Background
Lung cancer is a worldwide health problem, with more than 1.8 million new cases and almost 1.6 million deaths estimated in 2012 [
1,
2]. Inadequate early diagnosis is one of the major reasons for the fast-growing incidence of lung cancer in recent years, which is especially common in developing countries. Thus, exploiting new diagnostic methods is essential for extending the survival of patients with lung cancer.
Tumor biomarkers, which highly express in tumors tissues, are major indicators in auxiliary diagnosis for tumor. So far, several tumor biomarkers have been applied to the clinical diagnosis, such as AFP in hepatocellular carcinoma, CEA, and CA19-9 in colorectal carcinoma and CA125 in ovarian carcinoma [
3‐
8]. The biomarkers closely correlating with lung cancer mainly include NSE, CEA, CA19-9, CYFRA21, SCCA and PROGRP, which the specificity and susceptibility account for 20–62% [
9‐
11]. Since tumor biomarkers in the blood can be quickly and easily obtained in a noninvasive manner, the development of potential blood-based markers will be helpful for early diagnosis of lung cancer, monitoring of disease status, development of targeted therapies, evaluation of response to therapy and survival.
Follistatin (FST), a single chain glycoprotein, is originally isolated from the follicular fluid of ovary, which can suppress follicle-stimulating hormone (FSH) secretion from anterior pituitary cells and participate in various physiological and pathological processes [
12‐
15]. FST widely exists in gonads and extragonadal tissues, peripheral blood and cell culture supernatant [
16‐
19]. Serum FST levels were correlated not only with pregnancy but also with various solid tumors, including gonadal cancer, gastric cancer, hepatocellular carcinoma, basal cell carcinoma, and melanoma [
20‐
23]. The recent studies have reported that FST was aberrantly expressed in human lung adenocarcinoma cells, suggesting that FST might be a potential biomarker for diagnosis of lung adenocarcinoma [
22,
24]. However, it remains unclear whether serum FST expression is associated with lung cancer patients with different histological types, TNM staging, tumor progression, and recurrence.
In this study, we firstly investigated the association of serum FST levels with patients in two broad histological subtypes of lung cancers: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), and NSCLC was subdivided into lung adenocarcinoma (LADC), squamous cell carcinoma (SCC), lung adenosquamous cell carcinoma (LASC) and large cell lung cancer (LCLC). Next, we assessed FST expression in LADC according to TNM staging and category. Finally, we analyzed the relationship between serum FST expression and recurrence in LADC patients.
Materials and methods
Patients and healthy subjects
The subjects were chosen from both the patients with lung cancer (LC) and the patients with benign lung diseases (BLD) admitted into Tianjin Medical University Cancer Institute & Hospital between October 2014 and December 2016. All diseases were verified by pathological and cytological diagnoses. Tumor node metastasis (TNM) staging were based on the Criteria of Lung Cancer Staging from the 8th edition of the Union for International Cancer Control (UICC) and American Joint Committee on Cancer (AJCC) Cancer Staging Manual [
25‐
27]. The healthy subjects (HSs) were selected randomly from the Physical Examination Center of Tianjin Medical University Cancer Institute & Hospital. We collected the clinical data of all the subjects, including name, age, serology, imaging (ultrasound, CT, MRI, etc.), pathology, etc. The patients with benign lung disease excluded malignant tumors, and the health subject’s imaging excluded lung disease. All subjects excluded autoimmune diseases, cardiovascular diseases, severe liver and kidney diseases, blood diseases, infectious diseases, and other malignant tumors.
Serum sample processing
Peripheral blood was collected from each patient under an empty belly in the morning of the second day after hospitalization according to the previously described methods [
21]. The serum was obtained by centrifuging at 1500
g for 10 min at 4 °C then stored at − 80 °C 200 μL/tube separately. The control serum samples were similarly collected from the healthy subjects in the morning on the day of their routine examination.
ELISA for serum FST
Serum FST levels in patients with LADC, SCC, LASC, LCLC and SCLC were measured by using ELISA kits (R&D Systems, Minneapolis, USA) according to the manufacturer’s instructions. Absorbance at 450 nm was measured and the serum FST levels were calculated based on the standard curve.
Statistical methods
Data were expressed as the mean ± standard deviation and the differences among groups were compared via ANOVA. A value of P < 0.05 was considered statistically significant. The diagnostic performance of FST was evaluated by nonparametric receiver operating characteristic (ROC) curves, sensitivity, specificity, the area under the ROC curve (AUC), and with 95% confidence intervals (CI). The cut-off value of FST was calculated by Youden’s index, the peak point of ‘sensitivity + specificity − 1’, according to all points of a ROC curve, and served as a standard for choosing the most suitable cut-off value. Data analyses were performed by SPSS 16.0 for Windows (SPSS Inc, Chicago, IL, USA).
