an adult population after 7 years of follow-up. In the previous prevalence study, 847 people older than 18 years were randomly selected from all over the Basque Country and were invited to answer a medical questionnaire, followed by a physical examination and an oral glucose tolerance test. In the reassessment, the same variables were collected and the resulting cohort comprised 517 individuals of whom 43 had diabetes at baseline. The cumulative incidence of diabetes was 4.64% in 7 years and the raw incidence rate was 6.56 cases/1000 person-years (95%CI: 4.11-9.93). Among the incident cases, 59% were undiagnosed. The most strongly associated markers by univariate analyses were age >60 years, dyslipidaemia, prediabetes and insulin resistance. We also found association with hypertension, obesity, family history of diabetes and low education level. Multivariate analysis adjusted for age and sex showed that a set of risk factors assessed together (dyslipidaemia, waist-to-hip-ratio and family history of diabetes) had great predictive value (AUC-ROC=0.899, 95%CI: 0.846-0.953, p=0.942), which suggests the need for early intervention before the onset of prediabetes This work was partially supported by grants from the Department of Health of the Basque Country Government (2015111020); ISCIII (PI14/01104), co-funded by ERDF/ESF, "A way to make Europe"/"Investing in your future"; UPV/EHU (IT1281-19); Menarini Group Spain (BCA16/029); Endocrine-European Reference Network (EndoERN 739527); and CIBERDEM (Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders). The study funders were not involved in the design of the study; the collection, analysis, and interpretation of data; writing the report; and did not impose any restrictions regarding the publication of the report.

The transcription factor EVI1 plays an oncogenic role in several types of neoplasms by promoting aggressive cancer features. EVI1 contributes to epigenetic regulation and transcriptional control, and its overexpression has been associated with enhanced PI3K-AKT-mTOR signaling in some settings. These observations raise the possibility that EVI1 influences the prognosis and everolimus-based therapy outcome of clear cell renal cell carcinoma (ccRCC). Here, gene expression and protein immunohistochemical studies of ccRCC show that EVI1 overexpression is associated with advanced disease features and with poorer outcome&mdash, particularly in the CC-e.3 subtype defined by The Cancer Genome Atlas. Overexpression of an oncogenic EVI1 isoform in RCC cell lines confers substantial resistance to everolimus. The EVI1 rs1344555 genetic variant is associated with poorer survival and greater progression of metastatic ccRCC patients treated with everolimus. This study leads us to propose that evaluation of EVI1 protein or gene expression, and of EVI1 genetic variants may help improve estimates of prognosis and the benefit of everolimus-based therapy in ccRCC.

Insulin‐like growth factor levels and chronic lymphocytic leukaemia Spanish Ministry of Economy and Competitiveness–CarlosIII Institute of Health cofunded by FEDER funds/European Regional Develpment Fund (ERDF)–a way to build Europe (PI17/01280, PI11/01810, PI14/01219, PI11/02213, PI15/00966, RCESP C03/09, RTICESP C03/10, RTIC RD06/0020/0095, RD12/0036/0056, Rio Hortega CM13/00232, SV-09-CLINIC-1, CIBERESP and SAF2016-79347-R) and the Agència de Gestió d’Ajuts Universitaris i de Recerca AGAUR(2017SGR1085, 2014SGR756). The ICGC CLL-Genome Project was funded by Spanish Ministerio de Economía y Competitividad (MINECO) through the Instituto de Salud CarlosIII (ISCIII), PMP15/00007 and CIBERONC

Dormant or slow-cycling tumor cells can form a residual chemoresistant reservoir responsible for relapse in patients, years after curative surgery and adjuvant therapy. We have adapted the pulse-chase expression of H2BeGFP for labeling and isolating slow-cycling cancer cells (SCCCs). SCCCs showed cancer initiation potential and enhanced chemoresistance. Cells at this slow-cycling status presented a distinctive nongenetic and cell-autonomous gene expression profile shared across different tumor types. We identified TET2 epigenetic enzyme as a key factor controlling SCCC numbers, survival, and tumor recurrence. 5-Hydroxymethylcytosine (5hmC), generated by TET2 enzymatic activity, labeled the SCCC genome in carcinomas and was a predictive biomarker of relapse and survival in cancer patients. We have shown the enhanced chemoresistance of SCCCs and revealed 5hmC as a biomarker for their clinical identification and TET2 as a potential drug target for SCCC elimination that could extend patients' survival.

Uveal melanoma is considered a rare disease but it is the most common intraocular malignancy in adults. Local treatments are effective, but the systemic recurrence rate is unacceptably high. Moreover, once metastasis have developed the prognosis is poor, with a 5-year survival rate of less than 5%, and systemic therapies, including immunotherapy, have rendered poor results. The tumour biology is complex, but angiogenesis is a highly important pathway in these tumours. Vasculogenic mimicry, the ability of melanomas to generate vascular channels independently of endothelial cells, could play an important role, but no effective therapy targeting this process has been developed so far. Angiogenesis modulates the tumour microenvironment of melanomas, and a close interplay is established between them. Therefore, combining immune strategies with drugs targeting angiogenesis offers a new therapeutic paradigm. In preclinical studies, these approaches effectively target these tumours, and a phase I clinical study has shown encouraging results in cutaneous melanomas. In this review, we will discuss the importance of angiogenesis in uveal melanoma, with a special focus on vasculogenic mimicry, and describe the interplay between angiogenesis and the tumour microenvironment. In addition, we will suggest future therapeutic approaches based on these observations and mention ways in which to potentially enhance current treatments.

