Benzodiazepine drug use and cancer risk: a dose–response meta analysis of prospective cohort studies

Conflicting results identifying the relationship between benzodiazepine drug use and cancer risk. Therefore, we conducted a dose-response meta-analysis of prospective cohort studies to clarify and quantitative assessed the relationship between benzodiazepine drug use and cancer risk. Up to July 2017, 22 original publications were included in current meta-analysis. Our results showed statistically significant association between benzodiazepine drug use and cancer risk (RR:1.25; 95% CI, 1.15–1.36). Subgroup analysis showed benzodiazepine using was associated with significantly a higher risk of breast cancer (RR:1.15; 95% CI, 1.05–1.26), ovarian cancer (RR:1.17; 95% CI, 1.09–1.25), colon cancer (RR:1.07; 95% CI, 1.02–1.13), renal cancer (RR:1.31; 95% CI, 1.15–1.49), malignant melanoma (RR:1.10; 95% CI, 1.03–1.17), brain cancer (RR:2.06; 95% CI, 1.76–2.43), esophagus cancer (RR:1.55; 95% CI, 1.30–1.85), prostate cancer (RR:1.26; 95% CI, 1.16–1.37), liver cancer (RR:1.22; 95% CI, 1.13–1.31), stomach cancer (RR:1.17; 95% CI, 1.03–1.32), pancreatic cancer (RR:1.39; 95% CI, 1.17–1.64) and lung cancer (RR:1.20; 95% CI, 1.12–1.28). Furthermore, a significant dose-response relationship was observed between benzodiazepine drug use and cancer risk (likelihood ratio test, P < 0.001). Our results showed per 500 mg/year, per 5 year of time since first using, per 3 prescriptions and per 3 year of duration incremental increase in benzodiazepine drug use was associated with a 17%, 4%, 16% and 5% in cancer risk increment. Considering these promising results, increasing benzodiazepine using might be harmful for health.


INTRODUCTION
Cancer is the second most frequently leading cause that caused over 8.8 million deaths worldwide in 2015 [1], and the World Health Organization predicts 14.1 million people are expected to develop cancer annually [2]. Cancer represents a heavy social health problem and economic burden, and costs on patients. The etiology of cancer involves both genetic and environmental factors.
Multiple lines of evidence have demonstrated that benzodiazepine drug use is the risk factors for cancer regardless of laboratory studies or animal studies [3,4].
Benzodiazepines are the derivatives of 1, 4-benzodiazepines, including diazepam, flurazepam, chlorazepam, oxazepam and triazolam. The main function of benzodiazepine is inducing feelings of calm, drowsiness and sleep, and have been widely used in clinical, mainly to treat anxiety and insomnia. Benzodiazepines has been one of the most commonly used drugs in the general population for nearly 50 years, with the use of older people Meta-Analysis www.impactjournals.com/oncotarget worldwide ranging from 10 to 42 percent. In the United States, about 6 to 10 percent of adults use benzodiazepines in 2010, while in Europe, some regions have found a higher proportion [5,6]. Therefore, it is important to evaluate the benzodiazepines drugs safety.
Previous studies have examined the correlation between benzodiazepines drug use and cancer risk . However, the result remains controversial. Additionally, no study to quantitative assessed benzodiazepines drug use in relation to cancer risk. Thus, we performed this doseresponse meta-analysis to clarify and quantitative assessed the correlation between benzodiazepines drug use and cancer risk.

Study characteristics
The characteristics of the included studies of benzodiazepines drug use and cancer risk are shown in the Tables 1 and 2. Results in different subgroups were treated as two separate reports. Finally, twenty-seven independent reports from twenty two studies investigated the association between benzodiazepines drug use and cancer risk. Among the selected reports, twenty-two are from Caucasia and five from Asia. A total of 2482625 participants with 312203 incident cases from seven countries were included in this meta-analysis.
Benzodiazepine drug use and the risk of cancer by type of cancer Eleven independent reports from seven studies investigated the association between benzodiazepines drug use and breast cancer. Compared with no benzodiazepines drug use, benzodiazepines drug use is significantly associated with a higher risk of breast cancer (RR:1.15; 95% CI, 1.05-1.26; P < 0.001) (Table 4). Furthermore, benzodiazepines drug use is significantly associated with breast cancer risk in Caucasia (RR:1.17; 95% CI, 1.08-1.26; P < 0.001) ( Table 4) and Asia (RR:1.09; 95% CI, 1.03-1.16; P < 0.001) ( Table 4). Eight independent reports from six studies investigated the association between benzodiazepines drug use and ovarian cancer risk. Compared with no benzodiazepines drug use, benzodiazepines drug use is significantly associated with a higher risk of ovarian cancer (RR:1.17; 95% CI, 1.09-1.25; P < 0.001) (

Subgroup analyses
Subgroup analysis was performed to check the stability of the primary outcome. Subgroup meta-analyses in study design, study quality, number of participants and number of cases showed consistent findings (Tables 3, 4).

