HBcAb positivity increases the risk of postoperative complications after extended hemihepatectomy for hilar cholangiocarcinoma

Abstract Background Hepatitis B core antibody (HBcAb) positivity is considered a prior hepatitis B virus (HBV) infection. However, little is known about the effect of HBcAb positivity on surgical safety for hilar cholangiocarcinoma (hCCA). The present study aims to investigate the role of HBcAb positivity on postoperative complications of hCCA. Methods A retrospective analysis was performed on the status of HBcAb positivity, liver fibrosis, perioperative surgical complications, and long‐term outcomes of hCCA patients with Hepatitis B surface antigen (HBsAg) negativity who underwent surgical treatment in Tongji Hospital from April 2012 to September 2019. Results HBcAb positivity with negative HBsAg occurs in 137 hCCA patients (63.1%). A total of 99 hCCA patients with negative HBsAg underwent extended hemihepatectomy, of whom 69 (69.7%) and 30 (30.3%) were HBcAb‐positive and HBcAb‐negative, respectively. Significant fibrosis was detected in 63.8% of the patients with HBcAb‐positive, which was markedly higher than those with HBcAb‐negative (36.7%) (p = 0.016). The postoperative complications and 90‐day mortality rates were 37.4% (37/99) and 8.1% (8/99), respectively. The incidence of postoperative complications in HBcAb‐positive patients (44.9%) was significantly higher than that in HBcAb‐negative patients (20.0%) (p = 0.018). All the patients who died within 30‐day after surgery were HBcAb‐positive. Multivariate analysis showed that the independent risk factors for complications were HBcAb positivity, preoperative cholangitis, portal occlusion >15 min, and significant fibrosis. There were no significant differences in recurrence‐free survival (RFS) and overall survival (OS) between HBcAb‐positive and HBcAb‐negative patients (p = 0.642 and p = 0.400, respectively). Conclusions HBcAb positivity is a common phenomenon in hCCA patients from China, a country with highly prevalent HBcAb positivity. The status of HBcAb‐positive markedly increases the incidence of postoperative complications after extended hemihepatectomy for hCCA patients.


| INTRODUCTION
Cholangiocarcinoma (CCA) can be classified into intrahepatic CCA (iCCA), hilar CCA (hCCA), and distal CCA (dCCA). Among them, hCCA is an epithelial carcinoma, also known as Klatskin tumor, which likely originates from the biliary tree and arises between the secondary bile duct and the cystic duct, accounting for approximately 50%-75% of CCAs. 1 hCCA is closely associated with primary sclerosing cholangitis, biliary cysts, hepatolithiasis, and chronic liver disease. 2,3 Surgery is the most effective treatment for hCCA patients. 1,4 Due to the peculiar anatomical location and predisposition of hCCA to vascular invasion, hemihepatectomy combined with caudate lobectomy is considered as the standard surgical strategy in many medical centers to increase tumor resectability and improve long-term outcomes. [5][6][7][8] However, the incidence of postoperative complications and the mortality associated with such surgical resection are relatively noticeable. 7,9 Hepatitis B core antibody (HBcAb) positivity is an abnormal index that appears after infection with hepatitis B virus (HBV); it typically exists in the serum for a long-time; thus, it is regarded as a sign of a past infection. 10,11 Hepatitis B surface antigen (HBsAg) is the hallmark of infection, and it is positive in the early stage of acute infection and persistently in chronic infection. 11 HBcAb positivity with negative HBsAg is also called prior HBV infection. 10,12 China is one of the susceptible countries to HBV infection, and the overall prevalence of HBsAg positivity and HBcAb positivity is still remarkable (up to 7.2% and 43.2%, respectively). 13,14 Previous studies showed that HBV was one of the risk factors for iCCA, and HBsAg positivity will significantly increase the incidence of complications after liver resection. 15,16 However, the incidence of HBcAb positivity in hCCA patients and the impact of HBcAb positivity on the safety of hCCA patients who received surgery have never been reported. Therefore, the present study aims to evaluate the prevalence of HBcAb positivity in hCCA patients and explore the effect of HBcAb positivity on postoperative complications of hCCA patients after curative resection.

