Circulating mRNA Expression of astrocyte-Elevated Gene-1 Associated with Treatment Response and Survival in Non-Small Cell Lung Cancer Patients Treated with Pemetrexed

Backgrounds: Astrocyte-elevated gene-1 (AEG-1) functions as an oncogene and regulates angiogenesis in non-small cell lung cancer (NSCLC). In this prospective study, we assessed the values of plasma AEG-1 mRNA expression by liquid biopsy associated with tumor response and survival in NSCLC patients treated with pemetrexed. Methods: Patients diagnosed advanced NSCLC were enrolled to be treated with pemetrexed combined platinum as rst-line chemotherapy. All patients underwent blood sampling before any cancer treatment (C0) and at �rst response evaluation after two cycles (C2) treatments. Response to chemotherapy and survival were assessed. Plasma mRNA was extracted from peripheral blood mononuclear cell (PBMC) and quanti�cation of RNA was performed by real-time PCR. Results: A total of 50 patients with advanced NSCLC were included and 13 of 50 patients combined with bevacizumab. In patient groups of SD (n = 13) and PD (n = 10), the plasma mRNA of AEG-1, thymidylate synthase (TS) and CK19 were elevated signi�cantly at C2 compared to patients in treatment response group (PR, n = 27) (PR v.s. SD or PD, AEG-1: 1.22 ± 0.80 v.s. 4.51 ± 15.45, p = 0.043). NSCLC patients had elevated AEG-1 (AEG-1 ≥ 2) after 2-cycle chemotherapy had shorter PFS and OS (high AEG-1 v.s. low AEG-1, median, PFS: 5.5 v.s. 11.9 months, p = 0.021; OS: 25.9 v.s. 40.8 months, p = 0.019, respectively). In Cox regression analysis, increased plasma mRNA expression of AEG-1indicated poor prognosis in survival. Conclusion: Circulating mRNA


Introduction
Lung cancer is the world leading cause of death by 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 RECIST (response evaluation criteria in solid tumors) criteria (3).Although clinical image presentation is often delay to indicate 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 tumor cell growth, invasion, angiogenesis, and progression to metastasis (4,5).High expression of AEG-1 could promote the carcinogenesis and leading to a poor clinical prognosis of NSCLC (6-8).Recent ndings 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 tumor tissue via biopsy may be very di cult to perform at the onset of resistance after chemotherapy since small lesions or patients' poor conditions.Liquid biopsy analysis is currently a rapidly expanding eld in translational cancer research as it might be useful at different points of the diagnostic/therapeutic course of cancer patients (12).Because messenger RNAs (mRNA) possess a critical role in intracellular protein translation and, it is likely that extracellular mRNAs re ect 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 tumor response and a good candidate for a prognostic factor.Liquid biopsy analysis is currently a rapidly expanding eld 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 tumor 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 tumor 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 tumor response and survival according to circulating mRNA concentration by liquid biopsy in NSCLC patients treated with pemetrexed.

Patients and data collection
This prospective study was approved by the investigational review boards of the National Taiwan University Hospital (NO.201510131RINB), and all participating patients provided informed consent before chemotherapy.Patients with a diagnosis of locally advanced or metastatic non-squamous cell lung cancer, stage IIIB or IV (7th version of the TNM staging system for lung cancer of the International Association for the Study of Lung Cancer) (23), and those who were treated with pemetrexed plus platinum as rst-line chemotherapy regimen in the National Taiwan University Hospital Yunlin Branch were potentially eligible for inclusion.The diagnosis of NSCLC was based on histological examination of biopsy specimens or on cytological analysis of samples aspirated with a ne needle.Molecular testing for epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK) rearrangements were investigated from the pathologic or cytological specimens.Patients' basic data and medication usage were retrieved from electronic medical records.