Objective Monogenic diabetes can be misdiagnosed as type 1 or type 2 diabetes in children. The right diagnosis is crucial for both therapeutic choice and prognosis and influences genetic counseling. The main objective of this study was to search for monogenic diabetes in Spanish pediatric patients suspected of type 1 diabetes with lack of autoimmunity at the onset of the disease. We also evaluated the extra value of ZnT8A in addition to the classical IAA, GADA and IA2A autoantibodies to improve the accuracy of type 1 diabetes diagnosis. Methods Four hundred Spanish pediatric patients with recent-onset diabetes (mean age 8.9 +/- 3.9 years) were analyzed for IAA, GADA, IA2A and ZnT8A pancreatic-autoantibodies and HLA-DRB1 alleles. Patients without autoimmunity and those with only ZnT8A positive were screened for 12 monogenic diabetes genes by next generation sequencing. Results ZnT8A testing increased the number of autoantibody-positive patients from 373 (93.3%) to 377 (94.3%). An isolated positivity for ZnT8A allowed diagnosing autoimmune diabetes in 14.8% (4/27) of pediatric patients negative for the rest of the antibodies tested. At least 2 of the 23 patients with no detectable autoimmunity (8%) carried heterozygous pathogenic variants: one previously reported missense variant in the INS gene (p.Gly32Ser) and one novel frameshift variant (p.Val264fs) in the HNF1A gene. One variant of uncertain significance was also found. Carriers of pathogenic variants had HLA-DRB1 risk alleles for autoimmune diabetes and clinical characteristics compatible with type 1 diabetes except for the absence of autoimmunity. Conclusion ZnT8A determination improves the diagnosis of autoimmune diabetes in pediatrics. At least 8% of pediatric patients suspected of type 1 diabetes and with undetectable autoimmunity have monogenic diabetes and can benefit from the correct diagnosis of the disease by genetic study., This work was partially supported by grants from Menarini Group Spain (BCA/16/030), University of the Basque Country, UPV/EHU (IT795-13), Department of Health of the Basque Government (GV2016111035) and ISCIII (PI14/01104) integrated into the National R&D&I Plan 2013-2016 and co-financed by FEDER (European Funds for Regional Development). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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.

Purpose: Mutations in the PI3K pathway occur in 16% of patients with pancreatic neuroendocrine tumors (PanNETs), which suggests that these tumors are an exciting setting for PI3K/AKT/mTOR pharmacologic intervention. Everolimus, an mTOR inhibitor, is being used to treat patients with advanced PanNETs. However, resistance to mTOR-targeted therapy is emerging partially due to the loss of mTOR-dependent feedback inhibition of AKT. In contrast, the response to PI3K inhibitors in PanNETs is unknown. Experimental Design: In the current study, we assessed the frequency of PI3K pathway activation in human PanNETs and in RIP1-Tag2 mice, a preclinical tumor model of PanNETs, and we investigated the therapeutic efficacy of inhibiting PI3K in RIP1-Tag2 mice using a combination of pan (GDC-0941) and p110α-selective (GDC-0326) inhibitors and isoform-specific PI3K kinase-dead–mutant mice. Results: Human and mouse PanNETs showed enhanced pAKT, pPRAS40, and pS6 positivity compared with normal tissue. Although treatment of RIP1-Tag2 mice with GDC-0941 led to reduced tumor growth with no impact on tumor vessels, the selective inactivation of the p110α PI3K isoform, either genetically or pharmacologically, reduced tumor growth as well as vascular area. Furthermore, GDC-0326 reduced the incidence of liver and lymph node metastasis compared with vehicle-treated mice. We also demonstrated that tumor and stromal cells are implicated in the antitumor activity of GDC-0326 in RIP1-Tag2 tumors. Conclusions: Our data provide a rationale for p110α-selective intervention in PanNETs and unravel a new function of this kinase in cancer biology through its role in promoting metastasis. Clin Cancer Res; 22(23); 5805–17. ©2016 AACR.

The mechanisms of tumor growth and progression involve the activation of different processes such as neovascularization and angiogenesis. These processes involve tumoral cells and stromal cells. Hence, inhibiting angiogenesis affects tumor growth and proliferation in patients with different types of cancer. Nevertheless, tumoral cells and stromal components are responsible for the resistance to antiangiogenic therapies. The majority of tumors respond to this type of therapy; however, some tumors may be indifferent to antiangiogenic therapies (intrinsic resistance) and other tumors become resistant during treatment (acquired resistance). Different strategies have been proposed to prevent resistance. Preclinical studies and clinical trials are focused to fight this therapeutic approach in order to prevent or delay tumor resistance to antiangiogenic therapies.