| Patients' selection
A retrospective study was conducted on consecutive hCCA patients who underwent surgery in the Department of Hepatobiliary and Pancreatic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology from April 2012 to September 2019.
The inclusion and exclusion criteria were as followings. Inclusion criteria: (1) Patients were pathologically diagnosed with hCCA; (2) Underwent extended hepatectomy (hemihepatectomy combined with caudate lobectomy); (3) Availability of complete clinicopathological data. Exclusion criteria: (1) Underwent exploration and biopsy; (2) Underwent limited resection; (3) Patients had other malignant tumors or severe systemic dysfunction. Figure 1 shows the patient selection flowchart for the study.

| Data collection
Patients' demographic, clinical data, and the existence of postoperative complications were collected and analyzed. HBV serological markers were determined by HBcAb-positive markedly increases the incidence of postoperative complications after extended hemihepatectomy for hCCA patients.

K E Y W O R D S
HBsAg negative, hemihepatectomy, hepatitis B core antibody, hilar cholangiocarcinoma, postoperative complications, surgical outcomes F I G U R E 1 The flowchart of the patient selection. the chemiluminescent microparticle immunoassay method (Architect-i2000 SR, Abbott, Dublin, Ireland), in which HBsAg ≥0.05 IU/mL, hepatitis B surface antibody (HBsAb) ≥10 IU/mL, hepatitis B e antigen (HBeAg) ≥1 S/ CO, hepatitis B e antibody (HBeAb) ≤1 S/CO, and HBcAb ≥1 S/CO indicate the positive result. The patients were diagnosed with cholangitis when they showed fever, jaundice, and abdominal pain (Charcots triad). According to the Bismuth-Corlette classification of hCCA, 17 the tumor was classified as type I, II, IIIa, IIIb, and IV. Liver fibrosis was determined by Masson's Trichrome staining of the formalin-fixed paraffin-embedded samples. According to the Laennec staging system, liver fibrosis or cirrhosis was categorized into five stages: F0 (no definite fibrosis), F1 (minimal fibrosis), F2 (mild fibrosis), F3 (moderate fibrosis), and F4 (cirrhosis). 18 Significant fibrosis was defined as ≥F2 and advanced fibrosis as ≥F3.
Postoperative complications and criteria were defined as follows: postoperative pleural effusion refers to the Bultrasound exploration in sitting and standing positions and the depth of pleural effusion to be more than 3 cm, or a pleural fluid that requires a thorax puncture or drainage; massive ascites refers to postoperative daily ascitic fluid drainage from abdominal drains exceeding 10 mL/kg of preoperative body weight, at least 3 days, or ascites requiring an invasive procedure comprising percutaneous puncture or drainage (radiology or surgery). [19][20][21] Postoperative infection refers to signs of inflammation and blood or drainage fluid cultures positive for pathogenic bacteria. 22 According to the criteria presented by the International Study Group of Liver Surgery, postoperative bile leakage was defined as the drainage of intra-abdominal fluid with an increased bilirubin concentration (at least three times the serum bilirubin concentration) on or after postoperative Day 3. 23 Posthepatectomy liver failure (PHLF) was defined as an increasing international normalized ratio (INR; or decreasing prothrombin time) and elevating serum bilirubin concentration on or after postoperative Day 5 (compared with the values of the previous day). 24 In the present study, we only counted the occurrence of grade B/C PHLF.

| Preoperative assessment and surgical procedure
Preoperative liver function and residual liver volume were considered comprehensively, and timely adjustment and treatment of preoperative abnormalities. In the case of obstructive jaundice and cholangitis, biliary drainage was performed in the remnant liver until the total bilirubin concentration decreased by more than half. Percutaneous transhepatic cholangial drainage (PTCD) was used in most cases, while some patients underwent endoscopic biliary drainage (EBD). Assessment of liver function was consistent with the indocyanine green test (ICG) performed after bile drainage reduced serum total bilirubin concentration; we set the safe limit for the ICG retention value as <10% at 15 min for liver resection. In these patients, at least 30% of the functional liver volume (estimated by a preoperative three-dimensional CT scan) could be preserved after hemihepatectomy or trisectionectomy. Patients with a high level of HBV DNA were required to receive effective antiviral therapy and have a viral load of less than 100 IU/ mL at screening. Standardize surgical procedures to minimize surgical risks. All patients underwent left or right hemihepatectomy or trisectionectomy combined with caudate lobectomy, extrahepatic biliary resection, and local lymphadenectomy. Hemihepatic vascular inflow occlusion or the intermittent Pringle maneuver was used to minimize blood loss during parenchymal resection.