Evaluation of treatment responses and survival
The primary objective was to identify associations between speci c biomarkers and treatment response rate.The secondary objective was to identify biomarkers associated with patient survival.All patients underwent blood sampling before chemotherapy (C0) and after 2 cycles of chemotherapy (C2).
The patients were administered with cisplatin or carboplatin plus pemetrexed intravenously every three weeks in patients with wild-type EGFR and ALK mutation or patients harboring EGFR or ALK mutation with disease progression after target therapy.Furthermore, patients received 500 mg/m2 pemetrexed intravenously every three weeks as continuation maintenance therapy if the disease did not progress after 4 cycles of the rst-line treatment.All patients were adults with good performance status (Eastern Cooperative Oncology Group Performance Status: 0-1).Treatment was discontinued if there was evidence of progressive disease, or unacceptable toxicity.
Chest computed tomography was performed every 3 months as routine clinical practices, and as needed to con rm treatment response.Treatment responses were evaluated according to the Response Evaluation Criteria in Solid Tumors, version 1.1 (24), and were de ned as complete remission, partial response, stable disease, and progressive disease.Response rate (RR) was de ned as the percentage of patients who achieved a complete remission or partial response.Disease-control rate (DCR) was de ned as the percentage of patients who achieved a complete remission, partial response, or stable disease.
Progression-free survival (PFS) was de ned as the time from the rst cycle of pemetrexed plus platinum to documented progression or death from any cause, and was censored at the date of the last follow-up visit for patients who were still alive and who had not exhibited progression.The overall survival (OS) was measured from the date of the start of the treatment to the date of the last follow-up.Patients were followed up until December 2018.

Isolation of Human PBMCs
Collect 4 ml of human venous blood sample in heparinised vials (BD biosciences) and mix well by gently inverting the tube several times.Take 4 ml of Ficoll Histopaque in a 15 ml centrifuge tube.Gently layer the blood on the top of Ficoll Histopaque.The layering should be done very slowly that blood and Ficoll Histopaque should stay as two different layers.Centrifuge the tubes (without any delay) for 30 min at 1200g in 4°C in a swing-out bucket.Fixed angle rotors also can be used but would require more caution when separating cells in interphase.Aspirate the whitish buffy coat (about 1 ml) (PBMCs) formed in the interphase between histopaque and medium.Wash the cells (centrifuge in 1000g for 10 min) twice with 10 ml of sterile PBS or sterile Dulbecco's modi ed eagle medium.The approximate yield of cells added 1 ml Trizol and placed in the − 80℃ refrigerator for freezing.

Plasma mRNA Extraction and Quantitative Real-time PCR.
Total RNA (from PBMCs) was extracted using a QIAamp RNA Blood Mini Kit (Qiagen).First-strand complementary DNA (cDNA) synthesis was performed using 2 µg of total RNA using Invitrogen Super Script Reverse Transcriptase (Thermo Fisher Scienti c, Carlsbad, CA, USA).Real-time PCR was performed according to the manufacturer's protocol for Applied Biosystems Power SYBR® Green PCR Master Mix, and the relative mRNA expression level of AEG-1(Astrocyte elevated gene-1, Metadherin, MTDH), TS (Thymidylate synthetase, TYMS), CK19 (Keratin19) and Actin was determined as a ratio to the actin.

Statistical methods
Variables as plasma mRNA level including AEG-1, TS and CK19 were presented as mean ± SD.The mRNA expression level before chemotherapy was indicated as baseline as 1.0.For comparisons of these biomarker variables, we used the independent T-test and paired T-test, respectively.Categorical variables are expressed as percentages.The median value of plasma AGE-1, TS, and CK19 mRNA expression was captured as cut-off level.Correlation of patients' clinical fator and treatment response was estimated by using the Pearson Chi-Square.For survival analysis we used the Kaplan-Meier method and median survival (and corresponding 95% con dence intervals) were compared using the log-rank test.Then, we used a Cox regression model (enter method) in order to assess variables associated with survival.If p < 0.05 was considered signi cant.Statistical analysis was performed using MedCalc (Version 19.6.1).

