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Association between serum leptin levels and breast cancer risk

Abstract Background: Many studies have indicated that leptin is correlated with breast cancer occurrence and tumor behavior. However, this issue remains controversial. Therefore, we conducted an updated meta-analysis to investigate the role of leptin in breast cancer. Methods: We performed a systematic literature search and identified relevant papers up to 1 September 2017. Standardized mean differences (SMDs) with 95% confidence intervals (CIs) were used to evaluate effect sizes. Results: Thirty-five eligible studies were included in the current meta-analysis. Serum leptin levels were related to breast cancer risk as demonstrated by calculations of the overall SMD = 0.46 (95% CI = 0.31-0.60, I2 = 93.5%). A subgroup analysis of BMI identified an association between breast cancer and serum leptin levels in patients who are overweight and obese (overweight: SMD = 0.35, 95% CI = 0.13–0.57, I2 = 88.1%; obesity: SMD = 1.38, 95% CI = 0.64–2.12, I2 = 89.6%). Additionally, menopausal status subgroup analysis revealed a significant association in postmenopausal women (SMD = 0.26, 95% CI = 0.12–0.40, I2 = 77.9%). Furthermore, we identified a significant association between breast cancer and serum leptin levels in Chinese women (SMD = 0.61, 95% CI = 0.44–0.79, I2 = 40.6%). Conclusion: The results of this meta-analysis suggested that leptin could be a potential biomarker for breast cancer risk in women, especially overweight/obese or postmenopausal women. Therefore, it may be useful for identifying subjects with a high risk for breast cancer who may benefit from preventive treatments.


Introduction
Breast cancer is common cancer and the second leading cause of cancer-related death in women worldwide. [1] Multiple factors, including genetic, environmental, and hormonal factors, are related to breast cancer. Obesity is a well-recognized risk factor for breast cancer. [2,3] Data in the literature shows that in obesity adipocytes induces chronic inflammation, which increases the local and systemic levels of cytokines such as tumor necrosis factor a (TNF-a) and interleukin 6 (IL-6). [4] The adipokines secretion profile alters the condition of obesity, modulates the immune response, and in particular affects the production and activation of neutrophil, such as leptin, which regulates the total neutrophil count and has a granulocytestimulating effect. [5] Leptin, a 16-kDa protein, is a product of the obese (Ob) gene and is secreted into the bloodstream, where it circulates in both bound and free forms. [6] The potential pro-tumoral effects of leptin are facilitated by its mitogen actions. Leptin has been shown to exert neoplastic effects in breast cancer by acting directly on tumor growth, migration and invasion signaling pathways or by decreasing tissue sensitivity to insulin or regulating inflammatory responses and tumor angiogenesis. [7,8] Leptin is a pleiotropic molecule that influences energy balance, appetite control, angiogenesis, reproduction, the immune response, and bone development. Leptin also affects the proliferation of different cell types, including the cells of the breast. [9,10] The results of several studies strongly support the hypothesis that leptin activity is correlated with breast cancer occurrence and tumor behavior. A few studies noted no associations between these parameters, a finding attributed to small patient samples and the presence of concomitant pathologies. [11][12][13][14] To better clarify this issue, Niu et al [15] conducted a meta-analysis and confirmed that leptin is positively associated with breast cancer. They suggested that serum leptin levels vary by population and increase from low to high in the following order: healthy people < patients with benign breast diseases < patients with breast cancer < patients with positive lymph node metastasis.
However, we noted that the results of recent studies were controversial. Rodrigo et al [16] did not find any relationships between serum leptin levels and sporadic breast cancer in postmenopausal women. Moreover, Georgia et al [17] found that leptin levels in healthy women are slightly higher than those in premenopausal women with breast cancer. Besides, the association between leptin and cancer risk was weak in the study by Touvier et al. [18] Moreover, no updated meta-analysis and systematic reviews have been performed recently. Therefore, we conducted this meta-analysis to verify whether serum leptin plays a vital role in breast cancer pathogenesis.