Discussion
Lung cancer is the most frequent cancer diagnosed and the leading cause of mortality in the world. Reductions in lung cancer mortality can be attained through treatment, especially if the disease is diagnosed at a stage where curative therapy is possible. Thus, it is urgent for finding new molecular biomarkers for early diagnosis of lung cancer, monitoring of disease status, development of targeted therapies, evaluation of response to therapy and survival [
3].
FST is a monomeric, cysteine-rich polypeptide which suppresses pituitary FSH release in a similar manner to inhibin [
13,
29]. Subsequent discovers indicate that this molecule can also play a variety of roles in several reproductive and nonreproductive systems as potent tissue regulators in the gonad, pituitary gland, pregnancy membranes, vasculature, and liver [
30]. Recent studies suggest that FST, as a stress responsive protein, plays a protective role under a variety of stresses [
31]. In addition, inactivation of hepatic FST may contribute to improve glucose tolerance and alleviate hyperglycemia [
32,
33].
Accumulating evidence indicates that FST has been implicated in the development and progression of solid tumours [
23]. Overexpression of FST was found in several human tumors, including gastric cancer [
34], ovarian cancer [
35], prostate cancer [
36], basal cell carcinoma [
37] and hepatocellular carcinoma [
38]. Several recent studies revealed the closed relationship between FST and breast cancer, one of these studies by Zabkiewicz et al. showed that FST overexpression appears to promote breast cancer in vitro proliferation and reduce invasiveness [
39‐
41]. Furthermore, FST plays also a role in angiogenesis and metastasis of solid tumours. The effect of FST on tumour angiogenesis seems to be complex, some observations in both lung- and liver-derived tumours are strongly suggestive of FST inhibiting tumour angiogenesis [
42], but other evidence shows FST may have a promotory effect on tumour angiogenesis [
43,
44]. Additionally, some studies support the role of FST in controlling tumor metastasis [
40,
42,
45,
46]. Very recently, Seachrist et al. [
47] found FST is a metastasis suppressor in a mouse model of HER2-positive breast cancer.
FST has shown strong promise as a diagnostic or prognostic marker for solid tumours. Some studies reported that serum FST levels were significantly increased in patients with ovarian cancer [
21], hepatocellular cancer [
48], and breast cancer [
39,
49]. In the previous study, we have reported serum FST overexpression in lung adenocarcinoma [
22]. However, the prognostic value of FST in the serum of lung patients with different types, TNM staging, and recurrent lung cancer remains poorly investigated.
In this study, we firstly examined serum FST levels in lung cancer patients with different histological types. We found that serum FST levels in patient group with lung cancer were significantly higher than those in the healthy control group and lung benign disease group. ROC analysis revealed that when compared LC with HSs and BLD, the serum FST levels provided a diagnosis efficacy with AUC of 0.971 and 0.728 respectively, indicating that serum FST seemed likely to have a potential role in lung cancer diagnosis. Since LADC ranks first in the incidence of lung cancers with all histological types and the studies on individualized LADC treatments are gradually intensified, we further observed the correlation of serum FST levels with LADC according to the TNM staging. Our data showed that serum FST levels had a much higher expression in patients with stage III and IV LADC. Simultaneously, our results also showed that serum FST levels increased significantly in patients with different T category of LADC, but there was no significant difference among the T categories. Moreover, serum FST levels were also elevated in patients with LADC according to N and M categories. Notably, we found that serum FST was elevated in the diagnosed patients with recurrent as compared to the healthy subjects. Taken together, the above results indicated that serum FST levels modestly reflected the disease progression and metastasis of lung cancers.
To date, the potential mechanism for an increase of serum FST levels in human carcinogenesis is not clear, a possible mechanism of FST overexpression may represent a unique strategy of tumors to overcome the inhibitory action of activin by decreasing its local bioavailability [
50].
Although our data has shown a close relationship between FST expression and lung cancer with different histologic types, TNM staging and disease recurrence after surgery, thus suggesting the potential of FST as a biomarker for lung cancer diagnosis, we are aware that the sample size in this cohort is rather small, which limits the power of multivariate analyses, therefore, further validation by larger scale prospective trials is needed. Another limitation of our study is the use of one testing methodology, i.e., serum FST levels measurement by ELISA, it needs to be further corroborated by optimal tissue-based analysis of FST expression in lung cancer tissues. Furthermore, although rigorous screening in this experiment has been performed, future study needs to take into consideration of the possibility that the patients were previously treated, because local inflammatory response to therapy could also contribute to an increases of serum FST levels.
Authors’ contributions
PZ, YR, and FC collected the data; PZ and JX performed the statistical analysis; PZ and XZ conceived and designed this study. PZ, JX, and XZ wrote the manuscript. All authors read and approved the final manuscript.