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Full-length 66-kDa estrogen receptorα (ERα) stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal domain and AF-2 in the ligand binding domain. Another physiologically expressed 46-kDa ERα isoform lacks the N-terminal A/B domains and is consequently devoid of AF-1. Previous studies in cultured endothelial cells showed that the N-terminal A/B domain might not be required for estradiol (E2)-elicited NO production. To evaluate the involvement of ERαAF-1 in the vasculoprotective actions of E2, we generated a targeted deletion of the ERα A/B domain in the mouse. In these ERαAF-1zero mice, both basal endothelial NO production and reendothelialization process were increased by E2 administration to a similar extent than in control mice. Furthermore, exogenous E2 similarly decreased fatty streak deposits at the aortic root from both ovariectomized 18-weekold ERαAF-1+/+LDLR-/- (low-density lipoprotein receptor) and ERαAF-1zeroLDLR-/- mice fed with a hypercholesterolemic diet. In addition, quantification of lesion size on en face preparations of the ic tree of 8-month-old ovariectomized or intact female mice revealed that ERαAF-1 is dispensable for the atheroprotective action of endogenous estrogens. We conclude that ERαAF-1 is not required for three major vasculoprotective actions of E2, whereas it is necessary for the effects of E2 on its reproductive targets. Thus, selective ER modulators stimulating ERα with minimal activation of ERαAF-1 could retain beneficial vascular actions, while minimizing the sexual effects.

Although estradiol (E 2 ) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E 2 (80 μg/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E 2 increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E 2 , whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E 2 . To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ERα or ERβ has been disrupted were ovariectomized and treated, or not, with E 2 . Basal NO production was increased and the sensitivity to acetylcholine decreased in ERβ knockout mice in response to E 2 , whereas this effect was abolished in ERα knockout mice. Finally, these effects of E 2 on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E 2 in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ERα, but not ERβ, mediates the beneficial effect of E 2 on basal NO production.

Nitric oxide (NO) is produced by a family of three isoenzymes: the endothelial, inducible and neuronal NO synthases. L-Nitroarginine methyl ester (L-NAME) is the most commonly used inhibitor of NO synthase activity. The goal of the present study was to evaluate to what extent L-nitroarginine (L-NA), the in vivo circulating metabolite of L-NAME, blocks NO production in the rat aorta depending on the NO synthase isoform expressed (and evidenced by Western blotting) and on the presence or absence of the extracellular NO synthase substrate L-arginine (100 microM, i.e. the plasma concentration). Intact [endothelium present (E+)] control aortic rings express mainly endothelial NO synthase. L-NA (30--100 microM) induced a dose-dependent contraction (due to blockade of the relaxant properties of NO) irrespective of the presence or absence of L-arginine. In deendothelialized (E-) control aortic rings, the three isoforms of NO synthase are virtually absent (as demonstrated by Western blotting) and L-NA does not elicit any contractile effect. E- aortic rings from lipopolysaccharide (LPS)-treated rats express mainly inducible NO synthase. In these rings, L-NA induced a dose-dependent (0--100 microM) contraction in the absence of extracellular L-arginine, whereas L-arginine (100 microM) completely abrogated the contractile effect of the NO synthase inhibitor. Chronic L-NAME administration (50 mg/kg/day for 4 weeks) elicited the aortic expression of inducible NO synthase, but to a lesser extent (about 5-fold) than in LPS-treated rat aorta. The average plasma concentration of L-NA was 50 +/- 10 microM in these rats. In E- rings from these L-NAME-treated rats, L-NA induced a similar contractile response (but smaller in magnitude) to that observed in LPS-treated rat aorta. Altogether, these data suggest that (1) in the presence of a physiological concentration of extracellular L-arginine, L-NA fails to inhibit inducible NO synthase, and (2) chronic L-NAME administration, at a dose commonly given to block NO production in vivo, leaves the activity of inducible NO synthase unaffected.