Search strategy
The present meta-analysis followed the Meta-analysis of Observational Studies in Epidemiology (MOOSE) guidelines. [19] All analyses were based on previously published studies. year, or publication status restrictions were imposed. We also performed manual searches of the references cited in the retrieved articles and earlier reviews on the topic.

Inclusion and exclusion criteria
Case-control and cohort studies published as original articles, and reports containing relative risk estimates or raw data about serum leptin levels were eligible for inclusion in the study. We assessed the relevance of the corresponding abstracts and retrieved full copies of the manuscripts to determine whether they met our primary inclusion criteria. We ultimately selected only those studies: [1] reported sufficient information for estimating standardized mean differences (SMDs) or odds ratios (ORs) with 95% confidence intervals (CIs) [2] ; were independent and did not duplicate previously published results [3] ; included patients who were newly diagnosed and had histopathologically confirmed breast cancer and had not been treated via surgery, chemotherapy, or radiotherapy [4] ; and indicated that all blood specimens were collected early in the morning after a period of fasting and before the initiation of any therapeutic approach. In cases in which multiple publications pertained to the same study population, the study with the largest number of case subjects was used. Also, data from articles with more detailed reporting were used for sub-analyses. Breast cancer diagnoses were classified using the International Classification of Diseases for Oncology (Third Edition codes C50.5-C50.9).
The following studies were excluded from the analysis [1] : studies reporting recurrence risk estimates of breast cancer among patients [2] ; studies reporting mortality risk estimates of breast cancer [3] ; and studies with patients who had a prior malignancy or coexisting severe medical conditions. Review articles not reporting original data were also excluded from the study, although they were checked for useful references.

Data extraction
Two researchers independently extracted detailed information and data using a pre-designed data extraction form and assessed the quality of the individual studies. In the event of disagreement, a third researcher would assess the relevant articles further. Data regarding the following parameters were extracted from each study: the first author's name, the year of publication, countries, ethnicities, sample sizes, age of the participants, cancer types, cancer stages, body mass indices, serum leptin levels (means and standard differences), treatment status, test methods, leptin levels among controls, and sources of controls (hospitals or populations). Moreover, data pertaining to serum leptin levels among women of different menopausal statuses were also retrieved.

Quality assessment
The Newcastle-Ottawa scale was modified to capture information pertaining to the following relevant quality characteristics of the included reports: sampling representativeness, sample sizes, exposure definitions, famine severity assessments, confounding adjustments, outcome assessments and statistical methods. The total score ranged from 0 to 9 (studies with scores of 0-3, 4-6, and 7-9 were considered low-, moderate-, and high-quality studies, respectively).

Statistical analysis
The command metan in Stata 14.0 (StataCorp LLC, College Station, TX) was used to combine the results of the included studies to perform the meta-analysis. SMDs and 95% CIs were calculated for each study, based on the sample size and the mean (and standard deviation [SD]) serum leptin levels in the case and control groups. We used the I 2 statistic to investigate whether heterogeneity was present among the studies. [20] The pooled effect size (SMD) was evaluated using a random-effects model if the heterogeneity was considered statistically significant (I 2 >50% and P < .10); otherwise, a fixed-effects model was used. We calculated the heterogeneity with the x 2 based Q-test and the I 2 statistics test.
To identify the sources of heterogeneity, we using metaregression and sensitivity analysis to evaluate between-study heterogeneity by assessing the influence of different study features, such as quality, sample size, year of publication, test method, BMI, and menopausal status. [21] To examine the impacts of effect modifiers, we conducted subgroup analyses based on information from the primary studies. Publication bias was assessed by Begg and Egger tests (a= 0.05). [22,23] 3. Results