Background Antitumor activity of the combination of somatostatin analogues (SSAs) and the mammalian target of rapamycin (mTOR) inhibitor everolimus in patients with neuroendocrine tumors (NETs) has been reported but not confirmed in prospective trials. Materials and Methods This prospective, multicenter, single-arm phase II EVERLAR study evaluated everolimus 10 mg/day and the SSA octreotide 30 mg every 28 days in patients with advanced nonfunctioning well-differentiated gastrointestinal NETs (GI-NETs) that progressed in the last 12 months (ClinicalTrials.gov NCT01567488). Prior treatment with SSAs and any systemic or locoregional therapy was allowed except for mTOR inhibitors. Patients continued treatment until disease progression or unacceptable adverse events (AEs). The primary endpoint was progression-free survival (PFS) at 12 months; secondary endpoints included early biochemical response, objective response rate (ORR) by RECIST v1.0, overall survival (OS), AEs, activation of mTOR pathway (insulin-like growth factor 1 receptor [IGF1R] and phosphoS6 [pS6] expression). Results Forty-three patients were included in the intent-to-treat analyses. After 12 months of treatment, 62.3% (95% confidence interval [CI] 48%–77%) of patients had not progressed or died. The 24-month PFS rate was 43.6% (95% CI 29%–58%). The confirmed ORR was 2.3%, and stable disease was 58.1%. Median OS was not reached after 24 months of median follow-up. Dose reductions and temporary interruptions due to AEs were required in 14 (33%) and 33 (77%) patients, respectively. The most frequent AEs were diarrhea, asthenia, mucositis, rash, and hyperglycemia. No correlation was observed between IGFR1 and pS6 expression and PFS/OS. Conclusion The everolimus-octreotide combination provided clinically relevant efficacy in nonfunctioning GI-NETs, similar to the results of RADIANT-2 in functioning setting. Implications for Practice The EVERLAR study reports prospective data of somatostatin analogue in combination with everolimus in nonfunctioning gastrointestinal neuroendocrine tumors suggesting meaningful activity and favorable toxicity profile that supports drug combination in this setting.

Background The management of advanced neuroendocrine tumors (NETs) has recently changed. We assessed the activity of pazopanib after failure of other systemic treatments in advanced NETs. Methods This was a multicenter, open-label, phase II study evaluating pazopanib as a single agent in advanced NETs (PAZONET study). The clinical benefit rate (CBR) at 6 months was the primary end point. Translational correlation of radiological response and progression-free survival (PFS) with circulating and tissue biomarkers was also evaluated. Results A total of 44 patients were enrolled. Twenty-five patients (59.5%) were progression-free at 6 months (4 partial responses, 21 stable diseases) with a median PFS of 9.5 months [95% confidence interval (CI) 4.8–14.1]. The CBR varied according to prior therapy received, with 73%, 60% and 25% in patients treated with prior multitarget inhibitors, prior mTOR inhibitors and both agents, respectively. A nonsignificant increase in PFS was observed in patients presenting lower baseline circulating tumor cell (CTC) counts (9.1 versus 5.8 months; P = 0.22) and in those with decreased levels of soluble-vascular endothelial growth factor receptor-2 (sVEGFR-2) (12.6 versus 9.1 months; P = 0.067). A trend toward reduced survival was documented in patients with VEGFR3 rs307821 and rs307826 missense polymorphisms [hazard ratio (HR): 12.3; 95% CI 1.09–139.2; P = 0.042 and HR: 6.9; 95% CI 0.96–49.9; P = 0.055, respectively]. Conclusions Pazopanib showed clinical activity in patients with advanced NETs regardless of previous treatments. Additionally, CTCs, soluble-s VEFGR-2 and VEGFR3 gene polymorphisms constitute potential biomarkers for selecting patients for pazopanib (NCT01280201). Clinical trial number NCT01280201.

