Serum and tissue leptin in lung cancer: A meta-analysis

Many studies have found that leptin is involved in tumorigenesis and the progression of lung cancer. However, these studies were inconsistent. Therefore, we performed a meta-analysis to investigate the role of leptin in the patients with lung cancer. A systematic literature search in the several databases and on commercial Internet search engines was carried out to identify studies published up to July 8, 2016. The standardized mean difference (SMD) and odds ratio (OR) with 95% confidence interval (CI) were used to investigate the effect sizes. Finally, 21 eligible articles were included in the current meta-analysis. Overall, there is no relationship between levels of serum leptin and lung cancer. However, a subgroup analysis in high-study quality group found a weak association between serum leptin concentrations and lung cancer in Chinese (SMD=0.77, P=0.035). Additionally, the meta-analysis indicates that the serum leptin levels were lower in the weight-losing group than in the sustained weight group (SMD=-0.80, P=0.001). Further, there was evidence of a significant association between expression levels of leptin protein in tissue and lung cancer (OR=7.35, P<0.001). The present meta-analysis suggests that the serum and tissue leptin may be involved in the pathogenesis of lung cancer and tumor metastasis, especially among Chinese. However, the leptin may not appear to play an important role in cancer cachexia development.


INTRODUCTION
Lung cancer is the most lethal malignancy. The most recent cancer statistics report estimated that lung cancer accounts for approximately 26% of all female cancer deaths and 29% of all male cancer deaths [1]. Despite several significant advances in cancer treatment, the 5-year relative survival for lung cancer is still less than 18% [1]. The main reasons contributing to the poor prognosis are the large population with advanced-stage lung cancer at diagnosis and the unsuccessful treatment in metastatic disease. Thus, there is intense interest in identifying novel biomarkers involved in this aggressive disease, for early diagnosis and to optimize its medical management. Previous studies have indicated many biomarkers considered as prognosticators, as well as indicators of screening and potential therapeutic targets for lung cancer, such as carcinoembryonic antigen, cytokeratin 19 fragment (CYFRA21-1), circulating cell free DNA, C-reactive protein, interleukin-6 (IL-6), and repetitive A (A type 3) [2][3][4][5][6].
Leptin, the product of the ob gene, is a cytokine synthesized and secreted mainly by adipocytes [7]. It inhibits food intake and regulates energy metabolism by inducing anorexigenic factors and suppressing orexigenic neuropeptides [8]. The published studies have suggested that food consumption is associated with a transient increase in serum leptin levels, whereas fasting reduces ob gene expression [7]. However, serum levels of leptin can be disturbed by other factors as well, such as various pro-inflammatory cytokines, including TNF-a, IL-1, and IL-6 [9]. Currently, leptin www.impactjournals.com/oncotarget/ Oncotarget, 2017, Vol. 8, (No. 12), pp: [19699][19700][19701][19702][19703][19704][19705][19706][19707][19708][19709][19710][19711] Research Paper is also recognized as a polyfunctional cytokine, and it is considered to be involved in the pathogenesis of several cancer types, including breast cancer and pancreatic cancer [10][11][12]. Additionally, a growing body of studies has found that leptin is also involved in tumorigenesis and the progression of lung cancer. Among them, some studies have found that elevated serum leptin levels represent an independent risk factor for non-small-cell lung cancer (NSCLC) [13][14][15], whereas other studies have reported significant correlation between decreased serum leptin levels and prognosis in lung cancer [16,17]. Further, a few studies have indicated that the expression of leptin protein was significantly higher in lung cancer tissues than in normal lung tissues [18,19].
However, the observed relationships of these studies were inconsistent, and a single study may lack sufficient power to detect the possible small effect of the leptin levels on lung cancer, especially when the sample size is relatively small. Therefore, we carried out a most recent meta-analysis to accurately investigate the association between leptin and the risk of lung cancer, and to further assess the role of leptin in lung cancer.