Clinical Evaluation of Treatment E cacy
From the year 2015 to 2018, a total of 50 patients (29 male and 21 female) with advanced nonsquamous NSCLC received Pemetrexed/Platinum based doublets chemotherapy.The median of age was 65 years old (range: 37 to 92 years old).Twenty-two of them harbored EGFR mutation and three of them were ALK positive.Chemotherapy regimens included 36 cisplatin and 14 carboplatin with pemetrexed, thirteen of 50 patients combined with bevacizumab (Table 1).

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 signi cant correlation between AEG-1 and TS plasam mRNA expression levels and treatment e cacy in NSCLC.Furthermore, we demonstrated that circulating AEG-1 mRNA expression detected by liquid biopsy after chemotherapy were associated with disease prognosis and survival.
Circulating tumor cells (CTCs) are crucial to tumor metastasis and valuable for prediction of clinical outcome in patients with NSCLC using "liquid biopsies".(25,26).Chemotherapy is a standard mode of treatment for all cancers.CTC levels are determined before and after rounds of chemotherapy.(27, 28) However, isolation and enumeration of CTCs is di cult because of their low numbers, size, and heterogeneity.Novel techniques for CTCs capture and detection from peripheral blood mononuclear cells (PBMCs) had been developed (29,30), and CTCs were counted 0.01-0.04% of total PBMCs (31).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 (32)(33)(34)(35)(36)(37)(38)(39).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 (9).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 (40)(41)(42).Bastawisy AE et al. (43) demonstrated that high CK19 was found with prognostic value in cases showing progression disease.However, there was no statistically signi cant correlation between high CK19 and overall survival.This is in contrary to the results reported by Bréchot et al. (44) and Rosenblatt et al. (45) who showed signi cant correlation with survival.In our study, plasma CK19 mRNA expression were elevated signi cantly in patients with poor treatment response.Though, the role of CK19 expression was not signi cant in survival.This may be explained by the small sample size of the present study.
AEG-1 was con rmed 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 metalloprotease (MMP)-2, and HIF1-α and supporting a potential role of AEG-1 in tumor angiogenesis (46).AEG-1 mRNA expression upregulation indicated a signi cantly positive correlation with vascular endothelial growth factor (VEGF) expression and increased intratumoral microvessel density (iMVD, labeled by CD105) counting (47).Several studies have showed that over-expression of AEG-1 signi cantly associates with tumor aggressiveness and poor prognosis in NSCLC (48, 49).These results suggested that AEG-1 may play important roles in malignant transformation and tumor angiogenesis in NSCLC, and anti-AEG-1 mRNA expression may be a novel potential strategy for antiangiogenic therapy of NSCLC.In our study, bevacizumab-combined chemotherapy decreased more plasma AEG-1 mRNA expression, also had higher response rate, longer PFS and OS.However, the differences were not signi cantly.This may be limited by the small sample size of the present study.

Conclusion
AEG-1 functions as an oncogene and regulates angiogenesis in NSCLC.AEG-1 also promotes TS expression contributing to the development of resistance to pemetrexed.Bevacizumab-combined chemotherapy inhibits AEG-1 expression and could increase treatment bene ts and survivals.Plasma AEG-1 mRNA level could be used a predictive and prognostic biomarker in NSCLC patients treated with pemetrexed.Increased expression of AEG-1 leaded to chemoresistance and also caused lung cancer progression, which could be used for clinical evaluation combined with image studies. Figures

Figure 2 Dot
Figure 2

Figure 3 Expression
Figure 3

Table 1
Pemetrexed plus Platinum with and without Bevacizumab for NSCLC: Basic Characteristics and Plasma AEG-1 mRNA expression was lower in patients treated with bevacizumab at C2 but not signi cantly (with bevacizumab v.s.without, AEG-1: 1.25 ± 0.75 v.s.3.25 ± 12.20, p = 0.560).Moreover, the change of plasma mRNA level as AEG-1, TS and CK19 between different chemotherapy regimens including either cisplatin or carboplatin was not signi cant.