A Case-Control Study on the Relationship Between Cholecystectomy and Cholangiocarcinoma in China


 Aims: The present study aimed to explore the correlation between cholecystectomy and cholangiocarcinoma, and to provide preliminary clinical basis for precise cholecystectomy in China.Methods: We conducted a retrospective analysis of 9744 patients with cholangiocarcinoma, colon cancer, pancreatic cancer, femoral fracture, and hepatic hemangioma diagnosed in Xijing hospital from August 2008 to August 2018. They were divided into three groups: case group (1749 cases of cholangiocarcinoma), positive control group (3137 cases of colon cancer and 1950 cases of pancreatic cancer), negative control group (1794 cases of femoral fracture and 1114 cases of hepatic hemangioma). We collected the general information (gender, age), past medical history, cholecystectomy history from the patients, and these data were analyzed by chi-square test and logistic regression analysis. Results: The cholecystectomy rate of the case group was significantly higher than that of the positive control group and the negative control group by chi-square test (p<0.025). The cholecystectomy rate and the history of cholecystolithiasis were analyzed by logistic multivariate regression analysis. The OR values of cholecystectomy rate were 1.553 (95%CI: 1.311-1.840) and 3.181 (95%CI: 2.561-3.951), respectively, and the difference was statistically significant (p<0.000). The OR values of the history of cholecystolithiasis were 2.460 (95%CI: 2.093-2.890) and 5.426 (95%CI: 4.325-6.809), respectively, and the difference was statistically significant (p<0.000). In case group, the difference between cholecystectomy and cholecystolithiasis was statistically significant (p<0.000) by chi-square test. Conclusions: In conclusion, cholecystectomy is one of the risk factors of cholangiocarcinoma and the patients who undergo cholecystectomy have a higher risk of cholangiocarcinoma than the control groups. Cholecystectomy should be conducted with caution and the precise surgical treatment of gallbladder diseases is advocated.


Introduction
With the improvement of living conditions and the change of dietary habit of Chinese people, the incidence of cholecystolithiasis in China has reached from 2-7% to 8-10%, which leads to the increasing number of the cholecystectomy year by year. With the development of minimally invasive technology, endoscopic cholecystectomy has become one of the most effective therapies for cholangiocarcinoma [1][2][3]. And endoscopic cholecystectomy has become one of the most frequent hepatobiliary surgery and general surgery [4][5][6][7]. Cholecystectomy can remove gallstones, gallbladder polyps and other lesions, greatly reduce the incidence of gallbladder carcinoma, and can eliminate the pain caused by gallbladder stones or biliary pancreatitis [8][9][10]. Because of the large amount of cholecystectomy, the complications of it can't be ignored. The major short term complication is biliary tract injury, which seriously affects the patient's quality of life, and can cause a secondary operation. When it comes to the irreparable biliary stenosis, it could even threaten the patient's life [11,12]. Long term complications of cholecystectomy are easy to be ignored, which mainly included dyspepsia and the increased risk of gastrointestinal cancer [13,14]. It is known that the incidence of gastrointestinal cancer after cholecystectomy increased and the most common malignance is colon cancer [15,16]. And it has also been reported that the incidence of pancreatic cancer increased after cholecystectomy [17,18].
Cholangiocarcinoma is the second most common malignancy of the liver, originating from biliary epithelial cells [19,20]. In recent years, according to the clinical and basic research of cholangiocarcinoma, viral hepatitis plays a crucial role in the development of cholangiocarcinoma.
Hepatitis C was thought to be a risk factor of cholangiocarcinoma in USA and Europe [21][22][23]; studies showed that hepatitis B was a risk factor of cholangiocarcinoma in Asia [24,25]. Owing to the unconspicuous symptoms at the early stage, cholangiocarcinoma is usually diagnosed at advanced disease stages, leading to poor prognosis [26,27]. At present, the diagnosis of this disease mainly depends on imaging technology, endoscopy, the examination of tumor markers and pathological biopsy.
And surgery is the most effective therapy for cholangiocarcinoma [28,29].
In this study, for the rst time, we conducted a case-control study in China to explore the correlation between cholecystectomy and the incidence of cholangiocarcinoma, so as to provide suggestion for the precise surgical treatment of cholecystolithiasis.