| Statistical analysis
Statistical analysis and graphical illustration were undertaken using the software SSPS 24.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). Continuous variables with normal distribution were presented as mean (standard deviation [SD]), and those with abnormal distribution were reported as median (interquartile range [IQR]). As appropriate, the frequencies of categorical variables were compared using the Pearson χ 2 or Fisher's exact test. The independent-samples t-test compared mean values of continuous variables (normally distributed), and the Kaplan-Meier (K-M) method was used to estimate overall survival (OS). Uni-and multivariable analyses of risk factors for complications were performed using logistic regression, univariate comparisons of survival and multivariate analysis were performed using the Cox proportional hazards model, and all variables with a p-value of 0.10 or lower at univariable analyses were entered into the multivariable analyses with backward selection. Multivariable logistic model was fitted using backward stepwise regression. p < 0.05 was considered statistically significant.

| Patients' demographic and HBV infection status
A total of 217 consecutive hCCA patients were retrospectively studied. Among these patients, 99 patients with HBsAg negativity who underwent curative resection were enrolled in this study and divided into the HBcAb+ (n = 69) group and HBcAb-(n = 30) group according to the status of HBV infection. Figure 1 shows the flowchart of the patient selection. Of the 217 patients who underwent surgery, 160 (73.7%) patients were HBcAb+, 23 (10.6%) cases were HBsAg+, 3 (1.4%) cases were HBeAg+, and 73 (33.6%) cases were HBeAb+; Meanwhile, we found that all patients with HBsAg+ had HBcAb+, HBcAb+ with HBsAg-occurs in 137 hCCA patients (63.1%). Besides, 57 (26.3%) cases had HBVnegative markers (including all negative or only HBsAb+). There were only two patients with hepatitis C, and neither of them underwent extended hemihepatectomy.

| Clinical data and liver fibrosis, postoperative mortality, and complications
Data of 99 HBsAg-negative patients who underwent extended hemihepatectomy were analyzed, in which the incidence of postoperative complications and 90-day mortality were 33.3% (33/99) and 8.1% (8/99), respectively. There were 69 (69.7%) and 30 (30.3%) patients in HBcAb+ and HBcAb-groups, respectively. The surgical procedures and postoperative complications of the two groups are shown in Table 1 (some patients had simultaneously two or more complications).
Pathological analysis of liver fibrosis was performed in 99 patients enrolled in the study. The pathological staging of liver fibrosis was summarized according to the Laennec staging system; the incidence of cirrhosis (F4) in all patients was 2.0% (2/99). The rate of significant fibrosis (F2 + F3 + F4) in the HBcAb+ group (44/69, 63.8%) was significantly higher than that in the HBcAb-group (11/30, 36.7%) (p = 0.016). Advanced fibrosis (F3 + F4) was detected in 24.6% of the patients with HBcAb-positive, which was markedly higher than those with HBcAb-negative (3.3%) (p = 0.011). Overall, HBcAb-positive patients were more likely to develop more severe fibrosis or cirrhosis.
As shown in Table 1, the incidences of postoperative complications (p = 0.014) and massive ascites (p = 0.035) in the HBcAb+ group were significantly higher than those in the HBcAb-group (p < 0.05). Although not statistically significant, the incidence of liver failure (p = 0.077) in the HBcAb+ group (15.9%) was higher than that in the HBcAb-group (3.3%). The length of stay in hospital was significantly longer in the HBcAb+ group compared to in the HBcAb-group (p = 0.012). There was no statistically significant difference between the two groups in the surgical procedures and other postoperative complications (p > 0.05).
Most patients who died within 90-day after surgery were HBcAb-positive, the leading causes of death were liver failure (six cases) and hemorrhagic shock (three cases), and all the patients who died within 90-day after surgery had significant liver fibrosis (F2/F3/F4).
Univariate and multivariate analyses of risk factors for complications were performed by logistics regression, and the results were summarized in
In order to identify prognostic risk factors in patients with hCCA who underwent liver resection, Cox univariate and multivariate analyses were performed. Multivariate analysis indicated that positive surgical margin [adjusted hazard ratio (HR), 3 (Table S1). Kaplan-Meier survival curves of surgical margin, microvascular invasion, lymph node metastasis, and pathological differentiation are shown in Figure S1.

