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193 Background: The standard chemotherapy for unresectablebiliary tract carcinoma (BTC) is based on gemcitabine and platinum compounds. However, these combinations have not been shown to be effective in improving long-term survival. Thus, there is a real need to find new strategies that would impact in a significant way on clinical outcome. Ecteinascidin-743 (ET-743), a compound isolated from the marine tunicate Ecteinascidia turbinata. ET-743, is approved for the treatment of ovarian cancer and soft tissue sarcoma. Phase II and III clinical trials are ongoing for the treatment of different solid tumors. No preclinical data are available about the efficacy of ET-743 in BTC. In a phase I study, one patient received ET-743 plus capecitabine and experienced a long lasting complete metabolic response. Here, we investigated the antitumor activity of ET-743 in preclinical BTC models. Methods: Four BTC cell lines TFK1, EGI-1, HuH28 and TGBC1 were used to evaluate the effect of ET-743 on proliferation, cell cycle, apoptosis and on the activation of DNA damage proteins. The effect on proliferation was also investigated on a primary cell culture of a gallbladder carcinoma (GBC) resistant to gemcitabine and oxaliplatin. On the same cells, the inhibition of VEGF secretion mediated by ET-743 was analyzed by ELISA. The anti-tumor activity of ET-743 was tested on EGI-1 xenografts in NOD/SCID mice. Results: In vitro, ET-743 is able to markedly reduce cell proliferation of BTC cell lines through cell cycle blockage on G0/G1 phase and to inhibit the growth of primary cell culture derived from GBC patient. Moreover, ET-743 promotes apoptosis by caspase 3 activation, activates proteins involved in DNA damage and reduces VEGF secretion. In the in vivo model, ET-743 is able to slow tumor growth in BTC xenograft. The mechanism of anti-tumor activity involves DNA damage, the induction of hypoxia transcription factor-1, and angiogenesis inhibition. ET-743 has no significant effect on apoptosis in vivo. Conclusions: These data suggest that ET-743 could represent an alternative chemotherapy for BTC treatment and encourage the development of clinical trials of ET-743 in BTC patients.

231 Background: Biliary carcinoma (BC) has a poor prognosis and limited therapeutic options. The identification of new targets is essential. Among candidates we have studied the tyrosine kinase Src, which is involved in proliferation, invasion, and migration in many tumors.It has been demonstrated that the inhibition of Src significantly reduces proliferation and metastasis development in different solid tumors, such as pancreatic and head and neck cancers. Targeting Src tyrosine kinase may be therapeuticallyuseful in cholangiocarcinoma. We explored the antitumor activity of Src inhibitor saracatinib (AZD-0530) in BC preclinical models. Methods: Four cholangiocarcinoma cell lines, TFK1, EGI-1, HuH28, and TGBC1-TKB, were treated with saracatinib and analyzed by western blot for the expression of Src and its downstream principal transducer FAK. The inhibition of proliferation was evaluated by cell titer glo assay after treatment with scalar doses of saracatinib alone or in combination with gemcitabine for 72 hours. The effect on migration of BC cells was tested by wound healing assay. Finally, we investigated the antitumor activity in EGI-1 xenografts in NOD/Shi-scidIL2rgnull mice treated with saracatinib at 10 mg/kg/die. Results: Activation of Src pathaway was shown in all cell lines and saracatinib was able to inhibit its downstream molecules. Saracatinib inhibited BC cell proliferation in standard monolayer liquid culture only at very high concentration (median doses from 2.5 to 15 μM) and did not enhance the antiproliferative effect of gemcitabine. Evidence of anti-tumor activity of saracatinib was obtained in terms of migration inhibition: quantitation of the wound closure over time revealed a significant and dose-dependent inhibitory effect of saracatinib on BC cell motility. In the in vivo model, no reduction of tumor volumes was evidenced up to 14 days of treatment. Assays with prolonged treatments are ongoing. Conclusions: These results suggest that inhibition of Src kinase by saracatinib impairs the invasiveness of bile duct carcinoma. Further in vivo studies will provide information about the effect on metastasis inhibition by saracatinib. No significant financial relationships to disclose.

10058 Background: Targeting pathways involved in progression and metastatization might be a suitable way to overcome chemoresistance in OS. Monoclonal antibodies (anti-IGF1R) andMK-I (SOR, dasatini...

10540 Background: Osteosarcoma is the most common primary bone tumor in young adults. Despite improved prognosis, resistance to chemotherapy remains responsible for failure of OS treatment. The search for alternative agents focused on specific molecular mechanisms in OS is mandatory. Sorafenib (BAY 43–9006) is a multitarget antitumoral drug (RAF, VEGFR-2/3, FLT-3, KIT, FGFR-1, RET, MCL- 1; PDGFR-β). We explored the expression of main sorafenib targets in OS and its activity in preclinical models. Methods: In 30 samples from OS patients and in 7 OS cell lines we performed immunohistochemistry for MAPK-P and Mcl-1 expression as well as mutational analysis of B-RAF and K-RAS. We assessed the cell proliferation inhibition both in vitro (Cell Titer-Glo) and in vivo (xenografts in SCID mice) caused by Sorafenib treatment. The sorafenib molecular mechanism was investigated by Western blot (MAPK-P, MCL-1) and ELISA tests (VEGF, MMP2/9). Results: Expression of MAPK-P and MCL-1 was shown in 20/30 and 24/30 specimen...

