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Cholangiocarcinoma (CCA) is a rare, aggressive disease with poor overall survival. In advanced cases, surgery is often not possible or fails; in addition, there is a lack of effective and specific therapies. Multidisciplinary approaches and advanced technologies have improved the knowledge of CCA molecular pathogenesis, highlighting its extreme heterogeneity and high frequency of genetic and molecular aberrations. Effective preclinical models, therefore, should be based on a comparable level of complexity. In the past years, there has been a consistent increase in the number of available CCA models. The exploitation of even more complex CCA models is rising. Examples are the use of CRISPR/Cas9 or stabilized organoids for in vitro studies, as well as patient-derived xenografts or transgenic mouse models for in vivo applications. Here, we examine the available preclinical CCA models exploited to investigate: (i) carcinogenesis processes from initiation to progression; and (ii) tools for personalized therapy and innovative therapeutic approaches, including chemotherapy and immune/targeted therapies. For each model, we describe the potential applications, highlighting both its advantages and limits.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy with limited therapeutic options. Trabectedin has a high antitumor activity in preclinical models of biliary tract carcinoma (BTC), being a promising alternative treatment. Here, we studied the effect of trabectedin at transcriptomic level on an ICC patient derived xenograft (PDX) and on the derived cell line, MT-CHC01. Further, putative targets of trabectedin were explored in the in vitro model. In vitro, trabectedin inhibited genes involved in protein modification, neurogenesis, migration, and motility; it induced the expression of genes involved in keratinization, tissues development, and apoptotic processes. In the PDX model, trabectedin affected ECM-receptor interaction, focal adhesion, complement and coagulation cascades, Hedgehog, MAPK, EGFR signaling via PIP3 pathway, and apoptosis. Among down-regulated genes, we selected SYK and LGALS1; their silencing caused a significantly reduction of migration, but did not affect proliferation in in vitro models. In MT-CHC01 cells, 24 microRNAs were deregulated upon drug treatment, while only 5 microRNAs were perturbed by trabectedin in PDX. The target prediction analysis showed that SYK and LGALS1 are putative targets of up-regulated microRNAs. In conclusion, we described that trabectedin affected genes and microRNAs involved in tumor progression and metastatic processes, reflecting data previously obtained at macroscopically level; in particular, we identified SYK and LGALS1 as new putative targets of trabectedin.

Background Fluoropyrimidines (FP) are the backbone chemotherapy (CT) for colorectal cancer (CRC). Although the most common toxicities have been extensively studied, FP-induced cardiotoxicity (FIC), an infrequent but potentially life-threatening toxicity, still lacks of a comprehensive characterization. The correlation between FIC and known cardiovascular (CV) risk factors remains controversial and based on retrospective observations. Methods CRC patients (pts) treated for the first time with FP at Candiolo Cancer Institute have been enrolled since January 2016. All pts were screened for potential CV risk factors; if needed, the treatment of preexisting CV comorbidities was optimized before starting CT. During the first 3 CT cycles, a monitoring with CV symptoms collection, seriated electrocardiograms and brain natriuretic peptide (BNP) measurements was performed. Primary objective was to assess the incidence of FIC. Secondary objectives included the analysis of the relationship of FIC with known CV risk factors and BNP levels. Results An interim analysis was conducted on 101 pts (65% male, median age 71.6 years). We found high prevalence of CV risk factors (BMI ≥25 54.2%, smoker 50%, heavy drinker 23%, sedentary lifestyle 63.3%) and comorbidities (diabetes mellitus 19.2%, dyslipidemia 34.7%, arterial hypertension 54%, stroke 3%, coronary artery disease 6%, arrhythmias 8%, heart failure 4%). 19 pts (18.8%) experienced FIC: 1 acute coronary syndrome (ACS), 1 coronary vasospasm, 1 paroxysmal supraventricular tachycardia (PSVT), 1 complete left bundle branch block (LBBB), 2 syncope, 4 typical chest pain, 6 sudden dyspnea, 3 sudden palpitations. After treatment of the CV events, only 3 pts had to discontinue FP (ACS, LBBB and PSTV). Among symptomatic pts, only 47% had CV comorbidities and/or CV risk factors. BNP levels increased on average by 73% [CI 99%: +39% - +106%] after the first cycle. Conclusions A high incidence of CV events, with no apparent correlation with CV comorbidities or risk factors, was observed. Prompt identification and treatment of CV events allowed most pts to complete the treatment with FP. Clinical trial identification NCT02665312. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure M. Aglietta: Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Merck; Advisory / Consultancy: Roche. All other authors have declared no conflicts of interest.

