Serum pleiotrophin as a diagnostic and prognostic marker for small cell lung cancer

Abstract Pleiotrophin (PTN) is involved in tumour progression, angiogenesis and metastasis. The purpose of this study was to investigate the expression level of PTN in the serum of patients with small cell lung cancer (SCLC) and to explore the clinical significance of PTN. Serum samples from 128 patients with SCLC, 120 healthy volunteers (HV) and 60 patients with benign lung disease (BLD) were collected. The levels of serum PTN were determined with ELISA and its correlation with the clinical data was examined. The serum PTN levels in SCLC patients were significantly higher than that in BLD patients (P < 0.05) or HV (P < 0.05). With a cutoff value of 258.18 ng/mL, the sensitivity and specificity of PTN to SCLC patients and BLD patients, SCLC patients and HV were 79.2% and 91.7%, 86.7% and 95.8% respectively. An area under the curve for all stages of SCLC resulting from PTN, which was significantly better than the other tumour markers tested including progastrin‐releasing peptide and neuron‐specific enolase. High serum PTN levels appear to correlate with poor survival in patients with SCLC. These results suggest that PTN levels in the serum could be a new effective biomarker for the diagnosis and prognosis of SCLC.

progression, angiogenesis and metastasis. [14][15][16] However, there is limited clinical research on the over expression of PTN in lung cancer.
Jager et al detected the expression of PTN in 14 non-small cell lung cancer (NSCLC) cell lines and 12 SCLC cell lines by RT-PCR. The results showed that PTN mRNA was expressed in nine SCLC and three NSCLC cells. 17 In a subsequent small sample study, 87% of SCLC patients and 63% of NSCLC patients had elevated serum levels of PTN, suggesting the potential role of serum PTN levels in the diagnosis of lung cancer. 18 Recent studies suggest that serum PTN may be a useful marker for the diagnosis and prognosis of lung cancer. 19 Pleiotrophin is overexpressed in SCLC tissues and the levels of PTN expression are associated with the diagnosis and prognosis. 20 However, the potential role of serum PTN levels in early diagnosis and prognosis of lung cancer, especially SCLC, a type of lung cancer, still requires further investigation.
In this study, we first examined serum PTN levels in patients with SCLC with an ELISA kit. Additionally, we investigate the correlation of PTN levels and adverse clinic features of SCLC such as tumor stage. Our results show that an elevated level of serum PTN serves as both a poor clinical state and a valuable diagnostic and prognostic biomarker for SCLC.

| Patients
We consecutively enrolled 128 patients with SCLC, 60 patients with benign lung disease (BLD), and 120 healthy volunteers (HV). Serum samples from all subjects were collected at Nanjing chest hospital.
Patients with SCLC were included if they met the following criteria: confirmation of SCLC via a review of pathologic slides by two independent observers to classify the histologic subtype; no pro-surgical or pro-diagnostic history of antineoplastic therapy, radiotherapy, or chemotherapy. Before treatment, the patients were divided into limited stage or extensive SCLC. Limited disease is defined as a single radiation field that is limited to the ipsilateral chest, and widespread disease is defined as a disease other than the ipsilateral half thorax, including malignant pleural or pericardial effusion or blood transfer.
Contralateral mediastinum and ipsilateral supraclavicular lymphatic diseases are divided into limited stages. Sublateral supravalinal lymphnode disease and supraclavicular lymphnode disease are often classified as extensive diseases. 21 All the patients in this study received platinumbased chemotherapy combined with errisecan or etoposide. The characteristics of the SCLC patients are presented in Table 1.  Table 1.
All participants provided written informed consent, and the Nanjing Chest Hospital Ethics Committee approved the research project.

| Serum collection
Blood samples from 128 patients with SCLC were obtained from the patients after diagnosis but prior to any treatment. After two cycle of chemotherapy, serum samples were obtained again. The whole blood samples were promptly centrifuged at 1500 g for 15 minutes and the supernatant stored at −80°C until use.

