Increased Serum S-TRAIL Level in Newly Diagnosed Stage-IV Lung Adenocarcinoma but not Squamous Cell Carcinoma is Correlated with Age and Smoking

Lung cancer is the leading cause of cancer mortality in the world accounting for 31% (for men) and 26% (for women) of all cancer deaths. Trends in lung cancer incidence and mortality reflect smoking habits and/ or exposure to other environmental or occupational carcinogens. The incidence rate in men is 34.9 per 100,000 with the highest rates observed in more developed countries, while in women the incidence rates are lower (11.1 per 100.000) (Greenlee et al., 2000; Parkin, 2001; 2004). Lung cancers can be grouped into two major histological types, namely non-small cell and small cell lung cancer (NSCLC and SCLC respectively), a reflection of their different clinical behaviours and sensitivity to chemoand radiotherapy. NSCLC accounts for 75-85% of lung cancer patients and consists of several


Introduction
Lung cancer is the leading cause of cancer mortality in the world accounting for 31% (for men) and 26% (for women) of all cancer deaths. Trends in lung cancer incidence and mortality reflect smoking habits and/ or exposure to other environmental or occupational carcinogens. The incidence rate in men is 34.9 per 100,000 with the highest rates observed in more developed countries, while in women the incidence rates are lower (11.1 per 100.000) (Greenlee et al., 2000;Parkin, 2001; RESEARCH ARTICLE
TNF-related apoptosis inducing ligand (TRAIL) is a TNF family member expressed as either a type II transmembrane protein or, similarly to other membrane-bound ligands of the TNF superfamily, as a soluble protein, which is detectable in the serum under physiological conditions. Although the best characterized biological activity of TRAIL, also known as Apo2 ligand, is associated with a potent induction of apoptosis in a variety of cancer cell types, the wide expression of TRAIL and TRAIL receptors in many normal tissues suggests that the physiological role of TRAIL is more complex than merely activating the apoptotic pathway in cancer cells (Sanlioglu et al., 2006;Terzioglu et al., 2007;Griffith and Lynch, 2008;Aydin et al., 2010). In general, TRAIL has been studied in tumour immunology settings. It selectively induces apoptosis in transformed cells while leaving non-transformed cells unaffected (Ashkenazi et al., 1999;Walczak et al., 1999). Although it has been shown that TRAIL can activate both pro-apoptotic and anti-apoptotic pathways, the factors regulating which one of these pathways are triggered are not well understood (McGrath, 2011).
Vitamin D is a steroid hormone that has been shown to possess anti-tumorigenic and immune-modulatory effects in vitro and in vivo. Its potential in cancer prevention and treatment is currently under detailed investigation. Evidence from animal model studies and in vitro cell culture suggest that vitamin D may play a beneficial role in pulmonary inflammation. Vitamin D status may be an important issue for lung cancer prevention (Beumer et al., 2012;Norton and O'Connell, 2012;Zhang et al., 2012).
With the prospect of more effective therapeutic options for advanced stage disease, the characterization of new methodologies for monitoring patients' pre-/post-therapy is of interest. In the present study, we investigated the concentrations of soluble TRAIL in the peripheral blood of adenocarcinoma and squamous cell carcinoma patients as well as in healthy controls, and performed a preliminary analysis of the relationship of those levels to disease outcome.

Patients
Subjects consisted of 18 patients with adenocarcinoma and 22 patients with squamous cell carcinoma. Clinical specimens were obtained from NSCLC patients seen at the Department of Oncology with a new diagnosis of stage IV disease. Clinical data for the patients was also recorded. 20 healthy age-and sex-matched healthy individuals served as a control group.
Written informed consent was obtained from all participants according to the Declaration of Helsinki. The study was approved by Antalya Education and Research Hospital Local Committee on Ethics.

ELISA analysis
T R A I L a n d 1 , 2 5 -d i h y d r o x y v i t a m i n D ( 3 ) concentrations in serum samples from 40 NSCLC patients and 21 healthy donors were analyzed using a TRAIL/APO2L ELISA kit (Diaclone, France) and Roche kit for 1,25-dihydroxyvitamin D(3) according to the manufacturer's instructions. The absorbance values in all assays were measured using a spectrophotometer set at 450 nm, and the concentrations of sTRAIL (pg/ml) and 1,25-dihydroxyvitamin D(3) (ng/ml) calculated from OD readings of recombinant standards.

