Biliary stenting alone versus biliary stenting combined with 125I particles intracavitary irradiation for the treatment of advanced cholangiocarcinoma

We aimed to compare the efficacy of percutaneous transhepatic biliary stenting (PTBS) and PTBS combined with 125I particles implantation in the treatment of advanced extrahepatic cholangiocarcinoma (EHC). A total of 184 advanced EHC patients, who received PTBS (PTBS group) or PTBS combined with 125I particles implantation (PTBS + 125I group) from January 2012 to April 2017 in our department, were retrospectively reviewed. The improvement of jaundice and liver function was observed in both groups. The postoperative complications, risk of biliary re-obstruction, and overall survival (OS) were compared between the two groups. Amongst, 71 cases received PTBS and 113 had the additional implantation of 125I particles. The jaundice and liver function were significantly improved in all patients, especially in PTBS + 125I group. There was no significant difference in the risk of postoperative complications between the two groups. However, the risk of biliary re-obstruction significantly reduced in PTBS + 125I group (19.5% vs. 35.2%, p = 0.017). Kaplan Meier analysis showed that patients in PTBS + 125I group had a significantly better OS, both for hilar and distal cholangiocarcinoma. Univariate analysis demonstrated that preoperative levels of carbohydrate antigen 19-9 (CA19-9), total bilirubin, neutrophil count, lymphocyte count, and different therapeutic method were significant factors affecting OS. Multivariate analysis further identified the treatment of PTBS combined with 125I particles implantation as an independent protective prognostic factor (HR = 0.26, 95% CI: 0.17–0.39, p < 0.001). In conclusion, for patients with advanced EHC, PTBS combined with 125I particles implantation is superior to PTBS alone in improving liver function, inhibiting biliary re-obstruction, and prolonging survival time.

in advanced EHC due to the efficacy of improving biliary obstruction and relieving symptoms. However, because of the rapid tumor development, biliary stenting alone may easily cause the re-obstruction of biliary tract 9 . In contrast, the additional implantation of 125 I particles can effectively suppress tumor growth through the release of continuous γ rays 10 .
Recent studies have shown that biliary stenting combined with 125 I particles implantation is a safe and feasible palliative treatment for advanced EHC 11,12 . However, to date, few reports have compared the efficacy of biliary stenting alone with additional implantation of 125 I particles. Here, we summarized 184 cases of advanced EHC who underwent percutaneous transhepatic biliary stenting (PTBS) or PTBS combined with 125 I particles implantation in our department. The efficacy, complications, risk of biliary re-obstruction, and overall survival (OS) were compared between the two groups.

Methods
Patients. Advanced EHC patients who were admitted to our department from January 2012 to April 2017 were retrospectively analyzed. Inclusion criteria: (1) pathologically or clinically diagnosed as HCCA or DCCA; (2) unresectable or unwilling to surgery; (3) received PTBS (PTBS group) or PTBS combined with 125 I particles implantation (PTBS + 125 I group) for the first time. Exclusion criteria: (1) benign biliary stenosis; (2) distant metastasis; (3) previously received surgery, endoscopic stenting, or chemotherapy for EHC; (4) incomplete follow-up data. We reported this study in compliance with STrengthening the Reporting of OBservational studies in Epidemiology (STROBE). Our study was complied with Helsinki Declaration 13 , and was approved by the Ethics Committee of the First Affiliated Hospital of Bengbu Medical College. All patients signed informed consent prior to treatment.
Surgical procedures. Percutaneous transhepatic cholangial drainage (PTCD) was performed in advance for patients who had obstructive jaundice or biliary tract infection. About one week later, PTBS with or without 125 I particles implantation were performed under digital subtraction angiography. The process has been described previously 14,15 . The dose delivered by 125 I particles implantation was approximately at 1 cm from the center of the radioactive source.
All patients were followed-up until September 2017 or until the death. The follow-up contents included biochemical routine, tumor markers, abdomen ultrasound, and/or CT. Changes of serum ALT, AST, TBIL, DBIL, ALP, and ALB were observed at 1, 3, and 6 months postoperatively. When "P" type tube shifted, appropriate adjustment was performed. As 125 I particles have almost no anti-tumor effect at about 6 months after operation, re-implantation of new 125 I particles was performed every 6 months. Discarded 125 I particles were sent to Nuclear Medicine Center for centralized disposal.
Statistical analysis. We used SPSS 22.0 software for statistical analysis. Continuous variables with normal distribution were expressed as mean ± standard deviation and differences between groups were compared with t test. Or else, median (min-max) with Wilcoxon test was used. Postoperative changes of liver function were analyzed by using paired t test. The optimal cutoff value of continuous variable is determined by the largest value of Youden Index (sensitivity + specificity −1). The primary outcome was OS, which was assessed by Kaplan Meier curves compared with log-rank test. Variables with P value less than 0.05 in the univariate analysis were entered into a multivariate Cox regression model. P < 0.05 indicates statistically significant difference.