| Determination of PTN, ProGRP and NSE levels
The serum concentration of PTN was determined by using a commercial, two-site, sandwich-type ELISA (eBioscience, Santiago, USA) using streptavidin-coated microplate wells, a biotinylated-Fab monoclonal antibody, and an alkaline phosphatase-labeled polyclonal detection antibody. Serum NSE levels were measured by commercial ELSA-NSE kits (CIS Bio International, Gif-Sur Yvette, France); serum ProGRP was determined by ELISA Kit (ALSI, IBL-Hamburg, Germany). The intra-assay coefficient of variation (CV) was <5%, and the interassay CV was <6%. The normal upper limit of tumor markers is ProGRP 46 pg/mL and NSE 13 ng/mL. All tests were done in two copies and diluted properly, and technicians ignored the clinical data.

| Statistical analysis
Statistical software (SPSS for Windows, version 18) was used for data analysis. All values are given as mean ± SD was used. The Mann-Whitney U test was used to compare between serum sample groups, and the Kruskal-Wallis test was used to compare several groups. Chi

| Serum PTN, ProGRP and NSE levels in SCLC patients
As shown in Figure 1A, serum PTN levels in SCLC group were significantly higher than those in BLD group or HV group (P < 0.05).
Serum PTN levels in HV group were similar to those in BLD group (220.26 ± 41.59 ng/mL vs 239.39 ± 46.44 ng/mL, P > 0.05). The levels of serum ProGRP and NSE in patients with HV, BLD and SCLC were also shown in Figure 1B,C. Compared with HV and BLD, serum ProGRP and NSE levels in SCLC patients also increased significantly (P < 0.05).

| Diagnostic value of serum PTN
The sensitivity of the index in distinguishing SCLC patients, HV and BLD patients was calculated. As shown in Figure 2A  and yielded a better optimal diagnostic efficacy for SCLC patients (Table 2) than did DDH2 alone (P < 0.01).

| Relationship between PTN levels and clinicopathological characteristics
A cutoff point of 258.18 ng/mL, the patients were divided into high PTN and low PTN. The relationship between serum PTN level and clinicopathological characteristics in patients with lung cancer was nanlyzed. As shown in Table 3, PTN levels are associated with stage of disease (P < 0.05). However, there was no significant correlation between PTN level and age, sex, performance status and smoking status (P > 0.05).

| Association of serum PTN levels with OS of SCLC patients
Next, we examined the association between serum PTN levels with OS of patients with SCLC by performing the Kaplan-Meier survival analysis. We found that patients with higher levels of serum PTN level had significantly worse OS rates (P = 0.025, Figure 4).The follow-up time of these 120 patients was from 6 to 30 months, with a medium time of 12 months.  and invasion. 22 We have described for the first time the expression of serum Low group represents the levels of serum PTN < 258.18 ng/mL. *Statistically significant difference (P < 0.05) F I G U R E 4 Kaplan-Meier survival analysis of patients with small cell lung cancer (SCLC) based on serum pleiotrophin (PTN) levels. The overall survival rate of SCLC patients with high serum PTN level was significantly lower than patients with low serum PTN level (P = 0.025; log-rank test)

| CONCLUSIONS
In conclusion, we have confirmed the increase of serum PTN level in patients with SCLC in this study. High PTN levels appeared to correlate with poor survival in patients with SCLC. These results suggested that serum PTN could be a new effective biomarker for the diagnosis and prognosis of SCLC. Further studies are needed to clarify the precise role of PTN in tumor growth, and more large-scale prospective studies are needed to confirm this finding.

ACKNOWLEDGMENTS
The study was supported by the Major Program of Nanjing Medical Science and Technique Development Foundation (ZKX16064).

CONF LICT OF I NTEREST
The authors declare no any conflicts of interest in this work.