Search results
We identified 1482 records and screened 109 full texts for relevance based on the titles and abstracts. Thirty-five studies met our inclusion criteria. The excluded studies and the reasons for their exclusion are presented in detail in Figure 1. A total of 781 studies remained after exclusion of duplicated studies, and 452 articles were removed after the title and abstract screening because they were not relevant to our study aims. Fifty-nine items were excluded after full-text testing because they did not pertain to the relationship between breast cancer (n = 30) and serum leptin levels (n = 29). Five articles were excluded because they did not have sufficient information regarding baseline patient characteristics. Eight articles were excluded because they were meta-analyses. Two studies were excluded because they were 2 of multiple publications from the same study population, and the study with the largest number of case subjects was used. One study was composed of various ethnicities, which we stratified into different groups for subgroup analysis of ethnicity. All articles were in English and were published between 2000 and February 2017. Tables 1 and 2 present the descriptive characteristics of the eligible studies. Among the studies included in the analysis, 13 were performed in Asia. Five of those thirteen were conducted in China (2 in Taiwan and 3 in inland China; 2 articles pertained to the same study population, and the study with the smaller number of case subjects was used for supplemental information). [6,[24][25][26][27] Three studies were conducted in Turkey, [28][29][30] 2 in the Kingdom of Saudi Arabia, [11,31] 1 in Korea, [32] 1 in Sri Lanka, [16] and 1 in Iran. [33] Twenty-one studies involved Caucasian patients. Seven of these 21 studies involved patients living in the United States, [14,[34][35][36][37][38][39] 5 in Greece, [17,[40][41][42][43] 2 in Mexico, [44,45] 2 in France, [18,46] 1 in Portugal, [8] 1 in Germany, [47] 1 in Norway, [48] 1 in Australia, [49] and 1 in Italy. [50] Two studies were conducted among Africans, 1 study was conducted in Egypt, [13] and 1 United States study included persons of African descent. [39] Among the 35 studies, 2 included only premenopausal women, [36,38] and 4 contained only postmenopausal women. [34,35,40,41]

Pooling of studies and subgroup analysis
All 35 eligible studies were included in the meta-analysis, and the results of the overall and subgroup analyses of the studies providing data on the relationship between serum leptin levels and breast cancer are presented in Table 3. Serum leptin levels were related to breast cancer risk as demonstrated by calculations of the overall SMD = 0.46 (95% CI = 0.31-0.60). However, significant and non-ignorable heterogeneity (I 2 = 93.5%) was present. Therefore, we performed subgroup analyses of specific variables to determine if they were sources of the heterogeneity.
We conducted subgroup analyses of several factors that may modify the association between serum leptin levels and breast cancer, including country, ethnicity, BMI, test methods, quality, leptin levels among controls, sources of controls, and menopausal status. First, we performed a subgroup analysis in which the studies were organized into groups according to their countries.  www.md-journal.com Table 1 Characteristics of studies included in the meta-analysis.  Interestingly, analysis of the SMD of BMI showed that when the BMI was below 25 (SMD = 0.34, 95% CI = -0.03 to 0.71, I 2 = 86.5%), there was no difference in the leptin-breast cancer relationship between the case and control groups; however, we noted significant differences in the relationship between the case and control groups when the BMI was between 25 and 30 (SMD = 0.35, 95% CI = 0.13-0.57, I 2 = 88.1%) and when the BMI exceeded 30 (SMD = 1.38, 95% CI = 0.64-2.12, I 2 = 89.6%) (Fig. 3). Finally, we assessed menopausal status and found no significant association among premenopausal women (SMD = 0.18, 95% CI = -0.01 to 0.37, I 2 = 77.9%); however, a significant association was found between serum leptin levels and breast cancer in postmenopausal women (SMD = 0.26, 95% CI = 0.12-0.40, I 2 = 75.8%) (Fig. 4). Table 2 The levels of serum leptin in each primary study.  [17] 2010  [14] 1990

Sensitivity and meta-regression analysis
We conducted sensitivity analysis by sequentially excluding studies from the meta-analysis to further investigate the possible sources of the heterogeneity among the studies. The sensitivity analysis results suggested that our meta-analysis was stable (Fig. 5). We also performed multivariate meta-regression to evaluate the influence of several factors that may modify the association between serum leptin levels and breast cancer, including quality, sample size, year of publication, test method, BMI and menopausal status (the adjusted P values are .982, .230, 1.000, .700, .844, and .978, respectively). The results showed that these confounding factors did not substantially affect the heterogeneity. No publication bias was found by either Begg (P = .163) (Fig. 6) or Egger test (P = .093).

