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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.

In a search for new therapeutic targets for treating epithelial ovarian cancer, we analyzed the Transforming Growth Factor Beta (TGFβ) signaling pathway in these tumors. Using a TMA with patient samples we found high Smad2 phosphorylation in ovarian cancer tumoral cells, independently of tumor subtype (high-grade serous or endometrioid). To evaluate the impact of TGFβ receptor inhibition on tumoral growth, we used different models of human ovarian cancer orthotopically grown in nude mice (OVAs). Treatment with a TGFβRI&II dual inhibitor, LY2109761, caused a significant reduction in tumor size in all these models, affecting cell proliferation rate. We identified Insulin Growth Factor (IGF)1 receptor as the signal positively regulated by TGFβ implicated in ovarian tumor cell proliferation. Inhibition of IGF1R activity by treatment with a blocker antibody (IMC-A12) or with a tyrosine kinase inhibitor (linsitinib) inhibited ovarian tumoral growth in vivo. When IGF1R levels were decreased by shRNA treatment, LY2109761 lost its capacity to block tumoral ovarian cell proliferation. At the molecular level TGFβ induced mRNA IGF1R levels. Overall, our results suggest an important role for the TGFβ signaling pathway in ovarian tumor cell growth through the control of IGF1R signaling pathway. Moreover, it identifies anti-TGFβ inhibitors as being of potential use in new therapies for ovarian cancer patients as an alternative to IGF1R inhibition.

One of the main consequences of inhibition of neovessel growth and vessel pruning produced by angiogenesis inhibitors is increased intratumor hypoxia. Growing evidence indicates that tumor cells escape from this hypoxic environment to better nourished locations, presenting hypoxia as a positive stimulus for invasion. In particular, anti-VEGF/R therapies produce hypoxia-induced invasion and metastasis in a spontaneous mouse model of pancreatic neuroendocrine cancer (PanNET), RIP1-Tag2. Here, a novel vascular-targeting agent targeting semaphorin 4D (Sema4D) demonstrated impaired tumor growth and extended survival in the RIP1-Tag2 model. Surprisingly, although there was no induction of intratumor hypoxia by anti-Sema4D therapy, the increase in local invasion and distant metastases was comparable with the one produced by VEGFR inhibition. Mechanistically, the antitumor effect was due to an alteration in vascular function by modification of pericyte coverage involving platelet-derived growth factor B. On the other hand, the aggressive phenotype involved a macrophage-derived Sema4D signaling engagement, which induced their recruitment to the tumor invasive fronts and secretion of stromal cell–derived factor 1 (SDF1) that triggered tumor cell invasive behavior via CXCR4. A comprehensive clinical validation of the targets in different stages of PanNETs demonstrated the implication of both Sema4D and CXCR4 in tumor progression. Taken together, we demonstrate beneficial antitumor and prosurvival effects of anti-Sema4D antibody but also unravel a novel mechanism of tumor aggressivity. This mechanism implicates recruitment of Sema4D-positive macrophages to invasive fronts and their secretion of proinvasive molecules that ultimately induce local tumor invasion and distant metastasis in PanNETs., This work is supported by research grants from ERC (ERC-StG-281830) EU-FP7, MinECO (SAF2016-79347-R), ISCIII Spain (AES, DTS17/00194) and AGAUR-Generalitat de Catalunya (2017SGR771). Some of these include European Development Regional Funds (ERDF “a way to achieve Europe”). Vaccinex Inc. provided reagents and research money (

Activation of the tumor and stromal cell-driven angiogenic program is one of the first requirements in the tumor ecosystem for growth and dissemination. The understanding of the dynamic angiogenic tumor ecosystem has rapidly evolved over the last decades. Beginning with the canonical sprouting angiogenesis, followed by vasculogenesis and intussusception, and finishing with vasculogenic mimicry, the need for different neovascularization mechanisms is further explored. In addition, an overview of the orchestration of angiogenesis within the tumor ecosystem cellular and molecular components is provided. Clinical evidence has demonstrated the effectiveness of traditional vessel-directed antiangiogenics, stressing on the important role of angiogenesis in tumor establishment, dissemination, and growth. Particular focus is placed on the interaction between tumor cells and their surrounding ecosystem, which is now regarded as a promising target for the development of new antiangiogenics.

