Cell Free EGFR mRNA Expression and Implications for Survival and Metastasis in Non-Small Cell Lung Cancer Cases

Lung cancer is the major cause of cancer-related deaths and has the highest incidence, mortality rates amongst all malignancies worldwide (Siegel et al., 2013). Lung cancer has an incidence of over 1.6 million cases every year accounting for 13% of all new cancer diagnoses and 1.4 million deaths every year recorded for 18% of all cancer-related deaths (WHO, 2012; Lewis et al., 2013). Quantitative real-time RT-PCR (qPCR) was recently shown to be useful for early NSCLC diagnosis, prognosis prediction and gene expression analysis (Hayes et al., 2006). The molecular mechanisms involved in NSCLC are still relatively unknown and are being investigated extensively. Over the last decade, extensive research has been performed on tyrosine kinases (Engelman et al., 2005). All members have an extracellular ligandbinding region, a single membrane-spanning region and a cytoplasmic tyrosine-kinase-containing domain (Yarden et al., 2001; Riese et al., 2006). Ligand binding to EGFR receptors induces the


Introduction
Lung cancer is the major cause of cancer-related deaths and has the highest incidence, mortality rates amongst all malignancies worldwide (Siegel et al., 2013).Lung cancer has an incidence of over 1.6 million cases every year accounting for 13% of all new cancer diagnoses and 1.4 million deaths every year recorded for 18% of all cancer-related deaths (WHO, 2012; Lewis et al., 2013).Quantitative real-time RT-PCR (qPCR) was recently shown to be useful for early NSCLC diagnosis, prognosis prediction and gene expression analysis (Hayes et al., 2006).
The molecular mechanisms involved in NSCLC are still relatively unknown and are being investigated extensively.Over the last decade, extensive research has been performed on tyrosine kinases (Engelman et al., 2005).All members have an extracellular ligandbinding region, a single membrane-spanning region and a cytoplasmic tyrosine-kinase-containing domain (Yarden et al., 2001;Riese et al., 2006).
Ligand binding to EGFR receptors induces the formation of receptor homo and heterodimers and activation of the intrinsic kinase domain, resulting in phosphorylation on specific tyrosine residues within the cytoplasmic tail.These phosphorylated residues serve as docking sites for a range of proteins, the recruitment of which leads to the activation of intracellular signaling pathways (Schlessinger et al., 2004).EGFR is a critical component for signal transduction, blocking to EGFR can inhibit tumor growth and targeting EGFR tyrosine kinase activity could important therapeutic target in cancer therapy (Utsugi et al., 2013).Qing Li revealed that EGFR gene expression, and variations in gene copy number can be used in the differential diagnosis of high-grade and lowgrade CIN but also in the early diagnosis of cervical cancer (Qing et al., 2014).The EGFR receptors are implicated in the development of many types of cancer, and EGFR was the first tyrosine-kinase receptor to be linked directly in human tumors (Gschwind et al., 2004).The EGFR tyrosine kinase works through the auto-activation of the receptor via its homo/heterodimerization and autophosphorylation on tyrosine-rich cytosolic domains after the binding of the ligand (Hynes et al., 2005).There is evidence that the activated EGFR can also mediate signals through the STAT (Signal transducer and activator of transcription) transcription factors (Quesnelle et al., 2007;Hirsch et al., 2009).The EGFR is expressed in all cell types with the exception of hematopoietic cells.More importantly, the EGFR has been found to be over expressed in 50-70% of human primary colon, lung, and breast carcinomas as well as in other tumor types (Salomon et al., 1995).
Over-expression of EGFR-TK results in increased cell proliferation, survival, invasion and metastases.This has been implicated in the pathogenesis and progression of many malignancies as well as in the poor prognosis of patients (Wang et al., 2007(Wang et al., , 2008)).This study aimed to investigate the putative role of cell free EGFR gene expression with survival and metastases of NSCLC patients.

Study population and sample collection
Present study was approved by Institutional ethics committee of Maulana Azad Medical College & associated hospitals and All India Institute of Medical Sciences New Delhi.This study included 100 newly diagnosed NSCLC patients and 100 age and sex matched healthy controls.Patients' 3 ml blood was collected from each subject and serum was separated within 30 minute of the sample collection and stored at -80 o C until analyzed.Patients included in study were followed from the 2013 to 2014 for survival analysis.

Total RNA isolation and cDNA synthesis
Total RNA was extracted from serum using Trizol reagent according to the manufacturer's protocol (Invitrogen) and stored at -80 o C until further processing.cDNA was synthesised by using 100ng total RNA following manufacturers protocol (Verso, Thermo scientific,USA).

Quantitative Real time PCR
EGFR gene expression was studied by QRT-PCR (SYBR Green I technology) with β-actin gene as internal control.The primer sequences for EGFR gene expression were forward primer 5'-GGA GAA CTG CCA GAA ACT GAC C-3', reverse primer 5'-GCC TGC AGC ACACTGGTTG-3' (Ivan Bieche et al., 2003), for β-actin were forward primer 5'-CGACAACGGCTCCGGCATGTGC-3, reverse primer 5-GTCACCGGAGTCCATCACGATGC-3'.The expression of EGFR and β-actin was performed by PCR programme for 40 cycles, denaturation at 95°C for 40 s, annealing at 60°C for 40 s, extension at 72°C for 40s and reaction volume was 20 μl.A final extension step at 72°C for 5 min to complete the reaction and melting curve analysis was performed between the range of 35°C to 90°C to ensure the specific amplification.A control without cDNA was included in each experiment as non template control and all reaction were performed in duplicate.The relative quantification method (∆∆CT) was used to analyse the EGFR gene expression level by using β-actin as internal control and final results were expressed as mean fold change in EGFR gene expression in NSCLC patients as compared to control.

