Abstract
Background
This study aimed to elucidate the clinical characteristics of occult pancreaticobiliary reflux (OPBR) in benign gallbladder diseases, including gallbladder stone (GS), gallbladder polyp (GP), and gallbladder adenomyomatosis (GA).
Methods
We studied 578 patients with a normal pancreaticobiliary junction undergoing surgery for gallbladder diseases between December 2020 and January 2022. Of these, 80 patients had elevated bile amylase levels (> 110U/L) and were classified as OPBR patients; 498 comprised the control group. Patients fell into four groups: Group A (GA), Group B (GS alone), Group C (GS and GP), and Group D (GP alone).
Results
OPBR occurred in 16.2% of GS cases, 7.2% of GP cases, and 20.3% of GA cases. Patients with OPBR were typically older and more likely to have GS but less likely to have GP. In GS patients, OPBR incidence correlated with stone size, peaking at 48% in cases of gallbladder sludge (< 3 mm). OPBR incidence was highest in Group A (20.3%), followed by Group B (16.6%), Group C (8.9%), and Group D (4.8%). Patients over 50 and those in Groups A and B faced higher OPBR risk. In Group A, gallbladder sludge and hyper-gamma-glutamyltransferase were risk factors, while in Group B, gallbladder sludge and age over 50 were risk factors. No specific features associated with OPBR were found in Groups C and D.
Conclusion
In conclusion, GP isn’t associated with OPBR, irrespective of GS presence. However, in GS alone or GA patients, gallbladder sludge should raise OPBR concerns, necessitating tailored clinical attention.
Keywords: Occult pancreaticobiliary reflux, Gallbladder stone, Gallbladder polyp, Gallbladder adenomyomatosis, Gallbladder sludge
Introduction
Pancreaticobiliary reflux (PBR) is predominantly reported in individuals with pancreaticobiliary maljunction (PBM), a congenital anomaly characterized by the merging of the pancreatic duct and biliary duct outside the duodenal wall. This condition impairs the Oddi’s sphincter’s ability to regulate the pancreaticobiliary junction, leading to the backflow of pancreatic juice and bile [1–3]. In recent years, occult pancreaticobiliary reflux (OPBR), which is PBR occurring in individuals with a normal pancreaticobiliary junction (NPBJ), has garnered increasing attention, with its mechanism proposed to be associated with sphincter of Oddi dysfunction (SOD) or a relatively long pancreaticobiliary common channel [4–6]. Similar to individuals with PBM, patients with OPBR are at a heightened risk of developing gallbladder cancer [3, 6–8]. The reflux of pancreatic juice into the gallbladder, triggered by bile, can directly or indirectly damage the gallbladder epithelium, potentially leading to chronic inflammation and malignant transformations, including hyperplasia, metaplasia, atypical hyperplasia, carcinoma in situ, and invasive carcinoma [3, 4, 6].
There are still controversies regarding the treatment of patients with OPBR. Nevertheless, preventive cholecystectomy can be an efficient therapy for decreasing the risk of gallbladder cancer in patients with PBR [1, 9, 10]. In addition, the patient with OPBR would need long-term follow-up, because the reflux of pancreatic juice still exists after cholecystectomy, which is considered a risk factor for bile duct cancer [10]. However, there is a lack of accurate non-invasive diagnostic methods that can be used for OPBR. For patients with PBM, the abnormally long pancreaticobiliary common channel and/or an abnormal union can be diagnosed by imaging test, such as magnetic resonance cholangiopancreatography (MRCP), endoscopic retrograde cholangiopancreatography (ERCP), percutaneous transpehatic cholangiography (PTC), or intraoperative cholangiography [1, 2]. But in patients with OPBR, they have NPBJ, so it is difficult to preoperative diagnose by conventional imaging.
Gallbladder stone (GS), gallbladder polyp (GP) and gallbladder adenomyomatosis (GA) are common benign diseases of gallbladder and also the risk factors for gallbladder cancer [11–13]. In addition, the formation of partial GS and GA has been reported to be associated with PBR [6, 13, 14]. The aim of the present study was to clarify the clinical features of OPBR in patients with benign gallbladder diseases in order to identify OPBR in a timely and effective manner and thus avoid the risk of gallbladder cancer.