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.

Epithelial ovarian cancer is characterized by a low recovery rate because the disease is typically diagnosed at an advanced stage, by which time most patients (80%) already exhibit disseminated neoplasia. The cytokine receptor CXCR4 has been implicated in the development of metastasis in various tumor types. Using a patient-derived tissue macroarray and mRNA expression analysis, we observed high CXCR4 levels in high-grade serous epithelial ovarian carcinomas, the most metastatic tumor, compared with those in endometrioid carcinomas. CXCR4 inhibition by treatment with the CXCR4 antagonist AMD3100 or by expression of shRNA anti-CXCR4 similarly inhibited angiogenesis in several models of ovarian carcinomas orthotopically grown in nude mice, but the effect on tumor growth was correlated with the levels of CXCR4 expression. Moreover, CXCR4 inhibition completely blocked dissemination and metastasis. This effect was associated with reduced levels of active Src, active ERKs, the inhibition of EMT transition, and block of hematogenous ovarian cancer dissemination decreasing circulating human tumoral cells (CTC). In tumors, CXCR4-expressing cells also had more mesenchymal characteristics. In conclusion, our results indicate that CXCR4 expression confers a proinvasive phenotype to ovarian carcinoma cells. Thus, anti-CXCR4 therapy is a possible agent for a complementary treatment of advanced disseminated epithelial high-grade serous ovarian cancer patients., This study was supported by research grants from the Spanish Ministerio de Economía y Competitividad (SAF2013-46063R), The Spanish Institute of Health Carlos III (ISCIII) and the European Regional Development Fund (ERDF) under the Integrated Project of Excellence no. PIE13/00022 (ONCOPROFILE), and the Generalitat de Catalunya (2014SGR364) to F. Vinals. Work supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d'Oncología de Catalunya (XBTC), IDIBELL and PLATAFORMA BIOBANCOSPT13/0010/0013 and for the MD Anderson Foundation Biobank (B.0000745, ISCIII National Biobank Record). Grants from the AECC (Grupos Estables de Investigacion 2011-AECC-GCB 110333 REVE), the Instituto de Salud Carlos III (ISCIII), FEDER (PI16/00134), and CIBERONC (CB16/12/00295) to G. Moreno-Bueno. E. Alsina-Sanchís is a recipient of a predoctoral fellowship from the Ministerio de Economía y Competitividad.

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.