Discussion
The results of this meta-analysis were consistent with those of previous studies indicating that leptin plays a potential role in breast cancer progression. However, we noted non-ignorable heterogeneity among studies included in the meta-analysis (I 2 = 93.5%). We used a random-effects model to account for the heterogeneity; however, the model also increased the probability of a type-I error. To investigate the causes of the heterogeneity, we identified several characteristics, such as ethnicity, country, BMI, quality, measurement method, control source, breast cancer stage, cancer type, and menopausal status, as factors that may have added variability to the results.
To identify the source of the heterogeneity, we first conducted a multivariate meta-regression and sensitivity analysis by sequentially excluding each study. We did not observe any differences in heterogeneity among all the analyzed confounding factors, including ethnicity, quality, sample size, publication year, measurement method, BMI, and menopausal status. The other potential factors were not included in the meta-regression because we did not have sufficient information about them. The results of the sensitivity analysis indicated that the reliability of the meta-analysis was good. We found that none of the confounding factors introduced significant variability; however, we performed subgroup analysis to investigate the non-ignorable heterogeneity further. We used a random-effects model to account for the between-study variability.
Increasing amounts of evidence in the literature support the idea that an association exists between breast cancer risk and leptin.
In a previous meta-analysis, Niu et al [15] presented the combined-effect (d) values for serum leptin levels in different population groups. They showed that leptin levels vary among different groups and increase from low to high in the following Table 3 The pooled results of the serum leptin levels in breast cancer patients compared with control groups.  [51] and Dalamaga et al [52] ) that may have involved the same study population. We used the data from the study with the larger number of subjects and included the data from the survey with the smaller number of items as supplemental information when necessary. Moreover, our analysis included only patients who were newly diagnosed and had histopathologically confirmed breast cancer that had not been treated via surgery, chemotherapy, or radiotherapy. Thus, prior treatments were unlikely to have affected our results. Therefore, we may be able to improve the statistical power of the analysis. Some studies have suggested that BMI, ethnicity, and family history are closely related to breast cancer. [6,14,39,45,49] Besides, Crisostomo et al [8] showed that leptin is positively correlated with Figure 2. Subgroup analysis of the association between serum leptin levels and breast cancer in different countries. Zero is not included in this confidence interval, which indicates that the relationship between leptin levels and breast cancer is different between the case and control groups. The positive SMD value suggests that the mean circulating leptin level in the case group is higher than that in the control group in Asian and Caucasians, while the negative SMD value indicates that the mean leptin level in the case group is lower than that in the control group in Africans. Pan et al. Medicine (2018) 97:27 www.md-journal.com BMI in obese patients with breast cancer. In our analysis, we first evaluated the correlation between serum leptin levels and breast cancer risk and, found that serum leptin levels are significantly positively correlated with breast cancer risk. In our subgroup analysis in which BMI stratified patients, we found that serum leptin levels were not associated with breast cancer in healthy normal-weight individuals or normal-weight individuals with breast cancer. Interestingly, we noted a strong association between breast cancer risk and higher leptin levels in patients who are overweight or obese, a finding consistent with those of previous studies by Romero et al [45] and Cust et al. [49] Many authors have evaluated whether the association between leptin and breast cancer varies according to menopausal status. Interestingly, the relationship between breast cancer risk and leptin levels has been shown to be menopause status specific in several studies. [33,35,39,41,47] However, leptin levels have been shown to be inversely related to menopausal status in other studies. [8,11,13,[16][17][18]24,31,34,40,44] Based on the results of our metaanalysis, we agree that leptin levels are associated with breast cancer among postmenopausal women but not among premenopausal women.