Baseline information.
A total of 238 advanced EHC patients were admitted to our department from January 2012 to April 2017. Finally, 184 patients met selection criteria and were included in this study. Baseline data of the 184 patients are shown in Table 1. 44 (62.0%) out of the 71 cases in PTBS group versus 73 (64.6%) out of the 113 cases in PTBS + 125 I group were pathologically or clinically diagnosed as HCCA. There were no significant differences in gender, age, tumor location, serum levels of CA19-9, CEA, ALB, PT-INR, NC, LC, and CRP between the two groups (all p > 0.05). However, patients in the PTBS + 125 I group had significantly higher preoperative levels of ALT, AST, ALP, GGT, and TBIL (p < 0.05). The length of hospital stay was slightly longer in the PTBS + 125 I group, while the difference was not statistically significant (median: 15 vs. 13 days, p = 0.058). Moreover, hospital costs were relatively higher in PTBS + 125 I group compared with PTBS group (median: 36,631 vs. 28,821 CNY, p < 0.001).
Evaluation of clinical efficacy. All patients had significantly improved clinical symptoms (itching, fever, etc.) in both groups. In the PTBS group, the serum levels of ALT, AST, ALP, TBIL, and DBIL significantly decreased at 1, 3, and 6 months postoperatively, compared with preoperative levels (Fig. 1, all p < 0.05). However, no remarkable change of ALB was observed after operation in the PTBS group (p > 0.05). In contrast, the changes of all the above serum indices (including ALB) in the PTBS + 125 I group were more significant than that in the PTBS group.
Comparisons of complications and risk of biliary re-obstruction. After operation, a total of 9 (12.7%) patients in the PTBS group experienced complications, 6 of which had hyperamylasemia and 3 had biliary tract www.nature.com/scientificreports www.nature.com/scientificreports/ infection. In contrast, there were complications in 15 (13.3%) patients in the PTBS + 125 I group. Amongst, 10 cases had hyperamylasemia and 5 had biliary tract infection. No serious complications, radiation-related complications, or perioperative deaths were observed in both groups.
There was no statistically significant difference in overall complications between the two groups. However, compared with PTBS alone, the addition of 125 I particles reduced the risk of biliary re-obstruction (19.5% vs. 35.2%, p = 0.017).
The comparison of OS was further stratified according to tumor location. The median survival time was 9 months versus 10 months and the one-year survival rate was 37.2% versus 44.5% for patients with advanced HCCA and DCCA, respectively. Compared with PTBS alone, the addition of 125 I particles can significantly improve OS both in HCCA and in DCCA (Fig. 3, both p < 0.001).