The association of anti-EGFR to gemcitabine and oxaliplatin (GEMOX) chemotherapy did not improve survival in biliary tract carcinoma (BTC) patients. Multiple mechanisms might be involved in the resistance to anti-EGFR. Here, we explored the mutation profile of EGFR extracellular domain (ECD), of tyrosine kinase domain (TKD), and its amplification status. EGFR mutational status of exons 12, 18–21 was analyzed in 57 tumors by Sanger sequencing. EGFR amplification was evaluated in 37 tumors by Fluorescent In Situ Hybridization (FISH). Kaplan-Meier curves were calculated using the log-rank test. Six patients had mutations in exon 12 of EGFR ECD and 7 in EGFR TKD. Neither EGFR ECD nor TKD mutations affected progression free survival (PFS) or overall survival (OS) in the entire population. In the panitumumab plus GEMOX (P-GEMOX) arm, ECD mutated patients had a worse OS, while EGFR TKD mutated patients had a trend towards shorter PFS and OS. Overall, the presence of mutations in EGFR or in its transducers did not affect PFS or OS, while the extrahepatic cholangiocarcinoma (ECC) mutated patients had a worse prognosis compared to WT. Nineteen out of 37 tumors were EGFR amplified, but the amplification did not correlate with survival. ECC EGFR amplified patients had improved OS, whereas the amplification significantly correlated with poor PFS (p = 0.03) in gallbladder carcinoma patients. The high molecular heterogeneity is a predominant feature of BTC: the alterations found in this work seem to have a prognostic impact rather than a predictive role towards anti-EGFR therapy.

Biliary tract carcinomas (BTC) are a group of tumours arising from the epithelial cells of intra- and extra-hepatic biliaryducts and the gallbladder, characterised by a poor prognosis. Surgery is the only curative procedure, but the risk of recurrence is high and furthermore, the majority of patients present with unresectable disease at the time of diagnosis. Systemic therapy is the mainstay of treatment for patients who present recurrent or metastatic disease. Progress has been made in the last decade to identify the most effective chemotherapy regimens, with the recent recommendation of the combination of gemcitabine–cisplatin as the standard schedule. Comprehension of the molecular basis of cholangiocarcinogenesis and tumour progression has recently led to the experimentation of targeted therapies in patients with BTC, demonstrating promising results. In this review we will discuss the clinical experience with systemic treatment for BTC, focusing on future directions with targeted therapies. © 2012 Elsevier Ireland Ltd. All rights reserved.

Biliary tract carcinomas (BTC) are malignant tumors with limited therapeutic options. Clinical experiences with anti-EGFR therapies have produced unsatisfactory results. The strategies of combined inhibition of EGFR and MEK1/2 could be a promising therapeutic option in BTC treatment. Preclinical activity of Panitumumab and Trametinib was tested in in vitro (EGI-1, MT-CHC01 and WITT cells) and in in vivo (xenograft) BTC models with different K-RAS mutational status. Trametinib reduced MAPK phosphorylation in wild type (WT) WITT cells and in both K-RAS mutated cells; in EGI-1 was also able to switch off EGFR activation. Panitumumab reduced the activation of its target only in EGI-1 cells, and of MAPK only in WITT cells. While Trametinib inhibited cell growth in K-RAS mutated cell lines, Panitumumab had no effect on proliferation independently by K-RAS status. The addition of Panitumumab to Trametinib did not significantly potentiate its anti-proliferative effect also in mutated cells. In vivo, Trametinib was able to significantly slow the tumor growth in K-RAS mutated xenograft models, but did not have effect on K-RAS WT cells; the addition of Panitumumab potentiated the Trametinib efficacy in MT-CHC01 and overcame the resistance to the anti-EGFR in WITT cells, in which the monotherapy was ineffective. Only in K-RAS mutated xenografts Trametinib alone or in combination with Panitumumab significantly decreased Ki67 positive cell fraction and CD31 angiogenesis markers. In conclusion, this preclinical study provides a rational to plan clinical trials assessing the efficacy of Trametinib in K-RAS mutated BTC patients and the combination with anti-EGFR in WT BTC patients.