Serum leptin
Overall meta-analysis All 14 eligible case-control studies (773 cases and 594 controls) were included in the meta-analysis to investigate the association between serum leptin concentrations and lung cancer. As showed in Table 4, the overall meta-analysis results of the random-effect model suggested that no relationship exists between levels of serum leptin and lung cancer (SMD=-0.09, 95%CI=-0.70-0.53, P=0.780) ( Figure 2). However, a non-ignorable heterogeneity among studies was found (I 2 =96.2%). Therefore, we further conducted subgroup analyses of different specific effects that were expected to lead to heterogeneity.

Subgroup meta-analysis
At first, we carried out a subgroup analysis by language (English or Chinese), but we found no statistical significance between serum leptin concentrations and lung cancer regardless of whether the studies were published in English or in Chinese In the subgroup analysis of ethnicity, we found no association between serum leptin concentrations and lung cancer in Chinese people (SMD=0.50, 95%CI=-0.17-1.17, P=0.144) and in Europeans (SMD=-0.70, 95%CI=-1.79-0.39, P=0.208) ( Figure 2). Interestingly, a weak statistical significance was found in the Chinese people of high-study quality group (NOS score≥7, 532cases and 443 controls) (Chinese: SMD=0.77, 95%CI=0.05-1.48,     Table 4.

Sensitivity and meta-regression analysis
To further investigate the possible source of heterogeneity, we executed a sensitivity analysis by sequentially excluding studies from the meta-analysis to investigate the influence of each study on the pooled results. The result of sensitivity analysis found that the pooled ORs were not materially altered, suggesting the stability of our meta-analysis ( Figure 4). Moreover, we conducted a multivariate meta-regression analysis to assess the possible confounding factors. The results of multivariate meta-regression analysis showed that the publish year, ethnicity, cancer type, and study quality as confounding factors did not substantially affect heterogeneity (adjusted P value is 0.079, 0.816, 0412 and 0.366, respectively). Further, no publication biases were found in the Begg's (P=0.101) and the Egger's tests (P=0.185) ( Figure 5).

Serum leptin in weight-losing group
Only two case-control studies (81 cases and 67 controls) compared the difference of serum leptin concentration in the weight-losing group with that in the non-weight-losing group. The results of the meta-analysis indicated that the serum leptin levels were lower in the weight-losing group than in the non-weight-losing group (SMD=-0.80, 95%CI=-1.26-(-0.34), P=0.001) ( Figure 6).

Tissue leptin
In total, seven studies reported the role of leptin expression in lung cancer tissue, and all of these articles were conducted in China. For the meta-analysis, the fixedeffect model was used because there is no heterogeneity. The results indicated evidence for a significant association between expression levels of leptin protein in tissue and lung cancer (OR=7.35, 95%CI=5.21-10.39, P<0.001) (Figure 7). Interestingly, further analysis found that tissue leptin protein levels were statistically different between the lymph node metastases group and the non-lymph node metastases group (OR=3.83, 95%CI=2.18-6.72, P<0.001) (Figure 8). No publication bias was found in either the  include zero indicates no statistical difference between the case group and control group. Negative SMD value suggests the mean of serum leptin concentration of case group is lower than that of control group, while positive SMD value suggest the mean of case group was higher than control group.

Figure 3: The subgroup analysis results of association between the serum leptin levels and lung cancer in high-study quality group (NOS score≥7).
Failure of this confidence interval to include zero indicates no statistical difference between the case group and control group. Negative SMD value suggests the mean of serum leptin concentration of patients in high-study quality group is lower than that of control group, while positive SMD value suggest the mean of case group was higher than control group.    Begg's (P=1.0) or the Egger's test results (P=0.804) (Figure 9).