Statistical analysis
Data were analyzed using GraphPad Software (Prism 5.0, San Diego, CA) and were expressed as mean±standard error of the mean (SEM). GraphPad Software was used to plot data for all three groups (control, adenoC and SCC).
Comparison of parameters between the three groups used a one-way ANOVA (Tukey's multiple comparison).
Correlations between the patient characteristics and TRAIL concentrations were analyzed by Spearman correlation analysis. Values of p<0.05 were considered to indicate statistical significance.

Results
The demographics of the NSCLC patients were as follows: Adenocarcinoma group: mean age, 60.5±13.3 years; squamous cell carcinoma group: mean age, 65±11.7 years. Forty five percent of all patients were previous or ongoing smokers, and all patients were classified as stage IV.
1,25-dihydroxyvitamin D(3) levels were 15.9±2.1 ng/ ml (adenocarcinoma group); 16.9±1.3 ng/ml (squamous cell carcinoma group); and 20.0±2.0 ng/ml (control group) ( Table 1). Figure 1 illustrates the serum sTRAIL distribution for NSCLC and control groups. Both adenocarcinoma and squamous cell carcinoma patients had significantly higher serum levels of sTRAIL than the healthy controls, with a significant difference between the two patient populations. Investigation of potential correlations between levels of circulating TRAIL and other parameters, such as age, smoking status, survival and 1,25-dihydroxyvitamin D(3) in NSCLC patients are presented in supplementary Tables 1-3. Although the serum TRAIL concentrations were not correlated with survival in NSCLC patients, there was a correlation with age (p=0.016) and cigarette smoking (p=0.022) seen for adenocarcinoma patients but not squamous cell carcinoma patients (Supp . table 1 and  supp. table 2). In addition, there was a correlation between the circulating sTRAIL and 1,25-dihydroxyvitamin D(3) levels seen in the control group (Table 2).

Discussion
Tumor marker measurements, which may potentially provide sensitive and cost-effective early detection of recurrence, are becoming increasingly important in assessing the efficacy of novel therapy. Our study demonstrates that circulating sTRAIL levels were significantly higher in untreated NSCLC patients than in healthy controls although there was no correlation between sTRAIL levels and patient survival. One advantage to our study is that patients were newly diagnosed stage-IV NSCLC and none had undergone any treatment, so that sTRAIL levels were not influenced by previous drug treatment. However, the small size of the study population may in itself have been a contributing factor to our failure to establish a link between sTRAIL levels and disease outcome.
Age is a major determinant of cancer risk with adenocarcinoma diagnosed at younger ages (Charloux et al., 1997;Greenlee et al., 2000). Interestingly, we observed that serum concentrations of sTRAIL increased with age in adenocarcinoma patients.
Smoking obeys a dose-response relationship with risk for all types of lung cancer, (Curran et al., 2011), and approximately half of the patients, particularly those with adenocarcinoma, in our study reported a history of smoking. The connection between smoking and lung cancer was most evident among patients with squamous and SCLC, and weaker for adenocarcinoma (Barbone et al., 1997;Brambilla et al., 2001;Radzwikowska et al., 2002). We found a significant correlation between sTRAIL levels and smoking status.
Many studies have demonstrated a relationship between vitamin D and cancer, including malignancies of the breast, colorectum, prostate and lung (Pazdiora et al., 2011;Woloszynska-Read et al., 2011;Zhang et al., 2012). Vitamin D or its analogs, alone or in combination with cytotoxic drugs, may have some efficacy in the treatment of lung cancer (Ramnath et al, 2011). Vitamin D is converted to its active form locally in the lung, consistent with the idea that it plays a role in lung health. We observed a correlation between sTRAIL and 1,25-dihydroxyvitamin D(3) levels in healthy individuals, although no such correlation was seen in NSLC patients.
In conclusion, this is the first study to assess sTRAIL levels in NSCLC patients and to evaluate the relationship of those levels with clinical outcome. Our study demonstrates that sTRAIL levels are higher in newly diagnosed stage-IV NSCLC than in healthy controls. Further studies are needed to investigate whether the TRAIL system has a causal role in clinical outcome in adenocarcinoma, and its value as a marker for monitoring patients with NSCLC.