Study population
The Ethics Committee of Xi Jing Hospital approved this study. We collected patients with cholangiocarcinoma, colon cancer, pancreatic cancer, femoral fracture and hepatic hemangioma who were admitted to Xijing hospital from August 2008 to August 2018 through the Case data collection system of digital center from Xijing hospital. We collected patients' name, gender, age, admission time, discharge time, impatient department, operative name and the rst diagnosis after discharge, and then screened them. Patients with cancer and hepatic hemangioma were diagnosed pathologically, and patients with femoral fracture were diagnosed by X-ray, and the cases with ambiguous diagnosis were excluded. After the screening, 1749 cases of cholangiocarcinoma, 3137 cases of colon cancer, 1950 cases of pancreatic cancer, 1794 cases of femoral fracture and 1114 cases of hepatic hemangioma were included, a total of 9744 cases. We collected the general information of all patients (gender, age), past medical history (hypertension, diabetes, coronary disease, cholecystolithiasis) and the history of cholecystectomy, in which cholecystectomy was due to cholecystolithiasis (excluding the patients with cholecystectomy due to gallbladder polyps, gallbladder cancer and other gallbladder-related diseases).

Inclusion And Exclusion Criteria
Patients that met the following criteria could be included: (1) patients with cancer and hepatic hemangioma were diagnosed pathologically, and patients with femoral fracture were diagnosed by X-ray.
(2) the interval between the cholecystectomy and the diagnosis of related diseases after admission was more than 1 year. (3) the general information required in this study, the past medical history, the previous surgical history were complete, and the diagnosis was clear.
The exclusion criteria were based on the following: (1) patients with other malignancies at the same time or the related malignance was metastases of tumors derived from other sites. (2) the interval between the cholecystectomy and the diagnosis of related diseases after admission was less than 1 year (3) cases with cholecystectomy due to surgical treatment for other malignances. (Such as patients with pancreatic cancer underwent pancreaticoduodenectomy, and patients with liver cancer underwent partial hepatectomy) (4) cases of space-occupying lesions were not diagnosed as related malignant tumor. (5) cases of the general information required in this study, the past medical history, the previous surgical history were incomplete or the diagnosis was unclear.

Study Design
According to the above inclusion and exclusion criteria, 9744 cases were included in this study (1749 cases of cholangiocarcinoma, 3137 cases of colon cancer, 1950 cases of pancreatic cancer, 1794 cases of femoral fracture and 1114 cases of hepatic hemangioma). We collected the general information of all patients (gender, age), past medical history (hypertension, diabetes, coronary disease, cholecystolithiasis) and the history of cholecystectomy (operative time and postoperative pathological diagnosis). All the cases were divided into three groups: case group (1749 cases of cholangiocarcinoma), positive control group (3137 cases of colon cancer and 1950 cases of pancreatic cancer), and negative control group (1794 cases of femoral fracture and 1114 cases of hepatic hemangioma). Chi-square test and logistic regression analysis were used to study the factors, including gender, age, past medical history (hypertension, diabetes, coronary disease, cholecystolithiasis) and the history of cholecystectomy, that might affect the pathogenesis of the above diseases. To further determine whether cholecystectomy is one of the risk factors of cholangiocarcinoma and when is the high-risk period of cholangiocarcinoma after cholecystectomy, we explored the differences of the interval between cholecystectomy and the rst diagnosis of cholangiocarcinoma after cholecystectomy, and the differences between the cholecystolithiasis and the cholecystectomy in the case group.
Statistical analysis SPSS 22.0 statistical software was used for data analysis. The enumeration data was described by number and percentage. The normality test of the measurement data was performed by Shapiro-Wilk method. The measurement data conforming to the normal distribution was described by mean ± standard deviation, while the measurement data deviating from the normal distribution was described by means of median and quartile range. The chi-square test was used to compare the rate of cholecystectomy between groups. Logistic regression analysis was used to investigate the risk factors of cholangiocarcinoma and two-tailed P < 0.05 was considered statistically signi cant in this study.

Descriptive analysis
The normal test of the general information in three groups showed that age does not t the normal distribution (supplementary le1). So we used median and quartile range to describe the concentration and dispersion trend of age. The median of age of the case group was 63, 25 percentiles and 75 percentiles were 55 and 72, respectively. And the corresponding values of negative control group and positive control group were shown in Table 1. Then we used chi square test to compare the factors (gender, age, and past medical history) among the three groups. As shown in Table 2, among the three groups, the proportion of patients with hypertension in case group, negative control group and positive control group were 16.8%, 18.3% and 18.4%, respectively, and the chi square value was 2.391, p value was 0.303, the difference was not statistically signi cant. In addition, the difference of coronary disease among the three groups was not statistically signi cant. These evidence indicated that there was no difference in the history of hypertension and coronary disease among the three groups. However, there were signi cant differences in age, gender and diabetes history among the three groups.  Chi-square Test Of The Rate Of Cholecystectomy As shown in Table 3, the number of patients with cholecystectomy in the case group was 239, accounting for 13.7%; the number in the negative control group was 164, accounting for 5.6%; the number in the positive control group was 515, accounting for 10.1%. The chi square value was 88.586 and p value was 0.000, the difference of the rate of cholecystectomy among the three groups was statistically signi cant. The data indicated that effect of cholecystectomy on cholangiocarcinoma is different from that on the other diseases (colon cancer and pancreatic cancer, femoral fracture and hepatic hemangioma). Furthermore, the rate of cholecystectomy was compared in the case group and the positive control group, and compared in the case group and the negative control group separately, and the test level was adjusted with the partitions of X2 method. Comparing the rate of cholecystectomy between the case group and the positive control group, the chi square value was 16.631 and p value was 0.000, which means the difference was statistically signi cant (Table 4). Similarly, the difference of the rate of cholecystectomy between the case group and the positive control group was statistically signi cant too (Table 5).