Background So far, no biomarkers of response to anti-angiogenic drugs are available in colorectal cancer (CRC) treatment. Liquid biopsy technique identifies actionable targets in CRC patients (pts), tracking dynamic mutational changes. We and others described RAS mutant allele fraction in plasma (plMAF) as an independent prognostic marker in metastatic CRC (mCRC). Here, we explored the predictive value of plMAF in RAS mutant pts treated in first line with chemotherapy +/- bevacizumab (bev). Methods A multicentric prospective/retrospective analysis was conducted. We collected data from 226 mCRC pts and selected the subset not eligible for metastasis resection that had basal plMAF sample evaluable for RAS mutant MAF quantification using digital PCR (BEAMing). Pts were stratified as high (≥ 5.8%) or low ( Results From October 2017 to May 2019, BEAMing analysis from 62 basal plasma samples was performed. 42 pts (67.7%) were classified as high and 20 pts (32,3%) as low plMAF. Among high RAS plMAF, 24 pts received FOLFOX+bev (57%) and 20 pts FOLFOX alone (43%). In this high plMAF subgroup, a statistically significant longer PFS favouring FOLFOX+bev was observed when compared to FOLFOX alone (10.7 versus 6.9 months, respectively; HR: 0.30; p = 0.002). In the low RAS plMAF subgroup, no differences in terms of PFS were observed in either arm (8.9 versus 8.7 months, respectively; HR: 0.70; p = 0.6). Multivariate PFS model showed no association between RAS plMAF and clinicopathological variables, except for high RAS plMAF and treatment benefit with FOLFOX+bev. Conclusions Our results indicate that tumor-borne RAS plMAFs may constitute a potential predictive biomarker of efficacy for anti-angiogenic agents in mCRC. Confirmatory studies in randomized cohorts will be performed. Legal entity responsible for the study VHIO (Vall d’Hebron Institute of Oncology). Funding AECC (Asociacion Espanola Contra el Cancer). Disclosure G. Martini: Research grant / Funding (self), Research Project supported by ESMO with the aid of a grant from Amgen: ESMO-AMGEN. E. Elez: Honoraria (self): Merck Serono; Honoraria (self): Sanofi; Honoraria (self): MSD; Honoraria (self): Roche; Honoraria (self): Servier; Honoraria (self): Amgen; Honoraria (self): Array. G. Argiles Martinez: Honoraria (self), Honoraria (institution), Research grant / Funding (self), Travel / Accommodation / Expenses: Hoffmann-LaRoche, Bristol-Myers Squibb, Bayer, Servier, Amgen, Merck Serono, Menarini; Honoraria (self): Menarini; Honoraria (institution): Boston Pharmaceuticals; Honoraria (institution): Genentech; Honoraria (institution): Boehringer Ingelheim. M.J. Ortiz Morales: Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert Testimony: Sanofi. P.G. Nuciforo: Honoraria (self): BAYER; Honoraria (self): MSD; Honoraria (self): Novartis. R. Dienstmann: Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony: Symphogen, Ipsen, Amgen, Sanofi, MSD, Servier; Research grant / Funding (self): MERCK. J. Tabernero: Advisory / Consultancy: Array Biopharma, AstraZeneca, Bayer, BeiGene, Boehringer Ingelheim, Chugai, Genentech, Inc., Genmab A/S, Halozyme, Imugene Limited, Inflection Biosciences Limited, Ipsen, Kura Oncology, Lilly, MSD, Menarini, Merck Serono, Merrimack, Merus, Molecular Part. E. Aranda Aguilar: Advisory / Consultancy: Amgen; Advisory / Consultancy: Bayer; Advisory / Consultancy: Celgene; Advisory / Consultancy: Merck; Advisory / Consultancy: Roche; Advisory / Consultancy: Sanofi. A. Vivancos: Advisory / Consultancy: sysmex; Advisory / Consultancy: Novartis; Advisory / Consultancy: Merck; Advisory / Consultancy: Bristol-Meyers Squidd; Advisory / Consultancy: Guardant Health. All other authors have declared no conflicts of interest.

In this article, the effectiveness of a multi-targeted chemo-switch (C-S) schedule that combines metronomic chemotherapy (MET) after treatment with the maximum tolerated dose (MTD) is reported. This schedule was tested with gemcitabine in two distinct human pancreatic adenocarcinoma orthotopic models and with cyclophosphamide in an orthotopic ovarian cancer model. In both models, the C-S schedule had the most favourable effect, achieving at least 80% tumour growth inhibition without increased toxicity. Moreover, in the pancreatic cancer model, although 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 tumour growth was similar to that produced by the MTD followed by anti-angiogenic DC101 treatment. C-S treatment combined an increase in thrombospondin-1 expression with a decrease in the number of CD133+ cancer cells and triple-positive CD133+/CD44+/CD24+ cancer stem cells (CSCs). These findings confirm that the C-S schedule is a challenging clinical strategy with demonstrable inhibitory effects on tumour dissemination, angiogenesis and CSCs.

Interest in research on neuroendocrine tumors (NETs) has grown in the past 10 years, coinciding with improvements in our understanding of the molecular pathogenesis of NETs. In addition, NETs have become one of the most exciting settings for drug development. Two targeted agents for the management of advanced pancreatic NETs have been approved, but the development of targeted agents for NETs is limited by problems with both patient selection and demonstration of activity. In this review, we analyze these limitations and discuss ways to increase the predictive value of preclinical models for target discovery and drug development. The role of translational research and 'omics' methodologies is emphasized, with the final aim of developing personalized medicine. Because NETs usually grow slowly and metastatic tumors are found at easily accessible locations, and owing to improvements in techniques for liquid biopsies, NETs provide a unique opportunity to obtain tumor samples at all stages of the evolution of the disease and to adapt treatment to changes in tumor biology. Combining clinical and translational research is essential to achieve progress in the NET field. Slow growth and genetic stability limit and challenge both the availability and further development of preclinical models of NETs, one of the most crucial unmet research needs in the field. Finally, we suggest some useful approaches for improving clinical drug development for NETs: moving from classical RECIST-based response end points to survival parameters; searching for different criteria to define response rates (for example, antiangiogenic effects and metabolic responses); implementing randomized phase II studies to avoid single-arm phase II studies that produce limited data on drug efficacy; and using predictive biomarkers for patient selection.