Background mTORi usually induce stable disease but some patients present with a rapid objective response. In order to predict early those patients susceptible to respond to mTORi, we investigated the relationship between time to best response and time to progression. Methods Data were retrospectively collected in patients treated with mTORi (Ev:everolimus, Tem:temsirolimus) for mRCC in 1st to 4th line setting at the European G. Pompidou Hospital in Paris between 2007 and 2011. Time to best response was obtained from CT-scan and objective response assessed according to RECIST 2.0. Efficacy was assessed by time to progression (TTP) from initiation of 1st mTORi until disease progression/death. Overall survival (OS) was defined as the time from 1st mTOR to death/last contact. Kaplan-Meier estimates with Log-Rank test were used for categorical data. Cox model was used for continuous data and hazard ratios calculation. Only pts having received at least 1 cycle of mTORi were included. Results Among the 75 pts, 63 presented with a documented response: PR = 6, SD = 40, PD = 17. 12 pts were excluded for mTORi discontinuation due to toxicity before any response assessment or uncertain date of best response. The 63 eligible pts had a median age = 61 (range: 28-86), sex ratio = 51/12, Ev/Tem = 37/26. Line setting: 1st =7, 2nd =17, 3rd =19, 4th = 17, 5th = 3. Median follow-up time = 22.8m. Time to progression= 7.3m (95%CI: [4.3–9.1]). Time to best response: SD = 2.3 (0.7–8.2), PR = 7.6 (0.7–18.8), PD = 2.1 (0.9–5.0). Time to best response was significantly associated with TTP (N = 46, 35 events, HR = 0.86, 95% CI [0.76–0.97], p = 0.014), but also OS (N = 46, 31 events, HR = 0.83, 95% CI [0.70-0.98], p = 0.03]), an early response leading both to a more rapid progression and being of poor prognosis. Conclusions Time to best response was significantly associated to time to progression and overall survival in pts treated with mTORi for mRCC. Given the small sample and the rough assessment of RECIST criteria, further study is needed to investigate the real value of percentage in tumor decrease at each evaluation and velocity of response as a more precise predictive marker of efficacy for these drugs. Disclosure S. Oudard: Advisory board to disclose: Sanofi Aventis, Bayer, Novartis, Roche, Pfizer Compensated consultant relatioship to disclose: Novartis, Roche Honoraria to disclose: Sanofi Aventis, Bayer, Novartis, Roche, Pfizer All other authors have declared no conflicts of interest.

Background Study results demonstrated that IFN augments BEV activity and improves median PFS in pts with mRCC. Thus, combination BEV + IFN is a standard first-line treatment option for mRCC. Combining BEV with the mTOR inhibitor EVE may be an efficacious and well-tolerated treatment option. The open-label, phase II RECORD-2 trial compared first-line EVE + BEV and IFN + BEV in mRCC. Patients and methods: Therapy-naive pts with clear cell mRCC and prior nephrectomy were randomized 1:1 to BEV 10 mg/kg IV every 2 weeks with either EVE 10 mg oral daily or IFN (9 MIU SC 3 times/week, if tolerated). Tumour assessments were every 12 weeks. Primary objective was treatment effect on progression-free survival (PFS) per central review based on an estimate of the chance of a subsequent phase III trial success (50% threshold for phase II success). Results In EVE + BEV (n = 182) and IFN + BEV (n = 183) arms, median age was 60/60 years, 76/72% of pts were men, MSKCC risk was favourable/intermediate/poor in 36/57/7% and 36/57/7% of pts, and 43/46% of pts had >2 organs involved, respectively. For EVE + BEV and IFN + BEV, median treatment duration was 8.5/8.3 months, respectively; 23/26% of pts discontinued due to AEs. In EVE + BEV and IFN + BEV arms, median PFS by central review was 9.3/10.0 months (HRIFN/EVE, 0.91; 95% CI, 0.69-1.19; P =0.485), respectively; probability of subsequent phase III success was 5.1%. Results of central and local PFS analysis were consistent. Objective response rate was 27/28% in EVE + BEV and IFN + BEV arms, respectively. Median overall survival (OS) was not reached in the EVE + BEV arm and was 25.9 months (95% CI: 21.1, 30.2) in the IFN + BEV arm. Most frequent AEs (%) were stomatitis (63), proteinuria (49), diarrhoea (39), hypertension (38), and epistaxis (35) in EVE + BEV arm and decreased appetite (45), fatigue (41), proteinuria (37), and pyrexia (35) in IFN + BEV arm. Conclusions In RECORD-2, PFS and tolerability were similar for first-line EVE + BEV and IFN + BEV. Final OS analysis will occur after 2-year follow-up. Disclosure A. Ravaud: Alain Ravaud is a member of global, European, and/or French boards on urological tumors for Pfizer, Novartis, GlaxoSmithKline, Bayer-Schering, and Dendreon, and has received institutional grant support from Pfizer, Novartis, and Roche. O. Anak: Ozlem Anak is an employee of Novartis Pharma AG. D. Pelov: Diana Pelov is an employee of Novartis Pharmaceuticals Corporation. A. Louveau: Anne-Laure Louveau is an employee of Novartis Pharma S.A.S. T. M-H: Tay M-H is a speaker for an advisory board for Novartis Pharmaceuticals Corporation. B. Melichar: Bohuslav Melichar has received honoraria from Novartis and Roche and served on an advisory board for Roche. All other authors have declared no conflicts of interest.