DISCUSSION
Lung cancer is the most common cancer and the leading cause of cancer death in China [38,39] (and worldwide [1]). It is well known that invasiveness and immortalization are important characteristics of cancer tissues, and postoperative recurrence and metastasis are the main reasons for ineffective treatment and death in patients with cancers. Therefore, the researchers are searching for specific biomarkers for early diagnosis of cancer or to distinguish the high risk of disease recurrence to achieve a better survival. Many studies have concluded that leptin may play a important role in the pathogenesis of lung cancer [13,16,30], but these results are inconsistent. Therefore, we undertook a meta-analysis to determine the value of serum and tissue leptin in lung cancer.
In the current meta-analysis, there were 14 primary studies comparing the blood leptin concentrations in lung cancer patients and healthy controls. The overall results indicated that the serum leptin levels did not increase in the patients with lung cancer, as compared with the controls. However, we found a non-ignorable heterogeneity (I 2 =96.2%) between studies in the meta-analysis. Although the heterogeneity can be taken into account by using the random-effect model, it would increase the probability of type-I error. The following factors may have contributed to the significant heterogeneity: (1) the demographic characteristics and genetic backgrounds are different in European and Chinese populations; (2) the studied patients with lung cancer had different stages of cancer in each study; (3) different lung cancer types were included in the studies; (4) there were different ratios of patients with weight loss in each primary study; (5) different measuring methods were used in the primary studies; (6) the included studies were of differing quality; and (7) the included patients had different treatment statuses.
To identify the causes of heterogeneity, at first, we carried out a sensitivity analysis by sequentially excluding each study. Fortunately, the statistically similar results were obtained, suggesting the stability of the meta-analysis. Second, we also conducted a multivariate meta-regression analysis to further assess the possible confounding factors. However, the results suggested that the publish year, ethnicity, cancer type, and study quality, as the confounding factors, did not substantially affect the heterogeneity. Therefore, we finally conducted subgroup analyses of different specific effects.
The serum leptin levels still did not differ between the patients with lung cancer and the healthy participants metastases group and the non-lymph node metastases group. Negative OR value suggests the mean of leptin expression of lymph node metastases tissue is lower than that of non-lymph node metastases tissue, while positive OR value suggest the mean of lymph node metastases tissue was higher than non-lymph node metastases tissue. in the subgroups analyses (lung cancer types, published language, and treatment status). However, the subgroup analysis of ethnicity in the high-study quality group (NOS score≥7) indicated that the Chinese patients with lung cancer had higher serum leptin levels than the healthy population. Interestingly, our meta-analysis results were consistent with previous studies reporting that patients with hepatocellular carcinoma or prostate cancer have higher leptin levels than the normal population does [40,41].
In addition, high expression levels of tumorassociated leptin receptor are thought to promote tumor growth and progression [42]. Therefore, we also carried out a meta-analysis to investigate the role of tissue leptin protein expression in lung cancer. The results indicated that the positive expression of leptin protein is obviously higher in lung cancer tissue than in normal tissue, especially significantly higher in the lymph node metastases group. Surprisingly, an earlier study also suggested that the increased detection of ob-R in ovarian cancers was associated with decreased survival [43]. In animal experiments, deficiency of the leptin receptor resulted in failure of mammary tumor formation [44]. The integrin-dependent migration of chondrosarcoma cells by involvement of IRS-1/PI3K-dependent activation of Akt was canceled with the knockdown of the long form of the leptin receptor [45]. Thus, even though the mechanism remains completely unclear, we speculate that leptin may play an important role in the pathogenesis of lung cancer and tumor metastasis, and the increased serum leptin level may possibly predict the diagnosis and progression of lung cancer. Further, the results of previous studies and the current study may help us to identify a new molecular marker for lung cancer diagnosis and a target for treatment.
Moreover, cancer anorexia-cachexia syndrome causes health deterioration and is a negative predictive factor of treatment response [33]. In addition, a previous study has shown that leptin is a protein hormone that functions as the afferent signal in a negative feedback loop regulating body mass [46]. Therefore, we analyzed the role of serum leptin in lung cancer patients with weight loss. Interestingly, the results suggested that serum leptin levels were lower in the weight-losing group than in the patients without weight loss and in the normal participants. Similar results were found in Weryńska's [33] and Jamieson's [25] studies. These results and our meta-analysis results indicate that changes of serum leptin levels should be considered to be the result of cachexia and not the cause of it, because its concentration depends on the total body fat mass. However, because only a small number of included studies refer to these results, the evidence is relatively weak.
There were several limitations of this meta-analysis. First, even if no publication bias was observed using several tests, published studies were identified in only a Each circle represents a separate study for the indicated association. These circles in the funnel plot implied no asymmetrical distribution, which means no publication biases were found. few databases. Therefore, there may be other biases in the present study. Second, because sufficient data in primary studies are lacking, we failed to perform further subgroup analyses to investigate the other factors, such as gender, age, and cancer stage, which may have affected our results. Third, the subjects of this meta-analysis mainly came from China, so the results are possibly only applicable to the Chinese. Despite these limitations, we minimized the likelihood of bias through the whole process by creating a detailed protocol and by performing study identification, data selection, and statistical analysis, and we controlled for publication bias.
In summary, the current meta-analysis suggests that the serum and tissue leptin may play an important role in the pathogenesis of lung cancer and tumor metastasis, especially among Chinese. However, leptin may not be involved in cancer cachexia development. We recommend that researchers design more rigorous and uniform casecontrol or cohort studies to confirm the results in the future.

