## 4 Discussion  
In our previous study, AEG-1 was conferred to induce resistance to pemetrexed in non-small cell lung cancer by upregulating TS expression in vivo and in vitro [9]. Here, we reported that elevated circulating mRNA expression of AEG-1 and TS negatively affects treatment response and contributes to the poor prognosis in NSCLC patients treated with pemetrexed. Our data indicated a significant correlation between AEG-1 and TS plasma mRNA expression levels and treatment efficacy in NSCLC. Furthermore, we demonstrated that circulating AEG-1 mRNA expression detected by liquid biopsy after chemotherapy was associated with disease prognosis and survival. Circulating tumour cells (CTCs) are crucial to tumour metastasis and valuable for prediction of clinical outcome in patients with NSCLC using \"liquid biopsies\" [19; 24]. Chemotherapy is a standard mode of treatment for all cancers. CTC levels are determined before and after rounds of chemotherapy [25; 26]. However, isolation and enumeration of CTCs is difficult due to their low numbers, size, and heterogeneity. Novel techniques for CTCs capture and detection from peripheral blood mononuclear cells (PBMCs) had been developed [27; 28], and CTCs were counted 0.01-0.04% of total PBMCs [29]. Our study indicated that the change of AEG-1, TS, and CK19 plasma mRNA expression after chemotherapy were associated with disease prognosis and survival detected by RT-PCR from PBMC. Therefore, analysis of gene expression in PBMC could be used potentially to replace the techniques to capture CTC. Many studies have shown that TS expression level could be suggestive of the objective response of patients with NSCLC treated with pemetrexed containing chemotherapy [30; 31; 32; 33; 34; 35; 36; 37]. Our previous study revealed that increased expression of AEG-1 induces TS expression and contributes to the resistance of pemetrexed in cultured cells and in a few rebiopsy cases by IHC stain [11]. In this study, we investigated the plasma AEG-1 and TS mRNA expression by RT-PCR to detect that increased expression of AEG-1 and TS induces poor response to pemetrexed-contained chemotherapy. Furthermore, our analysis demonstrated about the negative correlation between AEG-1 and TS expression levels and prognosis in advanced NSCLC patients treated with pemetrexed. CK19 is expressed in NSCLC and is a prognostic determinant in lung cancer [38; 39; 40]. [PERSON] et al. [41] demonstrated that high CK19 was found with prognostic value in cases showing progression disease. However, there was no statistically significant correlation between high CK19 and overall survival. This is in contrary to the results reported by [PERSON] et al. [42], who showed significant correlation with survival. In our study, plasma CK19 mRNA expression was elevated significantly in patients with poor  
Figure 5: Cox regression multivariate analysis associated with overall survival (OS). AEG1: astrocyte-elevated gene-1; TS: thymidyllate synthase. treatment response. However, the role of CK19 expression was not significant in survival. This may be explained by the small sample size of the present study. AEG-1 was confirmed to have functions as an oncogene and regulates angiogenesis [4; 5]. AEG-1 expression directly correlates with increased expression of angiogenesis markers including angiopoietin-1 (Ang1), matrix metalloproteinase (MMP)-2, and HIF1-\(\alpha\) and supporting a potential role of AEG-1 in tumour angiogenesis [43]. AEG-1 mRNA expression upregulation indicated a significantly positive correlation with vascular endothelial growth factor (VEGF) expression and increased intratumoral microvessel density (iMVD, labelled by CD105) counting [44]. Several studies have shown that over-expression of AEG-1 significantly associates with tumour aggressiveness and poor prognosis in NSCLC [6; 45]. These results suggested that AEG-1 may play important roles in malignant transformation and tumour angiogenesis in NSCLC, and anti-AEG-1 mRNA expression may be a novel potential strategy for anti-angiogenic therapy of NSCLC.