e19530 Background: Advanced lung and gynecologic cancers are common in elderly pts but not optimally treated. Chemotherapy with PC is not recommended because of toxicity. The objective is to invest...

e16548 Background: A pilot program (PROCHE : Chemotherapy Optimization Program) to pre-emptively confirm scheduled chemotherapy treatment by collecting patient (pt) safety data was conducted. One a...

Background Biological toxicities of mTORi are well characterized. We investigate retrospectively if there is a link between biological toxicities and mTORi efficacy. Patients and methods Biological toxicities were retrospectively collected in patients treated with an mTORi (Everolimus [Ev], Temsirolimus [Tem]) for mRCC at the European G. Pompidou Hospital in Paris, France between 2007 and 2011. Only pts having received mTORi for at least 28 days were eligible. Lymphocytes, creatinaemia, glycaemia, phosphoraemia, liver transaminases, cholesterolaemia, and triglyceridaemia were recorded with onset date and higher grade (NCI-CTC 3.0). Time to onset and onset velocity defined as the absolute change from baseline (%) divided by the time to onset were calculated for each adverse event. Efficacy was assessed with time to progression (TTP) from the initiation of mTORi until disease progression/death and objective response according to RECIST criteria 2.0. Kaplan-Meier estimates with log-rank test were used to compare categorical data and Cox model for continuous data and hazard ratios. Results 75 pts were included. Median age = 56 y (37-86), sex ratio = 4/1 (M/F), pts on Ev (N = 44)/Tem (N = 31) according to setting: 1st = 2/6, 2nd = 9/11, 3rd = 14/9, ≥4th = 20/4. 10 pts on Ev had a dose reduction. Objective response: PR = 6, SD = 40, PD = 20. 7 patients discontinued due to toxicity and 2 for personal decision. Median follow-up time = 22.8 mo. Median TTP = 7.3 mo (95% CI, 4.7-10.8). Hyperglycaemia and hypophosphoraemia are less important in progressors (P Conclusions Lymphocytopaenia, hyperglycaemia, hypophosphoraemia, and hypertriglyceridaemia were significantly related to objective response or TTP. These events, together with their onset velocity, should be prospectively monitored to investigate their predictive value for efficacy. Disclosure All authors have declared no conflicts of interest.

Background Metabolic toxicities of mTOR inhibitors (mTORi) are well characterized. The purpose of the study was to investigate the relationship between these metabolic toxicities and mTORi efficacy. Methods From 2007 to 2011, metabolic toxicities were retrospectively collected in patients treated with an mTORi (everolimus, temsirolimus) for a metastatic renal cell carcinoma (mRCC) in a single institution. Patients were eligible if they have received an mTORi for at least 28 days. Changes in the following parameters were analyzed: lymphocytes, serum creatinine, glycemia, serum phosphate, liver transaminases, cholesterol, and triglycerides. The efficacy was assessed by progression-free survival (PFS) and tumor response. Results Data were collected from seventy-five patients (everolimus = 44 patients; temsirolimus = 31 patients). Six patients exhibited a partial response, 42 a stable disease and 15 had a progressive disease (12 missing). After a median follow-up of 12.8 months, the median PFS was 6.7 months (95% confidence interval: 4.0-9.1 months). Patients with CB had a statistically more severe absolute increase of glycemia and absolute decrease in phosphatemia (p = 0.002 and p = 0.02 respectively). The Progression Free Survival was significantly higher with the onset rate of hypophosphatemia (p = 0.03) and hyperglycemia (p = 0.001) and lower with the onset rate of lymphopenia (p = 0.004). Conclusions Hyperglycemia, hypophosphatemia and lymphopenia, were significantly associated with tumor response and/or PFS. Those events, as well as their onset rate, should be prospectively monitored as predictors of response to mTORi.