A scanning system that combines broadband diffuse optical spectroscopy and diffuse correlation spectroscopy for non-contact, large field-of-view imaging of small animal models and humans is present ...

A hybrid broadband diffuse optical and diffuse correlation spectroscopy system is used to measure local tissue hemodynamics, oxygen metabolism and gold nanorod concentration. The objective is to pr ...

Gahete et al., The truncated somatostatin receptor sst5TMD4 is associated with poor prognosis in breast cancer and increases breast cancer cell malignancy. Here, we examined the cellular/molecular mechanisms underlying this association, aiming to identify new molecular tools to improve diagnosis, prognosis or therapy. A gene expression array comparing sst5TMD4 stably-transfected MCF-7 cells and their controls (empty-plasmid) revealed the existence of profound alterations in the expression of genes involved in key tumoral processes, such as cell survival or angiogenesis. Moreover, sst5TMD4- overexpressing MCF-7 and MDA-MB-231 cells demonstrated increased expression/ production of pro-angiogenic factors and enhanced capacity to form mammospheres. Consistently, sst5TMD4-expressing MCF-7 cells induced xenografted tumors with higher VEGF levels and elevated number of blood vessels. Importantly, sst5TMD4 was expressed in a subset of breast cancers, where it correlated with angiogenic markers, lymphatic metastasis, and reduced disease-free survival. These results, coupled to our previous data, support a relevant role of sst5TMD4 in the angiogenic process and reinforce the role of sst5TMD4 in breast cancer malignancy and metastatic potential, supporting its possible utility to develop new molecular biomarkers and drug therapies for these tumors., This work has been funded by the following grants: BIO-0139, CTS-1406, PI-0639-2012, BFU2010-19300, BFU2013-43282-R, PI13/00651 and CIBERobn (to RML and JPC); PI-0541-2013 and “Miguel Servet” program (to MDG); PI13/00132, RETICC RD12/0036/0007, S2010/BMD-2303 (to GMB). CIBER is an initiative of Instituto de Salud Carlos III, Ministerio de Sanidad, Servicios Sociales e Igualdad, Spain.

Renal cell carcinoma (RCC) is a complex disease characterized by mutations in several genes. Loss of function of the von Hippel-Lindau (VHL) tumour suppressor gene is a very common finding in RCC and leads to up-regulation of hypoxia-inducible factor (HIF)-responsive genes accountable for angiogenesis and cell growth, such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Binding of these proteins to their cognate tyrosine kinase receptors on endothelial cells promotes angiogenesis. Promotion of angiogenesis is in part due to the activation of the phosphatidylinositol-3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway. Inhibition of this pathway decreases protein translation and inhibits both angiogenesis and tumour cell proliferation. Although tyrosine kinase inhibitors (TKIs) stand as the main first-line treatment option for advanced RCC, eventually all patients will become resistant to TKIs. Resistance can be overcome by using second-line treatments with different mechanisms of action, such as inhibitors of mTOR, c-MET, programmed death 1 (PD-1) receptor, or the combination of an mTOR inhibitor (mTORi) with a TKI. In this article, we briefly review current evidence regarding mechanisms of resistance in RCC and treatment strategies to overcome resistance with a special focus on the PI3K/AKT/mTOR pathway.

The traditional cytotoxic agents are of limited efficacy in the treatment of neuroendocrine tumors of the gastrointestinal tract (NETs). Recent investigations have brought up a number of biological features in this family of neoplasms that could represent targets for anticancer treatment. NETs seem to have an extraordinary tumor vascularization with high expression of proangiogenic molecules such as the vascular endothelial growth factor along with overexpression of certain tyrosine kinase receptors such as the epidermal growth factor receptor (EGFR), the insulin growth factor receptor (IGFR) and their downstream signaling pathway components (PI3K-AKT-mTOR). The rationale of an antiangiogenic approach in the treatment of NETs and the use of other pharmacological strategies such as EGFR, IGFR and mammalian target of rapamycin inhibitors are discussed. Additionally, the emerging results of recent clinical trials with these targeted drugs are presented.

Certain drugs that are used to treat cancer affect blood-vessel formation in tumours. But it seems that these antiangiogenic drugs can reduce the efficiency of other anticancer agents and increase the tumours' aggressiveness.