| DISCUSSION
To the best of our knowledge, this is the first study to demonstrate the incidence of HBcAb positivity in hCCA patients and the effect of HBcAb positivity on surgical complications for hCCA. We found that HBcAb positivity was common in hCCA patients (73.7%), and HBcAb positivity significantly increased the incidence of postoperative complications after extended hemihepatectomy for hCCA. China has a high prevalence of HBV infection, where 120 million cases are infected with chronic HBV, and about 300,000 patients die from HBV infection-related diseases (e.g., cirrhosis, liver failure, carcinoma, etc.) annually. 25 The epidemiological serosurvey of HBV infection conducted in 2006 in China showed that the positive rates of HBsAg, anti-HBs, and anti-HBc in Chinese cases aged 1-59 years old were 7.2%, 50.1%, and 34.1%, respectively. 13 The present research revealed that the prevalence rates of HBsAg, HBsAb, and HBcAb in hCCA patients were 10.6%, 48.4%, and 73.7%, respectively. The prevalence of HBcAb positivity in hCCA patients was remarkably higher than that in healthy individuals, suggesting a close relationship between hCCA and HBcAb positivity.
Numerous studies demonstrated that HBV infection was closely associated with hepatocellular carcinoma (HCC) and CCA. [26][27][28][29][30] Although the exact effect of HBV on tumorigenesis is unknown, integration of HBV DNA into the host genome induces genomic instability and direct insertion mutations of multiple cancer-related genes, which have been proven to be an essential risk factor for carcinogenesis. 28,30 As we all know, both hepatocytes and cholangiocytes have the same progenitor cells. Therefore, HBV is likely to induce HCC and CCA through similar mechanisms. 31 In addition, inflammatory changes caused by the continuous presence of the virus involve multiple signaling pathways leading to malignant transformation. 29 HBcAb positivity reflects prior HBV infection, which has recently attracted attention as a risk factor for liver carcinogenesis. [32][33][34] A previous meta-analysis suggested that HBcAb positivity increased the risk of cholangiocarcinoma. 32 However, no study has focused on surgical safety for hCCA in HBcAb-positive patients.
The current study revealed that the stage of liver fibrosis in HBcAb-positive patients was significantly higher than that in HBcAb-negative patients, and advanced fibrosis or significant fibrosis was more prevalent in HBcAb-positive than in HBcAb-negative patients. Previous studies have demonstrated an increased prevalence of advanced fibrosis/cirrhosis in prior hepatitis B patients, consistent with the present study's findings. 35,36 Liver fibrosis is characterized by excessive accumulation of extracellular matrix, which distorts the physiological architecture of the liver. 37

