Development of resistance is the major hindrance to successful chemotherapy treatment in oesophago-gastric (OEG) cancers. Platinum based chemotherapy prolong the survival however only 20 - 30 % of patients survive 5- years since diagnosis of the disease. Therefore understanding of the mechanisms that underlie this phenomenon and identification of novel biomarkers/targets that could predict the response to the treatment and sensitize these tumours to current therapy is needed. We established a panel of human cancer cell lines of oesophagus (OE21 and OE33) and gastric cardia (AGS) resistant to oxaliplatin, cisplatin and docetaxel. To study mechanisms of drug resistance we performed gene expression profiling on resistant and parental lines and differentially expressed genes involved in sphingolipids / lysosomes metabolism have been shown as associated with drug resistance. Selected markers were validated on mRNA and protein levels among the panel of OEG cell lines and clinical specimens. Cellular levels of sphingolipid species were determined in drug sensitive and resistant lines using mass spectrometry. This study revealed a positive correlation between over-expression of sphingosine kinase 1 (SPHK1) and increased levels of sphingosine -1-phosphate (S1P) associated with drug resistance in gastric cancer cell lines. Moreover it showed the predictive value of SPHK1 as high level of this protein correlates with poor survival of OEG cancer patients treated with cisplatin based chemotherapy, in contrast to those patients that received surgery alone. Additionally, lipidomic profiling data showed possibly distinct mechanisms of drug resistance between gastric and oesophageal tumours, indicating that mechanisms of drug resistance are likely cell type, rather than drug specific. In conclusions, this study proofs the clinical relevance of our in vitro experimental models to study mechanisms of drug resistance in OEG tumours and provides the source of novel biomarkers for targeted therapy strategies in this disease.

Introduction: The incidence of oesophageal cancer (OC) is increasing. Targeted therapies are being investigated, as chemotherapy in advanced OC achieves only 50% response rates and confers significant toxicity. TRANS-COG is a sub-study of COG, the only randomised trial of the Epidermal Growth Factor Receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib versus placebo in patients with advanced OC. Aim and Methods: The aim of TRANS-COG is to identify biomarkers for predicting response to gefitinib by investigating the frequency of mutations of EGFR pathway members and EGFR gene copy number gain (CNG) by Fluorescence In Situ Hybridisation (FISH). Results: No EGFR mutations were identified, frequency of other pathway member mutations are as follows: KRAS 3.6%, PI3KCA 3.2%, BRAF 0.6%. The frequency of EGFR CNG was 13.5%. There were two cases of combined EGFR CNG and KRAS mutation 0.3%.Patients with EGFR CNG tumours had improved OS with gefitinib compared to placebo, log rank p=0.033, HR 0.523 (95%CI 0.285-0.962). Those with EGFR high CNG (amplification) also had improved OS with gefitinib, median OS 4.40 months vs median OS 1.71 months for placebo log rank p=0.004, HR 0.184 (95% CI 0.052-0.653). Mutation of KRAS conferred poor prognosis, independent of treatment received; median OS 3.614 months in KRAS wild type vs 1.741 months in KRAS mutated tumours, log rank p=0.023, HR 1.8153 (95% CI 1.078 3.187). A novel KRAS codon 61 mutation, Q61L, was also identified. Conclusion: 21.18% of patients have dysregulation of the EGFR pathway, these abnormalities represent realistic therapeutic targets. This analysis demonstrates clinical utility of EGFR CNG as a predictive biomarker of gefitinib response.