Dose-response between benzodiazepines drug use and cancer risk
Using restricted cubic spline function, the test for a nonlinear dose-response relationship was significant (likelihood ratio test, P < 0.001), suggesting curvature in the relationship, increasing per 500 mg/year of benzodiazepine drug use was associated with a 17% increment of cancer risk, the summary relative risk of cancer risk for an per 500 mg/year of benzodiazepine drug use was 1.17 (95% CI: 1.02-1.35, P = 0.022) ( Figure 2). In addition, increasing benzodiazepine drug use (per 3 prescriptions increment) was associated with a 16% increment in cancer risk, the summary relative risk of cancer risk for an per 3 prescriptions of benzodiazepine drug use was 1.16 (95% CI: 1.11-1.22, P < 0.001) ( Figure 2). Furthermore, increasing per 3 year of duration of benzodiazepine drug use was associated with a 5% increment of cancer risk, the summary relative risk of cancer risk for an per 3 year of duration of benzodiazepine drug use was 1.05 (95% CI: 1.02-1.09, P = 0.042) ( Figure 2). Additionally, increasing per 5 year of time since first benzodiazepine drug use was associated with a 4% increment of cancer risk, the summary relative risk of cancer risk for an per 5 year of time since first benzodiazepine drug use was 1.04 (95% CI: 1.01-1.07, P = 0.003) ( Figure 2).

Sensitivity analysis
Sensitivity analysis was conducted to assess the stability of the results. The results show the results were stable in Supplementary Figure 1.

Publication bias
Each studies in this meta-analysis were performed to evaluate the publication bias by both Begg's funnel plot and Egger's test. P > 0.05 was considered no publication bias. The results show no obvious evidence of publication bias was found in the associations between benzodiazepine drug use and cancer risk (Supplementary Table 1).

DISCUSSION
Benzodiazepines are widely used to treat seizures, anxiety, insomnia, and panic disorder. In recent years there has been constant evidence that benzodiazepines are associated with cancer risk in vitro laboratory and animal studies. Several animal studies reported that benzodiazepines increase the risk of thyroid cancer [29] or liver cancer [3] through inhibiting apoptosis and stimulating tumor cell proliferation. In the meantime, several observational epidemiological studies reported that there was no link between benzodiazepines drug use and the risk cancer. However, recent cohort studies revealed that the use of benzodiazepines drugs was associated with an increased risk of cancer, and the benzodiazepine users were exposed to the risk of benign brain tumor about three times higher than the non-benzodiazepine users. Collectively, these data suggest that benzodiazepines drug use may play an important role in cancer risk, but presented controversial results. Thus, we performed this meta-analysis, aiming to study the role of benzodiazepines in cancer risk and to explain the possible reasons for controversial results.
In the current meta-analysis was based on 22 casecontrol or cohort study, with 2482625 participants with 312203 incident cases from seven countries. Thus, this meta analysis provides the most up-to-date epidemiological evidence supporting benzodiazepines drug use is harmful for health. A dose-response analysis revealed that increasing per 500 mg/year of benzodiazepine drug use was associated with a 17% increment of cancer risk. In addition, increasing benzodiazepine drug use (per 3 prescriptions increment) was associated with a 16% increment in cancer risk. Furthermore, increasing per 3 year of duration of benzodiazepine drug use was associated with a 5% increment of cancer risk. Additionally, increasing per 5 year of time since first benzodiazepine drug use was associated with a 4% increment of cancer risk. Subgroup meta-analyses by various factors also showed consistent findings.
Several plausible pathways may reasonable for the relationship between benzodiazepine drug use and cancer risk. The influence of chronic inflammation on cancer development is one possible pathway. Benzodiazepine drug use might increase the levels of inflammation mediators [30], which can increase the risk of cancer   Adjusted for age, sex, HMO, years of enrollment in the HMO, race at HFHS, and adjusted for use of drug classes other than the studied one.