4582 Background: Biliary tract carcinomas express EGFR and are potential candidates to EGFR target therapies. We recently described somatic mutations of EGFR that can enhance MAPK or Akt activation (Clin Cancer Res, 2006). Some of them are identical to those previously reported to confer sensitivity to some tyrosine kinase inhibitors (TKIs) like erlotinib or gefitinib in lung cancer. Here we report a molecular analysis of EGFR transducers potentially involved in TKI response. Methods: In 49 samples of biliary tract carcinoma we performed mutational analysis of exons from 18 to 21 of EGFR, exons 9 and 20 of phosphatidylinositol 3’-kinase (PI3K), exon 2 of K-Ras, exon 15 of B-Raf and exons from 5 to 8 of PTEN. Nuclear PTEN expression was analyzed by immunohistochemistry and the expression in cancer cells was compared to that of normal cholangiocites. Results: Mutations of EGFR have been detected in 7 out of 49 samples (14.3%). One of them was a new stop-codon mutation. Five hotspot mutations of PI3K (codon 545, 546, 1048 and 1059) were found in 4 cases (8.2%); 3 cases (6.1%) had single mutations in K-Ras and 4 (8.2%) had the V599E mutation in B-Raf. In some samples, mutations of multiple trasducers were present simultaneously. PI3K mutations were significantly more frequent in EGFR mutated samples compared to wild type (28% vs. 4.7% respectively, p No significant financial relationships to disclose.

TO THE EDITOR: A study by Philip et al published in the July 1, 2006, issue of the Journal of Clinical Oncology shows the therapeutic benefit of epidermal growth factor receptor (EGFR) blockade with erlotinib in patients with biliary tract carcinoma. The results are interesting because in a cohort composed of 24 chemotherapy-refractory patients and 18 chemotherapy-naive patients, erlotinib as a single agent can achieve disease control in 50% of patients with a median duration of disease control of 5.1 months. However, the authors conclude that the activity of erlotinib is modest because only 17% of the patients achieved the primary end point of the study, which was progression-free survival at 24 weeks. Although gefitinib and erlotinib are active in unselected patients with non–small-cell lung cancer (NSCLC), clinical characteristics and tumor molecular markers associated with enhanced benefit have been identified. Notably, mutations in the tyrosine kinase domain of EGFR were subsequently found to be associated with sensitivity to small molecule inhibitors in NSCLC. Emerging data also suggest that increased EGFR copy number is a predictor of response to gefitinib therapy, regardless of EGFR kinase domain mutational status. Furthermore, in contrast to wild-type EGFR, the EGFR mutations are mutually exclusive of K-Ras mutations, which are found in biliary tract carcinomas with a particularly high incidence in some geographical areas. Mutations of the EGFR kinase domain have recently been described by our group in 15% of biliary tract carcinoma specimens. One of these mutations was a T790M substitution, which is known to correlate with an acquired resistance to small molecule inhibitor treatment in NSCLC. Whereas 28% of the EGFR-positive patients were progression free at 24 weeks, the study by Philip et al, based on the lack of correlation between EGFR expression level and treatment outcome in other tumor types, includes six undetermined and seven EGFRnegative patients. This patient number is too small to provide evidence of any correlation. Thus, it would be of interest to know if other molecular analyses, such as sequencing of EGFR tyrosine kinase or of downstream members of the signaling pathway, like K-Ras, B-Raf or PTEN, or EGFR gene amplification, have been planned in this group of biliary tract cancer patients treated with erlotinib. This finding will have extraordinary implications and will serve as a critical step toward individualized patient-specific treatment plans based on the molecular constitution of the tumor of each individual.

9021 Background. Despite intensive chemotherapy and surgery treatment, lung and bone metastases develop in about 30% of OS patients. Mechanisms for this preferential metastatic behaviour are largely unknown. The CXCR4/SDF-1 system was shown to play a role in the homing of neoplastic cells in breast cancer as well as in rabdhomyosarcoma. This hypothesis was tested in vitro and in vivo in a mouse model. Methods. Human OS cell lines MG-63, SJSA, U2-OS and HOS were studied in standard colture conditions. Cell lines were evaluated for: CXCR4 expression by FACS analysis (mAb anti-CXCR4, BD Pharmingen); SDF-1 levels in conditioned medium (antibody sandwich ELISA, RD modulation of CXCR4 expression on OS cells by exposure to recombinant human SDF-1 (rhSDF-1). The adhesion and migration of SJSA cells were tested on a monolayer of bone marrow stromal cells in response to rhSDF-1. We evaluated lung metastasis development in mouse model (Balb-c nu/nu) with and without monoclonal antibody anti-CXCR4 (T134) t...