Patients and methods
Inclusion criteria and sample size
This prospective observational study was approved by the ethics committee of Shanghai East Hospital (Shanghai, China), and all participating patients provided their informed consent to obtain their bile samples during operation and participate in all necessary blood tests before surgery. As illustrated in the patient inclusion flowchart (Fig. 1), this study prospectively enrolled 578 consecutive patients with NPBJ who underwent surgery for benign gallbladder diseases between December 2020 and January 2022. The diagnosis of NPBJ was made using MRCP to assess the union of the common bile duct and the main pancreatic duct within the duodenal wall, where the sphincter of Oddi surrounds them with muscular fascicles to regulate the flow of bile and pancreatic juice [4].
Fig. 1.
Flowchart of patient selection for final analysis
Detection of bile amylase
One day before operation, blood samples were drawn from the patients for detection of amylase, lipase, bilirubin, γ-glutamyl transpeptidase (γ-GT) and alkaline phosphatase (ALP). 5 ml bile was extracted from the gallbladder with a sterile syringe during operation, stored at 4℃, and sent to the laboratory for analysis the following day by a technician who was blind to the study protocol. Bile amylase level was detected with Roche Cobas c702 (Roche, Basel, Switzerland). The normal range of serum amylase in our laboratory is 30–110 U/L. Previous studies have consistently attributed the presence of amylase in bile either to serum amylase passing through the liver or reflux from the pancreatic duct [4, 15, 16]. Donaldson et al. demonstrated that bile amylase levels were not significantly different from serum amylase levels in healthy individuals without liver disease [15]. Therefore, consistent with methods used in prior studie, we defined PBR as bile amylase levels exceeding normal plasma amylase concentrations [1, 2, 4, 16]. In total, 80 patients with the amylase level > 110U/L were assigned as the OPBR group, and the remaining 498 as the control group.
Patients grouping
Given that some patients presented with two or more benign gallbladder diseases simultaneously, we categorized patients into four groups based on postoperative pathological findings. Patients diagnosed with GA were assigned exclusively to Group A (n = 69), regardless of whether they had additional gallbladder diseases. The remaining patients, who only had GS and/or GP, were further classified into Group B (GS alone, n = 325), Group C (both GS and GP, n = 79), and Group D (GP alone, n = 105) (Fig. 2A). All diagnoses, including detailed characteristics of stones and polyps, were confirmed histologically.
Fig. 2.
Incidence and Distribution of OPBR in Patients with Different Benign Gallbladder Diseases.A Patients were divided into four groups according to gallbladder disease. B Comparison of the incidence of OPBR in patients with different benign gallbladder diseases. C Comparison of the incidence of OPBR in different groups of patients. *P < 0.05, **P < 0.01, ***P < 0.001
Statistical analysis
Data analysis was performed using SPSS 24 (IBM, Chicago, US). Qualitative variables are expressed as percentage (%), and comparison between groups was performed by χ 2 test or Fisher’s exact test. Quantitative data are presented as median (IQR). Normality was assessed using the Shapiro-Wilk test. Normally distributed variables were compared using the t-test or one-way ANOVA, whereas non-normally distributed variables were analyzed using the Mann-Whitney U test or Kruskal-Wallis H test. Variables with the P values < 0.1 in univariate analysis were subjected to multivariate logistic regression analysis. Values of P < 0.05 were considered statistically significant.
Results
Clinical characteristics and group-specific differences in benign gallbladder diseases
The clinical characteristics of patients with different benign gallbladder diseases were analyzed, and significant differences were found among the four groups (Table 1). Notably, bile amylase levels showed no significant differences across the groups (p > 0.05). Patients in Groups A, B, and C were generally older at the time of surgery compared to those in Group D, with median ages of 48 (IQR: 38–59), 48 (IQR: 36–58), and 45 (IQR: 36–59) years, respectively, compared to 39 (IQR: 32–52) years in Group D (p = 0.004). Groups A and B had a higher proportion of female patients, with males accounting for 40.6% in Group A and 30.8% in Group B, whereas Groups C and D had a more balanced gender distribution, with males comprising 49.4% and 48.6%, respectively (p = 0.001). The prevalence of abdominal pain was greater in Groups B (62.5%) and C (51.9%) compared to Groups A (33.3%) and D (15.2%) (p < 0.001). Since gallbladder wall thickening (> 3 mm) is a hallmark feature of GA [13], all patients in Group A exhibited gallbladder wall thickening (100%). Additionally, gallbladder wall thickening was more common in patients with GS—occurring in 37.2% of Group B and 38.0% of Group C—compared to those with only GP in Group D, where the incidence was 18.1% (p < 0.001). Furthermore, Group B patients were more likely to have elevated γ-GT levels, with 15.1% presenting with hyper-γ-GT, compared to 7.2% in Group A, 5.1% in Group C, and 4.8% in Group D (p = 0.004).