Pain is an issue that medicine considers of great importance. The treatment of pain and discomfort is essential during hospitalisation procedures, specially for newborn infants because, first, they are not able to communicate that they are in pain; and second large periods of pain or discomfort can lead to major issues. In order to assess the pain/discomfort suffered by patients, healthcare professionals use pain assessment scales: using physiologic parameters (e.g., heart beat, blood oxygen level, etc.) and observed behavioural parameters (e.g., cry, spasmodic movements, etc.) a value for pain is obtained. Current methods for pain assessment have some drawbacks, which could be overcome with the use of computerised systems. With this aim, we present a system that automatically analyses the pain or discomfort levels of newborns. The proposed system allows the remote monitoring of newborns and raises alarms upon specific conditions. Hence, caregivers (e.g., nurses and their assistants) can act accordingly to help relieving the pain. Moreover, the remotely monitoring is also useful for parents, since they cannot stay close to their babies hospitalised in neonate Intensive Care Units.

Tumor growth requires induction of an angiogenic program, and targeting of this program with antiangiogenic drugs shows an impact on tumor progression. However, although they are effective at reducing angiogenesis, these therapies have not produced widespread or enduring clinical benefit, which openly exposes their limitations. Here, we describe the current limitations of these therapies, including the known mechanisms and current controversies. Further, we present some of the recent approaches to predict these limitations and strategies to overcome them. With the development of meaningful predictive biomarkers and effective treatments that impede these limitations, longer and more robust efficacies will be achieved for a wider population of patients. Significance: The clinical benefit of antiangiogenic drugs is restricted because of intrinsic and acquired limitations. Acknowledging and understanding these limitations will not only allow the development of effective predictive biomarkers but also help in devising new therapeutic strategies that achieve longer efficacies for a wider population of patients. Cancer Discov; 4(1); 31–41. ©2013 AACR.

Background Cisplatin (CDDP) resistance in testicular germ cell tumors (GCTs) is still a clinical challenge, and one associated with poor prognosis. The purpose of this work was to test pazopanib, an anti-tumoral and anti-angiogenic multikinase inhibitor, and its combination with lapatinib (an anti-ErbB inhibitor) in mouse orthotopic models of human testicular GCTs. Methods We used two different models of human testicular GCTs orthotopically grown in nude mice; a CDDP-sensitive choriocarcinoma (TGT38) and a new orthotopic model generated from a metastatic GCT refractory to first-line CDDP chemotherapy (TGT44). Nude mice implanted with these orthotopic tumors were treated with the inhibitors and the effect on tumoral growth and angiogenesis was evaluated. Results TGT44 refractory tumor had an immunohistochemical profile similar to the original metastasis, with characteristics of yolk sac tumor. TGT44 did not respond when treated with cisplatin. In contrast, pazopanib had an anti-angiogenic effect and anti-tumor efficacy in this model. Pazopanib in combination with lapatinib in TGT38, an orthotopic model of choriocarcinoma had an additive effect blocking tumor growth. Conclusions We present pazopanib as a possible agent for the alternative treatment of CDDP-sensitive and CDDP-refractory GCT patients, alone or in combination with anti-ErbB therapies.

A scanning system for small animal imaging using non-contact, hybrid broadband diffuse optical spectroscopy (ncDOS) and diffuse correlation spectroscopy (ncDCS) is presented. The ncDOS uses a two-dimensional spectrophotometer retrieving broadband (610-900 nm) spectral information from up to fifty-seven source-detector distances between 2 and 5 mm. The ncDCS data is simultaneously acquired from four source-detector pairs. The sample is scanned in two dimensions while tracking variations in height. The system has been validated with liquid phantoms, demonstrated in vivo on a human fingertip during an arm cuff occlusion and on a group of mice with xenoimplanted renal cell carcinoma. (C) 2016 Optical Society of America

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.

Tumour progression requires the activation of a tumour and stromal cell-driven angiogenic programme, and the targeting of this process demonstrates an impact on tumour growth and progression. The results of preclinical studies have demonstrated a proinvasive/metastatic effect of antiangiogenic treatments with recent evidence supporting a contribution of the stroma to tumour aggressiveness and the short-term effects of antivascular endothelial growth factor therapy. Furthermore, hypoxia-dependent and -independent factors are considered as driving forces for tumour cell escape by altering both the tumour cells themselves and the stroma. This tumour–stromal cell alliance should be taken into consideration for the development of innovative therapeutic options targeting both tumour components to improve clinical benefits for cancer patients.

While traditional cytotoxic drugs have shown limited efficacy in neuroendocrine tumors (NETs), their biological features have been characterized and can be exploited therapeutically. Their most prominent trait is an extraordinary vascularization in low-grade NETs and an hypoxia-dependent angiogenesis in high-grade NETs, which is associated to a significant expression of many pro-angiogenic molecules. Therefore, several antiangiogenic compounds have been tested in these malignancies and among these, sunitinib has demonstrated activity in pancreatic NET patients by dually targeting the VEGFR and PDGFR pathways. In spite of these efficacious clinical results, apparent resistance to antiangiogenic therapies has been described in NETs animal models and in clinical trials. Therefore, overcoming anti-angiogenic resistance is a crucial step in the subsequent development of antiangiogenic therapies. Several strategies have been postulated to fight resistance, but pre-clinical studies and clinical trials will investigate and address these therapeutic approaches in the coming years in order to overcome resistance anti-angiogenic therapies in NETs.