Statistical analysis
Statistical analysis of data was performed using the SPSS 16 and Graph Pad version 6.0.Mann Whitney and Kruskal Wallis test were used to analyze the association with different variables.The Kaplan-Meier method was used to calculate the overall survival of NSCLC patients.A p value <0.05 was considered indicative of a statistically significant difference.

Demographics
All demographic features of the subjects are depicted (Table 1).In brief, total of 100 Non-small cell lung cancer patients (adenocarcinoma) were analyzed.This study included both males (71%) and females (29%) and mean age of 54.37 years.44% patients were in stage IV and 15%, 15%, 26% patients in stage I, II and III respectively while 44% patients had distant metastases .Patients with different pathological grade, grade 1 (well differentiated) includes 24%, grade 2 (moderately differentiated) includes 41% and grade 3 (Poorly differentiated) includes 35% cases.We included smoker 45% as well as non smoker 55% with different smoking type as cigarette, bidi, and hukka, 18% cases smoked cigarette, 16% cases smoked bidi and 11% cases smoked hukka.

EGFR gene expression and NSCLC patients:
We analyzed EGFR gene expression with several variables of Non-small Cell Lung Cancer patients in this study and observed mean value of increased gene expression was 13.54 fold in Non-small Cell Lung Cancer patients.Patients in stage I showed 4.07 fold increased EGFR gene expression, stage II showed 7.08 fold increased gene expression while in stage III showed 14.81 fold increased gene expression and stage IV showed 18.35 fold increased gene expression which is significantly associated (p<0.0001).Patients with metastases had 18.35 fold increased gene expression while patients without metastases had 9.86 fold increased gene expression also showed significant differences (p<0.0001) in gene expression.Patients who had pleural effusion showed 17.66 fold increased gene expression while patients without pleural effusion showed 12.88 fold increased gene expression.Patients with different smoking type is significantly associated (p=0.002) with EGFR gene expression.Patients who smoked bidi had 18.55 fold increased gene expression in comparison with cigarette (15.51fold) and hukka (7.22 fold) smokers.Patients with different smoking level had also significantly associated (p=0.03) with EGFR gene expression, data showed in Table 2.

EGFR gene expression and patients' survival
We assessed overall survival between two groups of NSCLC patients expressing above 13 fold and below 13 fold gene expression of EGFR in NSCLC patients.Patients with <13 fold increased gene expression had 15.8 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.15.6445Role of Cell Free EGFR mRNA Expression in Survival and Metastasis in Non-Small Cell Lung Cancer Cases month median survival time while >13 fold increased gene expression had only 6.7 month median survival time (Figure 1a).We further assessed the role of EGFR gene expression in survival of NSCLC patients with respect to their metastases status.Distant metastatic patients with <13fold increased gene expression had 7.9 months of median survival time while>13 fold increase gene expression had 5.0 months of median survival time (p=0.03)(Figure 1b).Non metastatic patients with<13 fold increased gene expression had 18 months of median survival time while patients with >13 fold increased gene expression had 7.1 months of median survival time (Figure 1c).

Discussion
In our present study we made an attempt to identify cell free EGFR mRNA expression in NSCLC patients.Amongst the lung cancers, non-small cell lung cancer (NSCLC) comprises 80% -85% of all cases and more than 70% patients are diagnosed in advanced stage (Siegel et al., 2013;Xu et al., 2014).Several experimental evidence suggests that the EGFR is involved in tumour  Salomon et al., 1995).Overexpression of EGFR has been reported and implicated in the pathogenesis of many human malignancies, including NSCLC (Inamura et al., 2010).Scagliotti et a. ( 2004) also have analysed that EGFR expression in NSCLC is associated with reduced survival (Scagliotti et al., 2004).In present study we also observed that higher EGFR gene expression has been found to be associated with poor survival of NSCLC patients.We observed a  (Bloomston et al., 2006) and EGFR expression has been correlated with local advanced and metastatic stage of disease (Tobita et al., 2003).In NSCLCs particularly adenocarcinomas, EGFR is overexpressed in 70% of the patients (Sekido et al., 2003).EGFR is overexpressed in the advanced NSCLC patients, and is associated with the poor survival and EGFR expression is clearly involved in the lung cancer pathogenesis (Dowell et al., 2005).
Travis et al in his study suggested that EGFR expression level may be used as prognostic and predictive marker in NSCLC (ADC) (Travis et al., 2013).EGFR was found to be a region for metastatic competency, and is thought to promote cancer cell migration and invasion (Masuda et al., 2012).Franklin WA and Hirsch FR et al. also observed that in lung carcinomas, EGFR is more commonly over expressed (Franklin et al., 2002;Hirsch et al., 2002).Its expression has been detected in a wide variety of human malignancies, including up to 50-78% of breast cancers (Ge et al., 2002).It has been revealed that high EGFR expression was found in many solid tumors such as, gastric cancer and breast cancer (Atmaca et al., 2012;Zhang et al., 2013).Lee et al. (2014) also suggested, EGFR overexpression may be the independent poor prognostic factor in primary breast carcinoma (Lee et al., 2014).
Here in this study we conclude cell free EGFR mRNA expression may be a predictive factor for patients' poor overall survival and metastatic behavior of NSCLC patients.Due to the small sample size in the present study our findings need to be validated by further independent and prospective studies on larger population.

Figure 1. Kaplan-Meier survival Curves with Respect to EGFR Gene Expression: (a) Overall Survival (b, c) Distant Metastases and no Metastases formation
and progression.Overexpression of EGFR has been correlated with prognosis and metastatic spreading in several carcinoma types, such as breast, head and neck, and lung malignancies (