The Scientific Foundation ‘Asociación Española Contra el Cáncer’ (AECC, Stable Coordinated Group, Hereditary Cancer); the BBVA Foundation; the Eugenio Rodríguez Pascual Foundation grant 2012; Generalitat de Catalunya AGAUR SGR 2012 grants 283, 290 and 312, and SGR 2014 grants 364, 530, and 535; Spanish Ministry of Health ISCIII FIS grants PI10/00057, PI10/00222, PI10/01422, PI12/01528, PI13/00132, and PI14/00336. ISCIII RTICC grants RD06/0020/1051, RD12/0036/0007, RD12/0036/0008 and RD12/0036/0063; Spanish Ministry of Science and Innovation, ‘Fondo Europeo de Desarrollo Regional (FEDER), una manera de hacer Europa’, MINECO grants SAF2010-20203 and SAF2013-46196; and the Telemaraton 2014 ‘Todos Somos Raros, Todos Somos Únicos’ grant P35 (...), Mateo, F.; Arenas, E. J.; Aguilar, H.; Serra-Musach, J.; de Garibay, G. Ruiz; Boni, J.; Maicas, M.; Du, S.; Iorio, F.; Herranz-Ors, C.; Islam, A.; Prado, X.; Llorente, A.; Petit, A.; Vidal, A.; Catala, I.; Soler, T.; Venturas, G.; Rojo-Sebastian, A.; Serra, H.; Cuadras, D.; Blanco, I.; Lozano, J.; Canals, F.; Sieuwerts, A. M.; de Weerd, V.; Look, M. P.; Puertas, S.; Garcia, N.; Perkins, A. S.; Bonifaci, N.; Skowron, M.; Gomez-Baldo, L.; Hernandez, V.; Martinez-Aranda, A.; Martinez-Iniesta, M.; Serrat, X.; Ceron, J.; Brunet, J.; Barretina, M. P.; Gil, M.; Falo, C.; Fernandez, A.; Morilla, I.; Pernas, S.; Pla, M. J.; Andreu, X.; Segui, M. A.; Ballester, R.; Castella, E.; Nellist, M.; Morales, S.; Valls, J.; Velasco, A.; Matias-Guiu, X.; Figueras, A.; Sanchez-Mut, J. V.; Sanchez-Cespedes, M.; Cordero, A.; Gomez-Miragaya, J.; Palomero, L.; Gomez, A.; Gajewski, T. F.; Cohen, E. E. W.; Jesiotr, M.; Bodnar, L.; Quintela-Fandino, M.; Lopez-Bigas, N.; Valdes-Mas, R.; Puente, X. S.; Vinals, F.; Casanovas, O.; Graupera, M.; Hernandez-Losa, J.; Ramon y Cajal, S.; Garcia-Alonso, L.; Saez-Rodriguez, J.; Esteller, M.; Sierra, A.; Martin-Martin, N.; Matheu, A.; Carracedo, A.; Gonzalez-Suarez, E.; Nanjundan, M.; Cortes, J.; Lazaro, C.; Odero, M. D.; Martens, J. W. M.; Moreno-Bueno, G.; Barcellos-Hoff, M. H.; Villanueva, A.; Gomis, R. R.; Pujana, M. A.

Angiogenesis and metabolism are entwined processes that permit tumor growth and progression. Blood vessel supply is necessary for tumor survival not only by providing oxygen and nutrients for anabolism but also by removing waste products from cellular metabolism. On the other hand, blocking angiogenesis with antiangiogenic therapies shows clinical benefits in treating several tumor types. Nevertheless, resistance to therapy emerges over time. In this review we discuss a novel mechanism of adaptive resistance involving metabolic adaptation of tumor cells, and we also provide examples of tumor adaptation to therapy, which may represent a new mechanism of resistance in several types of cancer. Thus, targeting this metabolic tumor adaptation could be a way to avoid resistance in cancer patients.

The vascular endothelial growth factor (VEGF) pathway is a clinically validated antiangiogenic target for non-small cell lung cancer (NSCLC). However, some contradictory results have been reported on the biological effects of antiangiogenic drugs. In order to evaluate the efficacy of these drugs in NSCLC histological subtypes, we analyzed the anticancer effect of two anti-VEGFR2 therapies (sunitinib and DC101) in chemically induced mouse models and tumorgrafts of lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC). Antiangiogenic treatments induced vascular trimming in both histological subtypes. In ADC tumors, vascular trimming was accompanied by tumor stabilization. In contrast, in SCC tumors, antiangiogenic therapy was associated with disease progression and induction of tumor proliferation. Moreover, in SCC, anti-VEGFR2 therapies increased the expression of stem cell markers such as aldehyde dehydrogenase 1A1, CD133, and CD15, independently of intratumoral hypoxia. In vitro studies with ADC cell lines revealed that antiangiogenic treatments reduced pAKT and pERK signaling and inhibited proliferation, while in SCC-derived cell lines the same treatments increased pAKT and pERK, and induced survival. In conclusion, this study evaluates for the first time the effect of antiangiogenic drugs in lung SCC murine models in vivo and sheds light on the contradictory results of antiangiogenic therapies in NSCLC.

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.