Open Access Article., Targeting the hypoxia response pathway and angiogenesis are two promising therapeutic strategies for cancer treatment. Their use as single strategies has important limitations. Thus, development of combined regimens has become an important step toward improving therapeutic efficacy. Also, non-invasive monitoring of the response to targeted biological therapies, as well as determination of the optimal schedule for combination regimens has become an active field of research over the last five years, with relevance for both preclinical and clinical settings. Here, we used an optical imaging method to non-invasively monitor the functional changes in HIF activity in response to antiangiogenic treatment in a xenograft model of human ovarian carcinoma. A bioluminescent reporter construct containing nine copies of the hypoxia response element upstream of the luciferase gene (9xHRE-luciferase) was characterized in vitro in a panel of tumor cell lines and in vivo in a subcutaneous xenograft model of ovarian carcinoma by means of optical imaging. We showed that in OVCAR-3 subcutaneous xenografts, the most abrupt change in the HIF functional reporter occurs before the onset of massive tumor growth. However, this system failed to detect hypoxia induced upon antiangiogenic treatment due to the compensating effects of increased hypoxia and decreased tumor cell viability caused by imbalanced neovascularization vs. tumor expansion. Therefore, the readout based on HIF functional reporter could be conditioned by the dynamics of tumor growth and angiogenesis, which is highly variable depending on the tumor type, tumor model and stage of progression., This study was supported by grants from the Ministerio de Ciencia y Tecnología/Ministerio de Ciencia e Innovación (SAF2008-03147 to LdP and SAF2010-19256 to BJ), Comunidad Autónoma de Madrid (S-SAL-0311_2006) and the 7th Research Framework Programme of the European Union (METOXIA, project ref. HEALTH-F2-2009-222741). B.M.P. and V.G. have been supported by a grant from the Comunidad Autónoma de Madrid (S-SAL-0311_2006).

Background Radiation-induced DNA double-strand break (DSB) repair can be tested by using pulsed-field gel electrophoresis (PFGE) in agarose-encapsulated cells. However, previous studies have reported that this assay is impaired by the spontaneous DNA breakage in this medium. We investigated the mechanisms of this fragmentation with the principal aim of eliminating it in order to improve the estimation of radiation-induced DNA repair. Methods Samples from cancer cell cultures or xenografted tumours were encapsulated in agarose plugs. The cell plugs were then irradiated, incubated to allow them to repair, and evaluated by PFGE, caspase-3, and histone H2AX activation (γH2AX). In addition, apoptosis inhibition was evaluated through chemical caspase inhibitors. Results We confirmed that spontaneous DNA fragmentation was associated with the process of encapsulation, regardless of whether cells were irradiated or not. This DNA fragmentation was also correlated to apoptosis activation in a fraction of the cells encapsulated in agarose, while non-apoptotic cell fraction could rejoin DNA fragments as was measured by γH2AX decrease and PFGE data. We were able to eliminate interference of apoptosis by applying specific caspase inhibitors, and improve the estimation of DNA repair, and apoptosis itself. Conclusions The estimation of radiation-induced DNA repair by PFGE may be improved by the use of apoptosis inhibitors. The ability to simultaneously determine DNA repair and apoptosis, which are involved in cell fate, provides new insights for using the PFGE methodology as functional assay.

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...

Introduction The role of antiangiogenic therapy in primary hormonal therapy in HER2-negative ER-positive early breast cancer is unknown. Potential biomarkers of response to antiangiogenic therapy are lacking. A phase I clinical trial was conducted with sunitinib and exemestane given at conventional dose (25 mg/d) during 6 months, before surgery. 18 patients were enrolled, 15 in dose level 0 of sunitinib (25 mg/d) and 3 in dose level 1 (37.5 mg/d). Results were presented in SABCS 2011. Main toxicities were: asthenia (50%), leucopenia (28%, all grade 2), diarrhea (28%), mucositis (22%), and hypertension (22%). 10 patients achieved radiological partial response (56%) and 8 patients stable disease (44%). 7 patients (38.89%) obtained a pathological downstaging. Potential biomarkers of response to antiangiogenic therapy are presented. Materials and methods Tissue samples were obtained by fine-needle aspiration or core needle biopsy before starting treatment, one month after and at surgery. All samples were formalin-fixed paraffin-embedded. Ki67, phospho-ERK and mean vessel density (by CD34), were analyzed by immunostaining. At the same time points, plasma levels of angiopoietin 2 (ANG2), soluble VEGFR2 and VEGF were analyzed by ELISA. Basal levels and its changes over time were evaluated and correlated with clinical outcomes. Results Changes in Ki67 were observed, with a median value of 16.44%pre surgery and 12.78% post surgery (p=0.062). A significant decrease in mean vessel density was not observed. Basal levels of plasmatic biomarkers are shown in the next table: Basal levels of plasmatic biomarkers SDPRpPath DownstNo Path DownstpANG 22396+/-6503455+/-13940.083184+/-16102818+/-7680.6VEGF110+/-12577+/-660.5794+/-6388+/-1210.9VEGFR210570+/-22512110+/-43940.3512140+/-432110980+/-31960.55 Values of biomarkers are average in ng/ml ± Standard Deviation. Differences analyzed by student's t-test. Abreviations: SD= stable disease. PR= partial response. p = p value. Path. Downst.= Pathological downstaging. Furthermore, there was a significant decrease in VEGFR2 mean levels after one month of treatment (p=0.0046): 12284±3449 ng/ml at baseline; 8148±3216 ng/ml at one month and 7732±3052 ng/ml at 6 months. Differences between basal and one month determination were significant (p Conclusions Baseline ANG2 levels have a promising predictive value of response in this phase I trial of neoadjuvant combination of sunitinib plus exemestane. There is a significant early decrease in VEGFR2 with treatment with sunitinib. These results should be validated in further studies to improve the selection of patients for antiangiogenic+hormonal therapy. Citation Format: Helena Verdaguer, Serafin Morales, Valentí Navarro, Alba Martinez Lopez, Anna Petit, Fina Climent, Oriol Casanovas, Sònia Pernas. Potential biomarkers of response to primary antiangiogenic and hormonal therapy in post-menopausal women with hormone-positive, HER2-negative primary breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-05.