Literature search
We performed a systematic literature search in the PubMed, Embase, Wanfang databases, and CNKI, to identify studies involving the role of leptin in lung cancer up to July 8, 2016. The key search terms were as follows: lung cancer, non-small-cell lung cancer, smallcell lung cancer, NSCLC, SCLC, and leptin. The metaanalysis was limited to studies published in English or Chinese. Additionally, we also conducted a web-based search using many commercial Internet search engines (such as Google and Baidu), using the same keywords. Further, the reference lists of the obtained articles were also reviewed.

Study selection
The inclusive criteria were as follows: (1) a study involving the role of serum leptin levels in lung cancer designed as a case-control study; (2) a study involving expression levels of leptin in tissue comparing lung cancer tissues with the normal tissues; (3) a primary study providing available data for calculating standardized mean difference (SMD) or odds ratio (OR) with 95% confidence interval (CI); and (4) the subjects of the study are human. The exclusion criteria were as follows: (1) lack of control cohort; (2) review and overlapping study; and (3) the study does not show the available data and is missing other essential information.

Study quality score evaluation
The qualities of included studies involving the role of serum leptin levels in lung cancer were assessed by using the Newcastle-Ottawa Scale (NOS) for case-control study, to investigate quality based on three aspects: selection, comparability, and exposure in the primary study. The total score ranged from 0 to 9 (0-3, 4-6, and 7-9 was considered low, moderate, and high quality, respectively).

Data extraction
Two independent authors (Xiang Tong and Yao Ma) collected the detail information and data from each study and used a predesigned data extraction Excel form. If there was any disagreement, the third author (Qilong Zhou) would further assess these articles. The information and data were extracted as follows: first author, publication year, country, ethnicity, sample size, age of participant, lung cancer type, stage of cancer, rate of weight loss, serum leptin levels (mean and standard deviation), leptin expression levels in tissue, treatment status, and test method.

Statistical methods
In the present study, the SMD with 95% CI was applied to compare the serum levels of leptin in the patients with lung cancer with the levels in healthy controls, whereas the OR and 95% CI were used to investigate the leptin expression levels in lung cancer tissue and in normal tissue. We calculated the heterogeneity by the χ 2 based Q-test and I-squared (I 2 ) statistics test. The pooled effect size (SMD and OR) would be assessed using a randomeffect model if heterogeneity was considered statistically significant (I 2 > 50% and P < 0.10); if not, the fixed-effect model was used. To determinate the ethnicity, lung cancer type, and study quality-specific effects, we also performed subgroup analyses of different specific effects.
In addition, publication bias was assessed using several methods [47,48]. The Begg's and Egger's tests were used to assess publication bias. The visual inspection of asymmetry in funnel plots was carried out to further investigate the publication bias. All data analyses were conducted using the STATA 12.0 software.