Table 1.
Clinicopathologic characteristics of four groups
| Variables | Group A (n = 69) |
Group B (n = 325) |
Group C (n = 79) |
Group D (n = 105) |
P-value |
|---|---|---|---|---|---|
| Bile amylase, U/L | 10 (0–75) | 10 (0–30) | 10 (0–20) | 20 (0–20) | 0.726 |
| Age, years | 48 (38–59) | 48 (36–58) | 45 (36–59) | 39 (32–52) | 0.004 |
| Male, n (%) | 28 (40.6%) | 100 (30.8%) | 39 (49.4%) | 51 (48.6%) | 0.001 |
| BMI, kg*m-2 | 22.94 (21.14–24.33) | 23.88 (21.76–26.24) | 24.05 (22.06–26.07) | 23.44 (21.48–25.68) | 0.079 |
| Abdominal pain, n (%) | 23 (33.3%) | 203 (62.5%) | 41 (51.9%) | 16 (15.2%) | < 0.001 |
| Hypertension, n (%) | 11 (15.9%) | 68 (20.9%) | 19 (24.1%) | 15 (14.3%) | 0.279 |
| Diabetes, n (%) | 3 (4.3%) | 29 (8.9%) | 6 (7.6%) | 9 (8.6%) | 0.655 |
| Diameter of CBD > 8 mm, n (%) | 4 (5.8%) | 22 (6.8%) | 2 (2.5%) | 1 (1.0%) | 0.058 |
| GWT > 3 mm, n (%) | 69 (100%) | 121 (37.2%) | 30 (38.0%) | 19 (18.1%) | < 0.001 |
| Hyper-bilirubinemia, n (%) | 7 (10.1%) | 13 (4.0%) | 2 (2.5%) | 4 (3.8%) | 0.153 |
| Hyper-γ-GT, n (%) | 5 (7.2%) | 49 (15.1%) | 4 (5.1%) | 5 (4.8%) | 0.004 |
| Hyper-ALP, n (%) | 2 (2.9%) | 17 (5.2%) | 2 (2.5%) | 3 (2.9%) | 0.665 |
Data are represented as the median (IQR) or percentage
Abbreviations: BMI body mass index, CBD common bile duct, GWT gallbladder wall thickness, Hyper-γ-GT hyper-γ-glutamyltransferase, Hyper-ALP hyper-alkaline phosphatasemia
Patients grouping: Group A (those with GA), Group B (those with GS alone), Group C (those with GS and GP) and Group D (those with GP alone)
Significant p-values are highlighted in bold
In summary, these findings highlight distinct clinical differences among the four patient groups, particularly in age, gender distribution, abdominal pain prevalence, gallbladder wall thickening, and elevated γ-GT levels, which may help to better characterize benign gallbladder diseases in this study.
Incidence and distribution of OPBR across different benign gallbladder diseases
Among the 578 patients with benign gallbladder diseases included in the study, the overall incidence of OPBR was 13.8% (80/578), with no significant difference observed between genders (Table 2). Of these patients, 451 had GS, 208 had GP, and 69 had GA, with OPBR incidences of 16.2%, 7.2%, and 20.3%, respectively. In patients with GS, the incidence of OPBR varied significantly based on stone size. Those with gallbladder sludge (maximum diameter < 3 mm) had a markedly higher OPBR incidence of 48.0%, compared to 15.7% in patients with stones between 3 and 10 mm, and 13.1% in those with stones ≥ 10 mm (p < 0.001). However, no significant correlation was found between OPBR occurrence and the number or size of stones or polyps (Table 2).