Logistic Univariate Regression Analysis
Then we analyzed a series of confounding factors such as gender, age, past medical history (hypertension, diabetes, coronary disease, cholecystolithiasis) and cholecystectomy by logistic univariate regression analysis, from which we screened out the differential factors among the groups for the subsequent logistic multivariate regression analysis. As shown in Table 6, we can conclude that except for coronary disease there were signi cant differences in gender, age, cholecystectomy, cholecystolithiasis, hypertension and diabetes among the groups. These evidence indicated that all these factors besides coronary disease may affect the incidence of cholangiocarcinoma. So we included all the factors except coronary disease into the logistic multivariate regression analysis to observe which were the risk factors of cholangiocarcinoma actually.  Logistic multivariate regression analysis between the case group and the negative control group.
Subsequently, we compared the six factors (gender, age, cholecystectomy, cholecystolithiasis, hypertension and diabetes) between the case group and the negative control group by logistic multivariate regression analysis. As shown in Table 8, we nd that the OR value of gender was 0.484 (95%CI = 0.427-0.549) and p value was 0.000, the difference was statistically signi cant. Which means compared with the incidence of cholangiocarcinoma, women were more likely to suffer from hepatic hemangioma or femoral fracture. Although the difference in age was statistically signi cant (0.000), however the OR value was 1.037 (95%CI = 1.032-1.042), basically showing no signi cant difference.
Since the median age in the case group and the negative control group were 63 and 56, respectively, indicating that the risk of cholangiocarcinoma, hepatic hemangioma and femoral fracture increased with age. Patients with hypertension were more likely to suffer from hepatic hemangioma or femoral fracture than cholangiocarcinoma. The OR value of hypertension was 0.688 (95%CI = 0.580-0.817) and p value was 0.000. Then we compared the diabetes between the case group and the negative control group. The OR value of diabetes was 0.659 (95%CI = 0.527-0.824) and p value was 0.000. The data showed that patients with diabetes have a higher risk of hepatic hemangioma or femoral fracture than those with cholangiocarcinoma. Refer to the difference of cholecystectomy, the risk of cholangiocarcinoma in patients with cholecystectomy was 3.181 times higher than that of hepatic hemangioma or femoral fracture (OR = 3.181, 95%CI = 2.561-3.951), and the difference was statistically signi cant. And the risk of cholangiocarcinoma was higher in patients with cholecystolithiasis, which was 5.426 times higher than that of hepatic hemangioma or femoral fracture (OR = 5.426, 95%CI = 4.325-6.809). Based on logistic multivariate regression analysis, we found that patients with cholecystolithiasis were more likely to suffer from cholangiocarcinoma than those with cholecystectomy. Therefore, we compared cholecystectomy and cholecystolithiasis by Chi-square test in the case group to determine if there was any difference between them. As shown in Table 9, the number of patients with cholecystolithiasis in the case group was 304, accounting for 17.4%; the number of patients with cholecystectomy in the case group was 239, accounting for 13.7%. The chi square value was 45.063 and p value was 0.000, the difference between cholecystectomy and cholecystolithiasis was statistically signi cant. These data showed patients with cholecystolithiasis has a higher risk of cholangiocarcinoma than those with cholecystectomy. Based on the above evidence, we can conclude that cholecystectomy has a close relationship with the incidence of cholangiocarcinoma. To further elucidate whether the risk of cholangiocarcinoma related to the interval between cholecystectomy and the diagnosis of cholangiocarcinoma the rst time after cholecystectomy, we divided the interval into three groups: "one to ve years" group, " ve to ten years" group, and "more than ten years" group. As shown in Table 10, the number of the patients in the "one to ve years group" was 105, accounting for 43.93%; the number of the patients in the " ve to ten years group" was 63, accounting for 26.36%; and the number of the patients in the "more than ten years group" was 71, accounting for 29.71%. We compared the "one to ve years group" with another two groups separately, and the difference was statistically signi cant. These data indicated that the incidence of cholangiocarcinoma is the highest within one to ve years after cholecystectomy.

