Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection.

Angiogenesis and signaling through the RAS/RAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK cascade have been reported to play important roles in the development of hepatocellular carcinoma (HCC). Sorafenib (Nexavar), a novel bi-aryl urea BAY 43-9006, is an orally administered multikinase inhibitor with activity against RAS/RAF kinases multikinase inhibitor with activity against RAF kinases and several receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-Kit. It is involved in angiogenic pathway and cell proliferation. Sorafenib has demonstrated potent anti-tumor activity in in vitro studies, preclinical xenograft models of different tumor types and human clinical trials. This review summarizes the history of sorafenib from its discovery by the medicinal chemistry approach through to clinical development and ongoing trials on the combination between sorafenib and trans-arterial chemoembolization (TACE) in HCC patients.

Angiogenesis and signaling through the RAS/RAF/mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK cascade have been reported to play important roles in the development of hepatocellular carcinoma (HCC). Sorafenib (Nexavar), a novel bi-aryl urea BAY 43-9006, is an orally administered multikinase inhibitor with activity against RAS/RAF kinases multikinase inhibitor with activity against RAF kinases and several receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), FLT3, Ret, and c-Kit. It is involved in angiogenic pathway and cell proliferation. Sorafenib has demonstrated potent anti-tumor activity in in vitro studies, preclinical xenograft models of different tumor types and human clinical trials. This review summarizes the history of sorafenib from its discovery by the medicinal chemistry approach through to clinical development and ongoing trials on the combination between sorafenib and trans-arterial chemoembolization (TACE) in HCC patients.

Abstract Tryptase, a serine protease stored and released from mast cells granules has been identified as a new non-classical angiogenetic factor. Tryptase is an agonist of proteinase-activated receptor-2 a G protein involved in cellular proliferation and angiogenesis. On the other hand mast cells can release tryptase following c-Kit receptor activation. We have evaluated the correlations among the number of MCs positive to tryptase (MCDPT), the number of c-Kit receptor expressing cells (C-KREC) and microvascular density (MVD) in a series of 91 primary T1-3, N0-2 M0 female breast cancer by means of immunohistochemistry and image analysis methods. Six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with primary antibodies: anti-tryptase (AA1; Dako, Glostrup, Denmark), anti-c-Kit receptor (A4502; Dako, Glostrup, Denmark) and anti-CD34 (QB-END 10; Bio-Optica Milan, Italy). In serial sections MVD, MCDPT and C-KREC were counted by means of image analysis (Quantimet 500 Leica) at x400 microscopic fields. Data demonstrated a significantly (r= ranging from 0.72 to 0.91; p: ranging from 0.001 to 0.003 by Pearson's analysis respectively) correlation between MVD, MCDPT and C-KREC to each other. Published in vitro data suggest that tryptase induce angiogenesis in vascular endothelial cells and breast cancer cells lines. According to these data we shown that MVD, MCDPT and C-KREC paralleled to each other suggesting a role in in vivo breast cancer angiogenesis. In this context the inhibition of c-Kit mast cells tryptase degranulation by several tyrosin kinase inhibitors might be evaluated in clinical trials. Finally available tryptase inhibitors such as gabexate mesilate or nafamostat mesilate might be evaluated in clinical trials as a new antiangiogenetic approach. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-05-03.

e17531 Background: Lung tumours seem to be good targets for radiofrequency ablation (RFA); nevertheless one limitation is the risk of local recurrence, not negligible in tumours larger than 2.0-2.5 cm. This study aimed to evaluate the feasibility, safety and basic efficacy of a new combined procedure (double track therapy) consisting of segmental pulmonary arterial chemoembolization (SPACE) followed by percutaneous RFA in patients with unresectable lung neoplasms. Methods: From January 2008 to May 2010, 16 patients (6 male/10 female, median age 69 years; range, 46-83) and 19 nodules were treated in 18 sessions. Patients had lung metastases from: uterine cancer (n=2), colorectal carcinoma (n=8), breast carcinoma (n=2), hemangiopericytoma (n=1) and 3 patients had primary unresectable NSCLC. The median diameter of neoplasms was 3.0 cm (range, 2.0-5.0). Antiblastic agents loaded on microspheres (Hepasphere) were selectively perfused in the subsegmental pulmonary parenchyma. Percutaneous CT-guided RFA of lung nodules was performed 48 hrs after SPACE. Pretreatment work-up included: contrast-enhanced CT-scan, 18F-FDG PET/CT and VL-SPET. Electrical impedance values were assessed in order to highlight any SPACE-related change. Results: The combined procedure was successfully completed in all patients. Overall, treatment was well tolerated. The major complications were 5 pneumothorax (26%) and 1 bronco-pleural fistula (5%). No treatment-related changes in general lung function were shown. According to modified RECIST criteria, complete response was achieved in 10 (62.5%) out of 16 patients, and in 12 (63%) out of 19 target lesions no contrast-enhancement was observed at the CT-scan after completed treatment. VL-SPET showed a modified sub-segmental alveolar ventilation after SPACE. A reduction of impedance (≤30%), corresponding to an increase of the delivered energy, was observed as a consequence of SPACE. Conclusions: Double track therapy with lung RFA after SPACE is technically feasible, relatively safe and effective. These preliminary data deserve further investigation.