T A B L E 1 (Continued)
Severe fibrosis or cirrhosis would impair the synthetic and metabolic functions of the liver, and it might involve a higher potential surgical risk. 38,39 HBcAb positivity can not only cause liver fibrosis and then decrease reserve function but also cause inflammatory liver injury, which can affect liver function. Therefore, HBcAb positivity and fibrosis have overlapping effects on liver reserve function. Still, HBcAb positivity could act as an independent factor, mainly because prior HBV infection can also impair liver reserve function through hepatitis, hepatocyte injury, virus reactivation, and other ways, thus affecting surgical safety. 28,35,40 Hemihepatectomy/trisectionectomy has widely been advocated as the preferred surgical treatment for hCCA, which has the advantage of obtaining a wide and negative surgical margin and thus improving the surgical outcomes. 41,42 Considering that hilar cholangiocarcinoma is likely to invade the parenchyma and the biliary branches of the caudate lobe, total caudate lobectomy was carried out for the radical treatment of hCCA. 42,43 Meanwhile, all patients underwent lymph node dissection and biliary reconstruction in our study. According to the NCCN guideline, liver resection is a potentially curative and safe option when the future remnant liver volume to the total liver volume ranges from 20% to 30% for patients with normal liver. 39,44 Hepatitis infection and severe liver fibrosis may affect liver functional reserve and postoperative remnant liver regeneration. 45,46 When the residual liver volume is insufficient to maintain the physiological function of the liver, it can lead to coagulation disorders, massive ascites, liver failure, severe infection, acute respiratory and circulatory failure, and even death. [47][48][49] Thus, preserving more liver parenchyma for patients with liver injury due to hepatitis infection or fibrosis is more critical to those receiving major liver resection because postoperative complication is a primary concern. 50 In the present study, the patients underwent surgery when the estimated residual liver volume was greater than 30%, and routine preoperative examinations were consistent with surgical indications. In the present study, the incidence of postoperative complications and massive ascites in HBcAb-positive patients were significantly higher than those in HBcAbnegative patients; similarly, the incidence of liver failure in the HBcAb-positive group (14.5%) was higher than that in the HBcAb-negative group (3.3%). In addition, HBcAbpositive patients had higher mortality within 30-day after surgery than HBcAb-negative patients (7.2% vs. 0%). Multivariate analysis indicated that the HBcAb positivity was a significant independent risk factor of postoperative complications (OR 4.534, 95% CI 1.19-17.28, p = 0.027). Moreover, liver fibrosis was another independent risk factor for postoperative complications, which might reflect prior HBV infection directly or indirectly caused liver damage and thereby increased the incidence of postoperative complications. Therefore, HBcAb positivity markedly increased the incidence of postoperative complications after liver resection for hCCA.
Previous studies on the relationship between HBV infection and the postoperative prognosis of iCCA are still controversial. Some reports suggested that current or previous HBV infection could improve the prognosis of iCCA, 51,52 while other studies indicated that HBV infection was associated with a worse prognosis. 53 However, no research has investigated whether HBV infection or HBcAb positivity will affect the prognosis of hCCA patients. In our study, another important finding was that there was no significant difference in OS or RFS between HBcAb-positive and HBcAb-negative patients, so the long-term prognosis of hCCA patients was not correlated with HBcAb status. Multivariate analyses revealed that the prognosis of hCCA is determined primarily by the more aggressive tumor and insignificantly by the status of HBcAb.
To ensure the R0 resection of hCCA while reducing the incidence of postoperative complications, we previously found that 54 hCCA had limited axial invasion. Thus, adjusting surgical strategies such as decreasing the extent of liver resection may provide a more safe and effective treatment for HBcAb-positive patients. In other words, reducing the volume of liver resection can be an appropriate solution for high-risk hCCA patients associated with underlying diseases such as prior HBV infection.
This study had some limitations. First, this was a single-center retrospective study with a small sample, meaning there is a high chance of it having selection bias. Second, heterogeneity of the included studies was induced by some factors, such as different surgical procedures. However, due to the small sample, we did not conduct subgroup analyses of surgical procedures. Third, we have not analyzed the impact of postoperative therapy on the long-term outcomes due to unavailable data. More prospective studies with a large sample size and RCT studies should be further carried out to explore the impact of HBcAb status on surgical outcomes of hCCA patients.

| CONCLUSIONS
In summary, HBcAb positivity is a common phenomenon in hCCA patients from China. HBcAb positivity markedly increased the risk of postoperative complications and mortality after extended hemihepatectomy for hCCA. Consequently, hepatobiliary surgeons should pay more attention to HBcAb status in hCCA patients with negative HBsAg, which could help them to select optimal surgical modalities.