Discussion
Many researches about the relationship between cholecystectomy and the incidence of cholangiocarcinoma have been reported in many countries, but the association is controversial and there is none in China. Ekbom,A conducted a cohort study in 1993, which indicated that gallstones are considered as a risk factor of cholangiocarcinoma and a reduced risk of cholangiocarcinoma 10 or more years after cholecystectomy [30]. A population based study was performed in Taiwan with 7938 cholelithiasis cases. They nd that patients who underwent cholecystectomy can reduce the incidence of subsequent cholangiocarcinoma while the increased risk of cholangiocarcinoma with the treatment of endoscopic sphincterotomy or endoscopic papillary balloon dilatation [31]. However, a systematic review and meta-analysis was performed including 16 articles comprising 220,376 patients with cholecystectomy, indicating that cholecystectomy was related to a signi cant 54% increase in the risk of cholangiocarcinoma [32]. And a case-control study was conducted to investigate the risk factors of cholangiocarcinoma, which concluded that cholecystectomy is one of the risk factors of extrahepatic cholangiocarcinoma [33]. Thus, to elucidate the relationship between cholecystectomy and the incidence of cholangiocarcinoma, we conducted this case-control study in China.
In this study, descriptive analysis of the general information of patients by Chi square test among the groups (case group, positive control group and negative control group) showed statistical differences in age, gender and diabetes. Based on logistic multivariate regression analysis, it was concluded that the difference of diabetes history between the case group and the positive control group was statistically signi cant, possibly because diabetes is one of the risk factors of pancreatic cancer [34,35]. Based on logistic multivariate regression analysis, we found that the difference of gender and diabetes between the case group and the negative control group was even more statistically signi cant. Which indicated that female patients are more likely to suffer from hepatic hemangioma and femoral fracture, and some studies supported this conclusion [36,37]. Furthermore, it has been reported that patients with type 2 diabetes have increased BMD (bone mineral density) but impaired structure and mineral properties, showing a unique bone phenotype that increases the risk of fracture [38], which is consistent with our conclusion.
The rate of cholecystectomy between the case group and the positive control group was analyzed by chisquare test, and it was statistically signi cant. Then we analyzed the rate of cholecystectomy between the case group and the negative control group the same way, and the difference was statistically signi cant too. Based on the chi-square analysis of the rate of cholecystectomy, we conducted logistic multivariate analysis between the case group and the positive control group, and the data showed that the OR value of the rate of cholecystectomy was 1.553 (CI95%: 2.093-2.890, p = 0.000), while the OR value of cholecystolithiasis between the case group and the negative control group was 5.426 (CI95%: 4.325-6.809, p = 0.000). These evidence indicated that patients with cholecystolithiasis have a higher risk of cholangiocarcinoma comparing with the patients who underwent cholecystectomy. However, the risk of cholangiocarcinoma remained high after cholecystectomy and did not fall to the level of the negative control group. Cholecystolithiasis as one of the risk factors of cholangiocarcinoma can be attributed to the chronic in ammatory stimulation to gallbladder and bile duct by gallstones. In ammation has become a recognized oncogenic mechanism, and pro-in ammatory cytokines could stimulate the expression of abnormal genes in biliary epithelial cells, which is closely related to the occurrence of cholangiocarcinoma [39][40][41]. Although cholecystectomy can eliminate the in ammatory stimulation of cholecystolithiasis, it can lead to bile acid metabolism disorder and increase the secretion of secondary bile acids, which has been shown to be involved in carcinogenesis [42][43][44]. In addition, cholecystectomy can lead to alteration of intestinal ora which is associated with the development of gastrointestinal tumor [45,46].

Conclusion
In conclusion, cholecystectomy has a close relationship with the incidence of cholangiocarcinoma and the patients who undergo cholecystectomy have a higher risk of cholangiocarcinoma than the control groups. So it is not recommended to perform cholecystectomy blindly. Therefore, we suggest that cholecystectomy should still be performed in patients, whose gallbladder lost the biological function or who have surgical indications, to eliminate the risk of gallstones-induced malignancy. With regard to the patients of cholecystolithiasis whose gallbladder is functional or the surgical indication of choledochotomy is not met, we should perform cholecystectomy with caution or the conservative treatment is practicable.

Declarations
Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
This study was approved by the ethics committee of the Xijing Hospital and performed in accordance with their guidelines. Because of the anonymous characteristics of the data, the informed consent was waived.

Consent for publication
Not applicable.

Con ict of interest
The authors declare that they have no con icts of interest.