Biliary tract carcinoma is a rare malignancy with multiple causes, which underlie the different genetic and molecular profiles. Cancer cell lines are affordable models, reflecting the characteristics of the tumor of origin. They represent useful tools to identify molecular targets for treatment. Here, we established and characterized from biological, molecular, and genetic point of view, an Italian intrahepatic cholangiocarcinoma cell line (ICC), the MT-CHC01. MT-CHC01 cells were isolated from a tumor-derived xenograft. Immunophenotypical characterization was evaluated both at early and after stabilization passages. In vitro biological, genetic, and molecular features were also investigated. In vivo tumorigenicity was assessed in NOD/SCID mice. MT-CHC01cells retain epithelial cell markers, EPCAM, CK7, and CK19, and some stemness and pluripotency markers, i.e., SOX2, Nanog, CD49f/integrin-α6, CD24, PDX1, FOXA2, and CD133. They grow as a monolayer, with a population double time of about 40 h; they show a low migration and invasion potential. In low attachment conditions, they are able to form spheres and to growth in anchorage-independent manner. After subcutaneous injection, they retain in vivo tumorigenicity; the expression of biliary markers as CA19-9 and CEA were maintained from primary tumor. The karyotype is highly complex, with a hypotriploid to hypertriploid modal number (3n+/−) (52 to 77 chromosomes); low level of HER2 gene amplification, TP53 deletion, gain of AURKA were identified; K-RAS G12D mutation were maintained from primary tumor to MT-CHC01 cells. We established the first ICC cell line derived from an Italian patient. It will help to study either the biology of this tumor or to test drugs both in vitro and in vivo. Electronic supplementary material The online version of this article (doi:10.1007/s13277-015-4215-3) contains supplementary material, which is available to authorized users.

Trastuzumab has changed the prognosis of HER2 positive breast cancers. Despite this progress, resistance to trastuzumab occurs in most patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show significant antitumor activity, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, since a high proportion of patients fail to respond to these alternative strategies, it is possible that cell escape from HER2 targeting may rely on HER2 independent pathways. The knowledge of these pathways deserve to be exploited to develop new therapies. We characterized two human HER2 overexpressing breast cancer cell lines resistant to trastuzumab and lapatinib (T100 and JIMT-1) from a molecular and biological point of view. Indeed, we assessed both in vitro and in vivo the activity of the multitarget inhibitor sorafenib. In both cell lines, the previously proposed mechanisms did not explain resistance to HER2 inhibitors. Notably, silencing HER2 by shRNA did not affect the growth of our cells, suggesting loss of reliance upon HER2. Moreover, we identified alterations in two antiapoptotic proteins Mcl-1 and Survivin which are known to be targets of the multikinase inhibitor sorafenib. Moreover, sorafenib, strongly inhibited the in vitro growth of T100 and JIMT-1 cells, through the downregulation of both Mcl-1 and Survivin. Similar results were obtained in JIMT-1 xenografts subcutaneously injected in NOD SCID mice. We provide preclinical evidence that tumor cells resistant to trastuzumab and lapatinib may rely on HER2 independent pathways that can be efficiently inhibited by sorafenib.

The recombinant monoclonal antibody trastuzumab has antiproliferative effect on breast cancer (BC) cells with ErbB2 overexpression. We postulated that a mechanism able to modify ErbB2 expression enhances the antitumor effect of trastuzumab. We analyzed whether granulocyte-colony stimulating factor (G-CSF), widely used in adjuvant cancer therapy to alleviate chemotherapy-induced myelotoxicity, could influence ErbB2 expression in BC cells and patients. The expression of ErbB2 (Herceptest) was analyzed in four BC cell lines (BT474, SKBR3, ZR75.1, and T47D) treated with G-CSF and in five samples biopsies from BC patients subjected to G-CSF rescue after chemotherapy. The effects of G-CSF and trastuzumab alone or their combination on cell growth and apoptosis were investigated. G-CSF receptor was detected on all cell lines and BC patients. G-CSF induced upregulation of ErbB2 in SKBR3, ZR75, and T47D cells. This modulation was not associated with an increase in tumor cell growth in vitro. Trastuzumab alone inhibited colony formation in soft agar but did not induce apoptosis on BC cells with no or low ErbB2 genomic amplification. The combination of trastuzumab and G-CSF enhanced the inhibition of tumor colony formation and induced apoptosis on these cells. This effect was further increased by G-CSF pretreatment. Five of nine BC patients showed an increase of Herceptest score after G-CSF administration. G-CSF treatment increases ErbB2 expression in vitro and in vivo enhancing the activity of trastuzumab on BC cell lines inducing apoptosis of BC cells with low or no ErbB2 genomic amplification.