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.

Background Pazopanib is an oral tyrosine kinase inhibitor of the VEGFR, PDGFR and KIT with demonstrated clinical activity in NETs. The aims of our study were to assess efficacy, safety and potential predictive biomarkers of pazopanib in pts with NETs who may have received previous antiangiogenic or mTOR inhibitor treatment. Methods All pts had pancreatic or extra-pancreatic metastatic NET disease documented as progressive. Pazopanib 800 mg was given daily until disease progression (DP) or unacceptable toxicity. Primary endpoint was Clinical Benefit Rate (CBR) at 6 months (m) defined as Complete Response (CR) plus Partial Response (PR) plus Stable Disease (SD) by RECIST-V-1.0. Optimal two-stage Simon design was utilized with H1 and H0 set at 70 % and 50 % respectively, Kaplan-Meier estimates were used for the analysis of time-to-event variables: 95% CI. Results 41 evaluable pts for response per protocol. 53.7% males, mean age 59.2 ± 10.3 years. At 6 months, 3 pts had PR (7.36%), 32 SD (78.0%), and 6 DP (14.6%), thus the CBR was 85.4%. Global median PFS 48,3 (13.9-82.7) weeks (w). By subgroups, CBR at 6 m and median PFS were 100% and 25.7 w in pts with no previous targeted therapy (9 pts), 88.9% and 48.3 w in pts with previous mTOR inhibitors (9 pts), 83.3% and 50.3 w in pts with previous antiangiogenics (16 pts) and 71.4% and 20.6 w in pts with previous antiangiogenics and mTOR inhibitors (7 pts) respectively. The sum of the longest diameter of target lesions had a decrease > 10% in 32.5 % of pts (37.5% without previous biological treatment, 22.2 % with previous mTOR inhibitor, 31.3% with prior multitarget inhibitor, and 42.9% with previous multitarget and mTOR, p = 0.506). Most frequent toxicities at of any grade were: asthenia (75%), diarrhoea (63%), nausea (42%). Translational studies on angiogenesis and inmunohistochemistry biomarkers and CTCs are ongoing. Conclusions Pazopanib 800 mg daily has promising activity in advanced NET regardless of previous treatment with other targeted therapies. This trial suggests the role that treatment sequencing with novel targeted agents may have in NETs. Disclosure All authors have declared no conflicts of interest.

427 The Major Vault Protein (MVP) Mediates Starvation Resistance of Human Glioblastoma Cells Via Deregulation of the PI3kinase Pathway D. Lotsch, S. Spiegl-Kreinecker, C. Pirker, J. Hlavaty, H. Petznek, M. Grusch, W. Berger. Medical University of Vienna, Internal Medicine I / Institute of Cancer Research, Vienna, Austria, Wagner-Jauregg Hospital, Department of Neurosurgery, Linz, Austria, University of Veterinary Medicine, Institute of Virology / Department of Pathobiology, Vienna, Austria

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.

The longitudinal effect of an anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibody (DC 101) therapy on a xenografted renal cell carcinoma (RCC) mouse model was monitored using hybrid diffuse optics. Two groups of immunosuppressed male nude mice (seven treated, seven controls) were measured. Tumor microvascular blood flow, total hemoglobin concentration and blood oxygenation were investigated as potential biomarkers for the monitoring of the effect of therapy twice a week and were related to the final treatment outcome. These hemodynamic biomarkers have shown a clear differentiation between two groups by day four. Moreover, we have observed that pre-treatment values and early changes in hemodynamics are highly correlated with the therapeutic outcome demonstrating the potential of diffuse optics to predict the therapy response at an early time point.