We also performed several subgroup analyses to assess whether several factors can modify the association between leptin levels and breast cancer risk. In the report in which the included studies were organized into groups by country, serum leptin levels were only weakly associated with breast cancer in Chinese people and were, not significantly associated with breast cancer in individuals from the United States, Mexico, Greece, or Turkey, possibly because the number of the studies evaluating patients from these countries were small. The analysis of the effect of ethnicity showed that serum leptin levels were significantly associated with breast cancer risk in Asians and Caucasians, this association was not identified in Africans because only a small number of studies evaluated African patients. The analysis of leptin levels among controls revealed that serum leptin levels were significantly associated with breast cancer in all groups. In the analysis of sources of controls, we found that the SMDs in hospital groups were consistent with the overall SMD; however, Figure 3. Subgroup analysis of the association between serum leptin levels and breast cancer in different BMI groups. Zero is not included in this confidence interval, which indicates a difference between the case and control groups. The negative SMD value suggests that the mean of the case groups is are lower than that of the control groups for the BMI below 25 group, while the positive SMD value suggests that the mean of serum leptin level in the case groups is higher than that in the control groups in both the BMI between 25 to 30 and the BMI exceeding 30 groups. the SMDs in population groups showed that serum leptin levels were not significantly associated with breast cancer, possibly because the studies evaluating patients from population groups were a few (2 articles). Moreover, the test method analysis showed that ELISA and RIA significantly affected the relationship between serum leptin levels and breast cancer. Finally, the subgroup analysis in which the included studies were stratified according to their quality showed no differences in the leptinbreast relationship between the studies with scores ≥7 and those with scores <7. The results indicate that this factor did not affect the association between breast cancer risk and leptin levels. There were several limitations to our meta-analysis. First, we were unable to perform additional subgroup analyses to investigate the effects of other factors, such as cancer stage, specific cancer classifications, and age of menarche, because the included studies lacked sufficient information regarding these factors. Second, we included only studies published in English in the analysis so that other biases may have affected the results of the present study. Third, the inclusion rates of hospital and population groups in the studies were 94.29% (33 articles) and 5.71% (2 items), respectively. The number of studies including population groups was small, so the power of this subgroup analysis was weak, and further research is required. Fourth, the participation rate of controls is an essential factor for assessing selecting bias. However, we found out only 1 study [40] had reported the number of the control group who agreed to take part in the concluded studies compared to the numbers who were asked to take part. Only 4 [26,28,40,50] of 35 studies stated that the controls were sources from volunteers, and 31 remained reported the controls were randomly selected from routine physical examination and were strictly matched for age. We were constrained by the lack of more detailed information to go Figure 4. Subgroup analysis of the association between serum leptin levels and breast cancer in different menopausal status groups. Zero is not included in this confidence interval, which indicates a difference between the case and control groups. A negative SMD value suggests that the mean of the case groups is lower than that of the control groups in the premenstrual group, while the positive SMD value suggests that the mean serum leptin level in the case groups is higher than that in the control groups in the postmenstrual group. further analyzation, and extensive sample size studies are needed to be conducted for eliminating the confounding factor of participation rates of controls. Despite these limitations, we minimized the possibility that a bias would affect our results by creating a detailed protocol and by carefully selecting and analyzing the data.
In summary, the current meta-analysis suggests that leptin may have a potential role as a biomarker for breast cancer risk, especially in overweight/obese and postmenopausal women. Thus, it may be helpful in identifying subjects at high risk for breast cancer who may benefit from preventive treatments.
However, some information, namely, information regarding specific cancer types and stages, was insufficient in the primary studies. In the future, the corresponding researchers will conduct additional prospective or longitudinal studies focusing on the above information to corroborate the association between leptin levels and breast cancer risk.