Friedman et al. (1998) Colon cancer (1960)
Population-based Duration of regular use 0 using, 1.0 (reference); less than 1years using, 0.9 (0.7, 1.3); 1to 5 years using, 1.2 (0. 8 Self-administered Duration of regular use 0 using, 1.0 (reference); less than 5 years using, 0.9(0.4, 1.7); more than 5 years using, 1.0 (0.4, 2.6) Age, geographical region, years of education, religion, parity, age at first pregnancy, menopausal status, age at menopause, history of breast cancer in the mother or sisters, alcohol consumption, number of visits to a doctor in the preceding year, total number of hospital admissions, and year of interview All cancers (3520) Self-administered Duration of regular use 0 using, 1.0 (reference); less than 1years using, 1.03 (1.02, 1.05); 1to 3 years using, 1.05 (1.02, 1.08); 3to 7 years using, 1.07 (1.03, 1.12); more than 7 years using, 1.11 (1.01, 1.23) Age, gender, use of aspirin, non-aspirinNSAIDs, 5-areductase inhibitors, statins, angiotensin-II antagonists, oral contraceptives and hormone supplements, antidepressants, antipsychotics, diagnoses of inflammatory bowel disease, COPD, diabetes, alcohol abuse and Charlson Comorbidity Index score  by inhibiting apoptosis and stimulating tumor cell proliferation [31]. Simultaneously, the treatment of benzodiazepine drug use before surgery can influence the depolarization of the mitochondrial membrane to inhibit the apoptosis of neutrophil cells, neutrophil apoptosis plays an important role in maintaining the homeostasis of the immune system and prevents damage to the host organs by promoting an immune response [32]. Addition, benzodiazepines are used to enhance the neurotransmitter of γ-aminobutyric acid by interacting with the chlorine ion channel that binds to GABA receptors. Besides as an inhibitory neurotransmitter effect, γ-aminobutyric acid is also thought can regulate cell proliferation and differentiation of brain and peripheral at various stages, and may participate in benign tumor growth in a variety of ways [33,34]. However, the potential mechanisms of benzodiazepines and tumor growth still remain unclear and controversial.
To our knowledge, this is the first study to identify and quantify the potential dose-response association between benzodiazepine drug use and cancer risk in a large cohort of both men and women. Although, we performed this meta-analysis very carefully, however, some limitations must be considered in the current metaanalysis. First, different sex of population should be included in this meta-analysis to explore the impact of different sex of population on benzodiazepine drug use and cancer risk. Second, we only select literature that written by English, which may have resulted in a language or cultural bias, other language should be chosen in the further. Third, in the subgroup analysis in cancer type, there has no insufficient statistical power to check a doseresponse in different cancer type, large data in different cancer type is warranted to validate this association.
In conclusion, our findings underscore the notion that benzodiazepine drug use was significantly associated with cancer risk increment. In the future, large-scale and population based association studies must be performed in the future to validate the risk identified in the current meta-analysis.

MATERIALS AND METHODS
This meta-analysis was conducted according to the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) checklist [35].

Search strategy
We included eligible studies to investigate the relationship between benzodiazepines drug use and cancer risk. To develop a flexible, non-linear, r meta-regression model, we required that an eligible study should have categorized into 3 or more levels.
PubMed, EMBASE and Web of Science databases were searched for studies that contained risk estimates for the outcomes of cancer and were published update to July 2017, with keywords including "benzodiazepine" [

Study selection
Two independent researchers investigate information the correlation between benzodiazepines drug use and cancer risk: outcome was cancer. Moreover, we precluded non-human studies, reviews, meta-analyses, editorials and published letters. To ensure the correct identification of qualified research, the two researchers read the reports independently.

Data extraction
Use standardized data collection tables to extract data. Each eligible article information was extracted by two independent researchers. We extracted the following information: first author; publication year; age; country; sex; cases and participants; the categories of benzodiazepines drug use; the relative risk or odds ratio (OR). We collect the risk estimates with multivariableadjusted. According to the Newcastle-Ottawa scale, quality assessment was performed for non-randomized studies [36].

Statistical analysis
We pooled relative risk estimates to measure the association between benzodiazepines drug use and cancer; the hazard ratio were considered equivalent to the relative risk [37]. Results in different subgroups of benzodiazepines drug use and cancer risk were treated as two separate reports.
Due to different definitions cut-off points in the included studies for categories, we performed a relative risk estimates by increaseper 500mg/year of benzodiazepine using or per 3 prescriptions increment or per 3 year of duration of benzodiazepine using or per 5 year of time since first benzodiazepine use using the method recommended by Greenland, Longnecker and Orsini and colleagues. In addition, using restricted cubic splines to evaluate the non-linear association between benzodiazepines drug use and cancer risk, with three knots at the 10th, 50th, and 90th percentiles of the distribution. A flexible meta-regression based on restricted cubic spline (RCS) function was used to fit the potential non-linear trend, and generalized least-square method was used to estimate the parameters. This procedure treats benzodiazepines drug use (continuous data) as an independent variable and logRR of diseases as a dependent variable, with both tails of the curve restricted to linear. A P value is calculated for linear or non-linear by testing the null hypothesis that the coefficient of the second spline is equal to zero [38].
We use STATA software 12.0 (STATA Corp, College Station, TX, USA) to evaluate the relationships between benzodiazepines drug use and cancer risk. By using Q test and I 2 statistic to assess heterogeneity among studies. Random-effect model was chosen if P Q < 0.10 or I 2 > 50%, otherwise, fixed-effect mode was applied. Sensitivity analysis was conducted to assess the stability of the results. Begg's and Egger's tests were to assess the publication bias of each study. P < 0.05 was considered signifcant for all tests.

CONFLICTS OF INTEREST
Authors have disclosed no conflicts of interest.

FUNDING
This study received no specific external funding.