Intrahepatic cholangiocarcinoma (ICC) is the second most common liver cancer. Conventional chemotherapy and radiotherapy have only a marginal impact on the natural history with a median survival of 12 months. Encouraging results obtained on ICC cell lines prompted us to evaluate the antitumor activity and mechanism of action of ET-743 on an ICC patient-derived xenograft (PDX) model, currently representing an innovative tool to personalize cancer therapy. The antitumor activity of ET-743 was tested on a cohort of mice carrying an ICC PDX. At the achievement of about 150 mm3 of volume, mice were randomized in two arms (n=7) of therapy in three independent experiments: 150 mg/Kg/weekly i.v. of ET-743 or drug vehicle for three administrations. Tumors were monitored weekly. Mean volumes of treated and untreated xenografts were compared by two-way Anova, considering a p-value ET-743 slowed tumor growth of PDX-treated compared to PDX-untreated mice in a statistically significant manner (p=0.04). Interestingly, we found a decrease in the number of proliferating cells (p=0.05) and a significant decrease in the number of new vessel formation (p=0.007). Upon ET-743 treatment, 1346 differentially expressed probes were found: 628 down-regulated and 718 up-regulated in ET-743 treated vs control PDX mice. Gene Ontology analysis revealed that the most significant deregulated processes were biological and cell adhesion, tissue development and inflammatory/immune response. Among the 20 most down-regulated genes, we found TFF3, MUC5B, MUC6A, WISP1, previously associated with cholangiocarcinoma. miRNA profiling revealed a deregulation of 6 miRNAs, 3 up-regulated and 3 down-regulated. Using the Mirpath program, we found that the down-regulated miRNAs affect important cancer related pathways (p53, Akt-PI3K, MAPK pathways) in a statistically significant way. In conclusion, ET-743 is able to control tumor growth, inhibiting proliferation and neoangiogenesis; moreover ET-743 perturbs gene and miRNA profiling affecting important cancer related pathways. Taken together these data are a strong rational for the design of a clinical trial to evaluate the activity of ET-743 in ICC patients. Citation Format: Caterina Peraldo Neia, Giuliana Cavalloni, Giovanna Chiorino, Paola Ostano, Massimo Aglietta, Francesco Leone. Antitumor activity, gene and miRNA modulation upon ET-743 treatment in an intrahepatic cholangiocarcinoma patient-derived xenograft model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5551. doi:10.1158/1538-7445.AM2014-5551

Tumor cell contamination might induce relapse after autologous transplantation in breast cancer patients. We used an ex vivo purging strategy to decrease the number of contaminating breast tumor cells in leukaphereses without altering the engraftment potential of the hemopoietic progenitor cells. This method is based on immunoselection of CD34+ cells derived from mobilized peripheral blood of patients with metastatic breast cancer and expansion in the presence of flt3 ligand, stem cell factor, interleukin 6, and thrombopoietin. Tumor contamination before and after culture was monitored by mammaglobin messenger RNA amplification by quantitative polymerase chain reaction. We analyzed both adherent and suspended cells obtained after 2 weeks of culture. Hemopoietic progenitors were increased among suspended cells. In this fraction, tumor cell contamination was decreased, whereas it increased within the adherent cell fraction. Experimental models using CD34+ cells from healthy donors spiked with breast cancer cells were also constructed to investigate whether treatment with anti–ErbB-receptor drugs could further reduce the tumor load without affecting the clonogenic potential of hemopoietic cells. For this purpose, we successfully assayed trastuzumab, a monoclonal antibody against ErbB-2, and gefitinib, an epidermal growth factor receptor tyrosine kinase receptor inhibitor. These results suggest that positively selected CD34+ cells from cancer patients contain tumor cells and that ex vivo expansion can reduce the tumor load of the suspended fraction. Target-based agents against ErbB-2, epidermal growth factor receptor, or both—such as trastuzumab or gefitinib—might increase the efficiency of purging.