e13543 Background: Tryptase is a serin protease stored in mast cell granules that plays a role in tumour angiogenesis. Data from experimental tumour models suggest that mast cells accumulate near tumour cells before the onset of angiogenesis and that they are required for the macroscopic expansion and metastatic spread of primary tumour cells. Hepatocellular cancer (HCC) is a well-established hypervascular tumour. This preliminary study aims to assess tryptase serum levels in 30 HCC patients before and after hepatic trans-arterial chemoambolization (TACE). Methods: In this study patients with intermediate stage (B) HCC (according to BCLC staging system) and adequate liver functional reserve (A or B status according Child-Pugh classification) were enrolled. Samples of blood were taken from HCC patients between 7 and 9 a.m.1 day before and 1 day after TACE. Venous blood was dispensed into a tube for serum (Becton Dickinson Hemogard Vacutainer Systems, Plymouth, UK). Serum blood samples were centrifuged at 1,500g for 10 minutes and then aliquoted and frozen at -80 °C. Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia,Uppsala, Sweden). TACE was performed using doxorubicin loaded on microspheres (Dc-Bead; Biocompatibles, Farnham [GB]). Results: Mean ± s.d. tryptase level pre-TACE was 7.74 ± 3.62 μg/L, and mean ± s.d. tryptase level post TACE was 4.67 ± 2.79 μg/L. A statistically significant difference between pre-TACE and post-TACE tryptase level concentrations was found: p=0.000 by t-test. No correlation among tryptase levels and other important clinical-pathological features of patients were found. Conclusions: This is the first preliminary report that analyzes the possible significance of serum tryptase levels in HCC patients. Tryptase is one of the most powerful angiogenic mediators released by mast cells and it may be angiogenic via several mechanisms. On the other hand HCC is an hypervascular tumour. Our results demonstrated higher serum tryptase levels in HCC patients suggesting the release of tryptase from HCC tissue. As expected, after TACE, serum tryptase levels had decreased. We suggest that tryptase may play a role as a biomarker of response to TACE treatment in HCC patients.

In this study, the production of storage polymers is made when cheese production wastewater is treated. Cheese plants typically produce high-strength wastewaters on an intermittent basis and are particularly suitable for treatment in Sequencing Batch Reactor (SBR) systems. The SBRs were operated in triplicate under two filling scenarios: 'React Fill' (RF) with mixing/aeration; 'Static Fill' (SF) with no mixing/aeration. The results suggest that an 'SF' approach outperforms an 'RF' with respect to storage products production (50% more PHB, 15% more glycogen). Additionally, the famine phase duration will vary depending on wastewater strength, suggesting optimisation per waste stream.

activity under hypoxic condition than normoxic condition. Furthermore, the small GTPase Ras is also activated in response to hypoxia, which then aids stabilization of HIF-1a, and in turn up-regulates MMP-9 expression. We also demonstrate that MMP-9 is up-regulated concurrently with HIF-1a in tumor tissues from breast cancer patients. Conclusion: These results suggest that HIF-1a promotes cell invasion through a MMP-9-dependent mechanism and that future anti-tumor agents could be used to target HIF-1a and MMP-9. Disclosure of Interest: None Declared

Typically, microbes associated with biological wastewater treatment processes are subjected to dynamic organic and nutrient loading conditions. This constantly changing environment imposes a stress, referred to as "feast-famine" that selects for microbes capable of biologically storing substrates as polymers during high organic concentration periods (i.e., feast) for use during periods of low organic availability (i.e., famine). In this study, we monitored the production of biostorage polymers generated with actual cheese wastewater treatment by way of sequencing batch reactors (SBRs). SBRs were employed and operated in duplicate under two long (i.e., hours) filling scenarios (1) "react fill" with mixing/aeration and (2) "static fill" with no mixing/aeration. Despite comparable effluent water quality levels, the results reveal that a "static fill" approach outperforms a "react fill" with respect to maximum biostorage polymer production (50% more poly-beta-hydroxybutyrate, 15% more glycogen). The presence of biostorage polymer production has been shown to be indicative of a more stable and robust process.