Discussion
In recent years, the incidence of EHC has increased year by year worldwide. Currently, there are limited treatment options for advanced EHC. It is reported that the 5-year survival rate of advanced EHC is less than 5% 3,16 . Therefore, the exploration of more effective therapeutic methods is critical to improve the outcomes of advanced EHC.
It is generally acknowledged that local progression or metastasis is considered advanced and unresectable for EHC. PTCD can improve the quality of life and has become one of the main treatment for advanced EHC 3 . Compared to palliative surgery, biliary stenting reduces postoperative complications and shortens length of hospital stay 9 . However, due to the rapid growth of tumor, biliary stent alone can easily cause re-obstruction of biliary tract and re-implantation of stent may be needed. In addition, biliary stenting alone shows a limited efficacy in improving survival of EHC 9 . www.nature.com/scientificreports www.nature.com/scientificreports/ Radiotherapy is one of the effective treatments for advanced EHC. Recent study has shown that, compared with biliary stenting alone, the addition of external radiotherapy can extend stent patency time (median: 326 vs. 196 days, P = 0.022) and improve survival time (median: 367 vs. 267 days, p = 0.025) 17 . The implantation of radioactive particles is an effective form of brachytherapy 11,18 . Compared with external radiation, the implanted particles can directly act on tumor, and the radioactivity in the tumor is much higher than that in surrounding normal tissues. In addition, brachytherapy can significantly reduce radiation-induced complications such as peptic ulcers, hemorrhage, and radiation enteritis.
Since 2012, PTBS with or without 125 I particles implantation has been routinely applied for advanced EHC in our department. In this study, we summarized the relevant clinical data of 184 cases with advanced EHC. We found that, compared with PTBS alone, the addition of 125 I particles significantly reduced the risk of biliary re-obstruction and improved survival, while it did not increase the risk of postoperative complications. No radiation-related complications, such as duodenal ulcer and enteritis, were observed in the PTBS + 125 I group.  www.nature.com/scientificreports www.nature.com/scientificreports/ It is suggested that, the brachytherapy treatment is safe and may be not limited by some external considerations such as the proximity to the duodenal wall. However, the technique still has several disadvantages. Firstly, as the "P" type tube contained 125 I particles is implanted in the intra-cavity of biliary stent, 125 I particles implantation will be performed selectively (usually several days later) provided the stent expansion is insufficient even with balloon assisted dilation. Secondly, the tube contained 125 I particles may slide or even remove from the stent due to inappropriate postoperative activity.
Multivariate analysis further showed that treatment of PTBS combined with 125 I particles were an independent favorable factor of OS, and could reduce the risk of death by 74%. Preoperative TBIL and CA19-9 were also identified as important factors affecting the prognosis of EHC, which are consistent with previous reports by Cai et al. and Waghray et al. 19,20 . However, the effects of postoperative levels as well as dynamic changes of TBIL and CA19-9 in the prognosis of EHC need further research.
The reasons of the addition of 125 I particles significantly improves outcome in advanced EHC may be manifold. It is reported that 125 I particles can continuously release X and γ rays to effectively kill tumor cells and inhibit tumorigenesis 10 . Recently, Du et al. and Kubo et al. showed that, after the implantation of radioactive 125 I particles, the percentages of CD3 + T, CD4 + T, natural killer (NK), and regulatory T cells significantly increased in peripheral blood of tumor patients 21,22 . In addition, the concentrations of IgM, IgG, and IgA, and complements C3 and C4 also increased, indicating that 125 I particles may stimulate not only cellular immunity but also humoral immunity 21 .
There are several limitations in the current study. Firstly, because of the relatively small sample size and single-center retrospective design, larger multi-center prospective studies are still required to validate our findings. Secondly, as sampling poses a huge challenge for EHC, the pathological diagnosis is impossible in the majority of cases. www.nature.com/scientificreports www.nature.com/scientificreports/ Chemotherapy and radiotherapy are the two major options for advanced EHC. The combination of 125 I particles brachytherapy with chemotherapy (or chemoradiation) is a promising treatment and the safety and efficacy need to be further studied.

conclusions
In conclusion, for patients with advanced EHC, PTBS combined with 125 I particles implantation is superior to PTBS alone in improving liver function, inhibiting biliary re-obstruction and prolonging survival time.
Ethical considerations. This