Identification of sensitive techniques for breast cancer cell detection might be relevant for high-dose chemotherapy programs with autologous stem cell transplantation. We investigated the feasibility of Maspin, Mammaglobin and c-ErbB-2 amplification by real-time quantitative polymerase chain reaction (RQ-PCR) for the detection of breast cancer cells in leukaphereses. Expression of the three markers was determined in primary breast cancers and cell lines. Peripheral blood (PB), bone marrow (BM), and leukapheresis samples from patients with malignancies other than breast cancer were used as controls. Sensitivity was evaluated by dilution of primary tumors and cell lines with mononuclear blood cells. We found expression of the three markers in all primary tumors and most cell lines. No blood specimen from control patients had the Maspin transcript, while only one was positive for Mammaglobin. Weak c-ErbB-2 expression was detectable in most PB, all BM and all leukapheresis samples from controls. We observed a low sensitivity of Maspin RQ-PCR and a sensitivity of Mammaglobin RQ-PCR up to one tumor cell in 106 mononuclear cells. One out of 18 leukaphereses from breast cancer patients screened for the presence of Mammaglobin mRNA was positive. We conclude that Mammaglobin RQ-PCR might be a useful tool for detection of leukapheresis contamination. Bone Marrow Transplantation (2001) 27, 517–523.

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...

HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly. In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells. In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A82.

Osteosarcoma (OS) is the most common primary bone tumor in children and young adults. Despite improved prognosis, metastatic or relapsed OS remains largely incurable and no significant improvement has been observed in the last 20 years. Therefore, the search for alternative agents in OS is mandatory. We explored phospho-ERK 1/2, MCL-1, phospho-Ezrin/Radixin/Moesin (P-ERM) as potential therapeutic targets. The activation of these pathways was shown by immunohistochemistry in about 70% of cases and in all OS cell lines analyzed. Mutational analysis revealed no activating mutations in KRAS. While, BRAF gene was found mutated in 4/30 OS samples from patients. Based on these results we tested the multikinases inhibitor sorafenib (BAY 43-9006) in preclinical models of OS. Sorafenib inhibited OS cell lines proliferation, induced apoptosis and downregulated P-ERK1/2, MCL-1, P-ERM in a dose dependent manner. ERM dephosphorylation is not due to ERK inhibition. The downregulation of MCL-1 increased apoptosis in OS cell lines. In chick embryo chorioallantoic membrane the angiogenesis induced by OS supernatants was blocked by sorafenib treatments. Indeed, sorafenib reduced VEGF and MMP2 production. Sorafenib treatment dramatically reduced tumor volume of OS xenografts and lung metastasis in SCID mice. In conclusion, ERK1/2, MCL-1 and ERM pathways are active in OS. Sorafenib is able to interrupt their routes in vitro and in vivo displaying antitumoral activity, antiangiogenic effect and reducing metastasis colonies formation in lungs. These data support the testing of sorafenib as a potential therapeutic option in metastatic or relapsed OS patients failing standard treatments. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C213.

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.

Background Intrahepatic cholangiocarcinoma (ICC) is an aggressive, highly lethal tumors and lacks of effective chemo and targeted therapies. Cell lines and animal models, even partially reflecting tumor characteristics, have limits to study ICC biology and drug response. In this work, we created and characterized a novel ICC patient-derived xenograft (PDX) model of Italian origin. Methods Seventeen primary ICC tumors derived from Italian patients were implanted into NOD (Non-Obese Diabetic)/Shi-SCID (severe combined immunodeficient) mice. To verify if the original tumor characteristics were maintained in PDX, immunohistochemical (cytokeratin 7, 17, 19, and epithelial membrane antigen) molecular (gene and microRNA expression profiling) and genetic analyses (comparative genomic hybridization array, and mutational analysis of the kinase domain of EGFR coding sequence, from exons 18 to 21, exons 2 to 4 of K-RAS, exons 2 to 4 of N-RAS, exons 9 and 20 of PI3KCA, and exon 15 of B-RAF) were performed after tumor stabilization. Results One out of 17 (5.8 %) tumors successfully engrafted in mice. A high molecular and genetic concordance between primary tumor (PR) and PDX was confirmed by the evaluation of biliary epithelial markers, tissue architecture, genetic aberrations (including K-RAS G12D mutation), and transcriptomic and microRNA profiles. Conclusions For the first time, we established a new ICC PDX model which reflects the histology and genetic characteristics of the primary tumor; this model could represent a valuable tool to understand the tumor biology and the progression of ICC as well as to develop novel therapies for ICC patients. Electronic supplementary material The online version of this article (doi:10.1186/s12885-016-2136-1) contains supplementary material, which is available to authorized users.