In this work, we have analyzed the expression of different members of the ErbB family in human samples of testicular germ cell tumors (GCTs). We observed expression of ErbB1 or ErbB2 in different tumor subtypes, but we also found high expression of ErbB3 in all GCTs tested. This pattern of expression was maintained when primary tumors were orthotopically implanted in nude mice. We have chosen a choriocarcinoma model characterized by high levels of ErbB1, but also of ErbB2 and ErbB3, to assay the in vivo effect of ErbB inhibitors on tumoral growth. Our results showed a complete lack of effect (refractoriness) to the pure ErbB1 receptor inhibitors cetuximab and gefitinib. While these inhibitors blocked ErbB1 phosphorylation, ErbB2 phosphorylation was not affected, suggesting an ErbB1-independent activation of this receptor. To confirm the importance of ErbB2 activation, animals were treated with lapatinib, a dual ErbB1 and ErbB2 inhibitor. Lapatinib treatment caused a 50% inhibition in tumor growth, an effect correlated with a blockade of both ErbB1 and ErbB2 phosphorylation levels, and of downstream signaling pathways (Akt, ERKs and Stat3). ErbB2 activation could still occur due to the formation of ErbB2/ErbB3 heterodimers, and ErbB3 activation was completely inhibited by lapatinib. Finally, combined inhibition of ErbB1 (gefitinib) and ErbB3 activities (knockdown expression by shRNA) inhibited tumoral testicular cells proliferation in a similar way to lapatinib. Our results explain why lapatinib but not anti-ErbB1 agents might be effective for treatment of testicular GCT patients.

Genetic deficiency for endoglin leads to increased metastatic capability by weakening the endothelial cell barrier., Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-β in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.

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.

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.

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.

Neuroendocrine tumors (NETs) represent a heterogeneous group of diseases with varied natural history and prognosis depending upon the organ of origin and grade of aggressiveness. The most widely used biomarker to determine disease burden and monitor response to treatment is chromogranin A (CgA), but it is far from being the optimal predictive and prognostic biomarker in NETs. Biological understanding and derived treatment options for NETs have changed markedly in recent years. Over the last decade, the genomic landscape of these tumors has been extensively investigated. This has resulted in the discovery of mutations and expression anomalies in genes and pathways such as the PI3K/Akt/mTOR, DAXX/ATRX, and MEN1, which are promising predictive and prognostic biomarkers and future candidates for targeted therapies. Additionally, the study of tumor stroma and environment are one of the most promising fields for discovery of potential new targets and biomarkers.

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.

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.

Cancer development, progression, and metastasis are highly dependent on angiogenesis. The use of antiangiogenic drugs has been proposed as a novel strategy to interfere with tumor growth, but cancer cells respond by developing strategies to escape these treatments. In particular, animal models show that antiangiogenic drugs currently used in clinical settings reduce tumor tissue oxygenation and trigger molecular events that foster cancer resistance to therapy. Here, we show that semaphorin 3A (Sema3A) expression overcomes the proinvasive and prometastatic resistance observed upon angiogenesis reduction by the small-molecule tyrosine inhibitor sunitinib in both pancreatic neuroendocrine tumors (PNETs) in RIP-Tag2 mice and cervical carcinomas in HPV16/E2 mice. By improving cancer tissue oxygenation and extending the normalization window, Sema3A counteracted sunitinib-induced activation of HIF-1α, Met tyrosine kinase receptor, epithelial-mesenchymal transition (EMT), and other hypoxia-dependent signaling pathways. Sema3A also reduced tumor hypoxia and halted cancer dissemination induced by DC101, a specific inhibitor of the VEGF pathway. As a result, reexpressing Sema3A in cancer cells converts metastatic PNETs and cervical carcinomas into benign lesions. We therefore suggest that this strategy could be developed to safely harnesses the therapeutic potential of the antiangiogenic treatment.

