## 1 Introduction  
Lung cancer is the world's leading cause of death from cancer [1], and it is usually diagnosed at an advanced or metastatic stage. Treatments for advanced stages are based on cytotoxic chemotherapy and other systemic therapies [2]. Response to treatment is assessed by computed tomography (CT) using the response evaluation criteria in solid tumors(RECIST) criteria [3]. Although clinical image presentation is often delayed, indicating cancer progression, to date, there is no reliable biomarker for the evaluation of response to standard chemotherapy treatment in lung cancer. Therefore, there is a need for more prognostic factors to manage patients with advanced non-small cell lung cancer (NSCLC).  
Expression of astrocyte-elevated gene-1 (AEG-1), a novel oncoprotein, is elevated in multiple cancers and plays a vital role in tumour cell growth, invasion, angiogenesis, and progression to metastasis [4; 5]. High expression of AEG-1 could promote the carcinogenesis and lead to a poor clinical prognosis of NSCLC [6; 7; 8]. Recent findings suggest that AEG-1 contributes to broad-spectrum resistance to various chemotherapeutics [9; 10]. Our previous data demonstrated that thymidylate synthase (TS) expression might be regulated by AEG-1 and that increased expression of these proteins contributes to lung cancer disease progression and may be associated with the development of resistance to pemetrexed [11]. AEG 1 could be used as a biomarker to identify subgroups of patients who require more intensive treatments. However, the acquisition of tumour tissue via biopsy may be very difficult to perform at the onset of resistance after chemotherapy due to small lesions or patients' poor conditions.  
Liquid biopsy analysis is currently a rapidly expanding field in translational cancer research as it might be useful at different points of the diagnostic/therapeutic course of cancer patients [12]. Messenger RNAs (mRNA) possess a critical role in intracellular protein translation, and it is likely that extracellular mRNAs reflect the status of the intracellular process and are conceivably potential biomarkers for cancer diagnosis or therapeutic monitoring [13]. Recent studies reported various coding RNAs in plasma or serum from patients with cancer, and levels of circulating cell free mRNAs were found to be predictive of clinical outcome [14; 15] and disease prognosis [16; 17].  
It has been hypothesized that circulating mRNA of AEG-1 expression could be a predictive factor of tumour response and a good candidate for a prognostic factor. Liquid biopsy analysis is currently a rapidly expanding field in translational cancer research as it might be useful at different points of the diagnostic/therapeutic course of cancer patients. Some studies have shown that circulating mRNA concentration could be a predictive factor of tumour response to surgery and to radiation therapy in cancer [18; 19], but very few studies have investigated circulating mRNA concentration as a predictive factor of tumour response to chemotherapy [20; 21; 22]. In this prospective study, we will assess the predictive and prognostic values of AEG-1, combined with TS and CK19 expression, on tumour response and survival according to circulating mRNA concentration by liquid biopsy in NSCLC patients treated with pemetrexed.