The successful introduction of rationally targeted agents into standard cancer care is a testimony of the vast knowledge base in tumor biology. However, in order to provide individually tailored therapy to patients and to identify small subsets of patients with a high likelihood to benefit from treatment, the identification of biomarkers for response or resistance to a particular therapeutic regimen is imperative. Herein, by the use of a genetically engineered mouse model of pancreatic neuroendocrine tumors, we have assessed the utility of pericyte characteristics in terms of differential marker expression to serve as surrogate markers for response or evasive resistance to anti-angiogenic therapy. We found that tumors refractory to therapy following long-term treatment with a vascular endothelial growth factor receptor-2 blocking antibody contained blood vessels with a prolific investment of pericytes expressing α-smooth muscle actin. Further analysis by simultaneous immunostaining for different pericyte markers led to the conclusion that the increased abundance of this particular subtype of blood vessels most likely occurred by co-option of vessels from the surrounding exocrine pancreas. Our findings may form the basis for retrospective analysis of pancreatic neuroendocrine tumors from patients having undergone treatment with anti-angiogenic agents in order to validate the occurrence of pericytes expressing α-smooth muscle actin as a biomarker for tumors refractory to therapy.

VEGF antagonists are now widely used cancer therapeutics, but predictive biomarkers of response or toxicity remain unavailable. In this study, we analyzed the effects of anti-VEGF therapy on tumor metabolism and therapeutic response by using an integrated set of imaging techniques, including bioluminescence metabolic imaging, 18-fluorodeoxyglucose positron emission tomography, and MRI imaging and spectroscopy. Our results revealed that anti-VEGF therapy caused a dramatic depletion of glucose and an exhaustion of ATP levels in tumors, although glucose uptake was maintained. These metabolic changes selectively accompanied the presence of large necrotic areas and partial tumor regression in highly glycolytic tumors. In addition, we found that the central metabolic protein kinase AMP-activated protein kinase (AMPK)—a cellular sensor of ATP levels that supports cell viability in response to energy stress—was activated by anti-VEGF therapy in experimental tumors. AMPK-α2 attenuation increased glucose consumption, tumor cell sensitivity to glucose starvation, and tumor necrosis following anti-VEGF therapy. Taken together, our findings reveal functional links between the Warburg effect and the AMPK pathway with therapeutic responses to VEGF neutralization in tumor xenograft models. Cancer Res; 71(12); 4214–25. ©2011 AACR.

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.

SummaryMultiple angiogenesis inhibitors have been therapeutically validated in preclinical cancer models, and several in clinical trials. Here we report that angiogenesis inhibitors targeting the VEGF pathway demonstrate antitumor effects in mouse models of pancreatic neuroendocrine carcinoma and glioblastoma but concomitantly elicit tumor adaptation and progression to stages of greater malignancy, with heightened invasiveness and in some cases increased lymphatic and distant metastasis. Increased invasiveness is also seen by genetic ablation of the Vegf-A gene in both models, substantiating the results of the pharmacological inhibitors. The realization that potent angiogenesis inhibition can alter the natural history of tumors by increasing invasion and metastasis warrants clinical investigation, as the prospect has important implications for the development of enduring antiangiogenic therapies.

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.

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...

Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT. Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed. Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model. Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.

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.

Gemcitabine shows a marked antitumor effect as a result of its cytotoxic action toward proliferative cells. In this article, we aim to investigate the potential antitumor and antiangiogenic effect of gemcitabine following a metronomic schedule that involves the regular administration of cytotoxic drugs at doses lower than standard treatment. In vitro results showed that human endothelial cells are more sensitive to gemcitabine (IC50 3 nmol/L) than pancreatic tumor cells (IC50 20 nmol/L). For in vivo studies, we used an orthotopic implantation model of human pancreatic carcinoma in nude mice. Gemcitabine was administered i.p. following a low-dose schedule (1 mg/kg/d for a month) and compared with the conventional schedule (100 mg/kg days 0, 3, 6, and 9 postimplantation). Metronomic treatment effect on established tumor was equivalent to standard administration. The measure of CD31 endothelial marked area allowed us to show an in vivo antiangiogenic effect of this drug that was further enhanced by using metronomic administration. This effect correlated with an induction of thrombospondin-1, a natural inhibitor of angiogenesis. Our results allow us to hypothesize that, in addition to a direct antiproliferative or cytotoxic antiendothelial cell effect, a secondary effect involving thrombospondin-1 induction might provide an explanation for the specificity of the effects of metronomic gemcitabine treatment. [Mol Cancer Ther 2008;7(3):638–47]