Table 2.
OPBR occurrence in relation to gender and specific benign gallbladder disease features
| Variables | Patients, n | Incidence of OPBR | |
|---|---|---|---|
| OPBR, n (%) | P value | ||
| All | 578 | 80 (13.8%) | |
| Gender | 0.650 | ||
| Male | 218 | 32 (14.7%) | |
| Female | 360 | 48 (13.3%) | |
| Diagnosis a | |||
| Gallstone | 451 | 73 (16.2%) | |
| No. of stones | 0.405 | ||
| Solitary | 136 | 25 (18.4%) | |
| Multiple | 315 | 48 (15.2%) | |
| Maximum diameter (mm) of stones | < 0.001 | ||
| < 3 (gallbladder sludge) | 25 | 12 (48.0%) | |
| ≥ 3 and < 10 | 204 | 32 (15.7%) | |
| ≥ 10 | 222 | 29 (13.1%) | |
| Gallbladder polyp | 208 | 15 (7.2%) | |
| No. of polyps | 0.777 | ||
| Solitary | 55 | 3 (5.5%) | |
| Multiple | 153 | 12 (7.8%) | |
| Maximum diameter (mm) of polyps | 0.468 | ||
| < 10 | 142 | 12 (8.5%) | |
| ≥ 10 | 66 | 3 (4.5%) | |
| Gallbladder adenomyomatosis | 69 | 14 (20.3%) | |
Abbreviations: OPBR occult pancreaticobiliary reflux
aA total of 728 benign gallbladder disease diagnoses were recorded in 578 patients, as some individuals presented with more than one condition
Significant variations in OPBR incidence were also observed among different benign gallbladder diseases (p = 0.001, Fig. 2B). Specifically, the incidence of OPBR in patients with GP alone was 4.8%, which was notably lower compared to those with GS alone (16.6%). Furthermore, patients with both GS and GA had a significantly elevated OPBR incidence of 31.4%, compared to patients with GS alone (16.6%), GP alone (4.8%), or those with both GS and GP (8.9%). Further subgroup analysis revealed disparities in OPBR incidence across different groups: Group A had the highest incidence at 20.3%, followed by Group B at 16.6%, Group C at 8.9%, and Group D at 4.8% (p = 0.004, Fig. 2C). These findings suggest that the presence of multiple benign gallbladder conditions, particularly GS in combination with GA, is associated with an increased risk of OPBR.
Risk factors and distinct clinical features of OPBR across patient groups
The comparison of clinical features between control and OPBR patients highlighted several distinctions (Table 3). OPBR patients exhibited a significantly higher prevalence of GS (91.3% vs. 75.9%, p = 0.002), a lower incidence of GP (18.8% vs. 38.8%, p = 0.001), and markedly elevated bile amylase levels (median: 480 U/L vs. 10 U/L, p < 0.001). Additionally, OPBR patients were generally older than control patients (median age: 52 vs. 44 years, p < 0.001). However, no significant differences were observed in gender distribution, body mass index (BMI), or the prevalence of other conditions. Univariable and multivariable logistic regression analyses identified several independent risk factors for OPBR (Table 4). Below are the results from the multivariable regression analysis. Age over 50 years was significantly associated with OPBR (OR: 1.96, 95% CI: 1.20–3.19, p = 0.007). Additionally, patients in Group A (OR: 4.05, 95% CI: 1.26–13.99, p = 0.019) and Group B (OR: 3.59, 95% CI: 1.38–9.35, p = 0.009) had a significantly higher risk of OPBR compared to Group D (reference group). BMI ≥ 24 kg/m² was inversely associated with OPBR risk (OR: 0.60, 95% CI: 0.37–0.99, p = 0.047).
Table 3.
Clinical features of control patients and OPBR patients
| Variables | Control patients (n = 498) |
OPBR patients (n = 80) |
P-value |
|---|---|---|---|
| Diagnosisa | |||
| Gallstone, n (%) | 378 (75.9%) | 73 (91.3%) | 0.002 |
| Gallbladder polyp, n (%) | 193 (38.8%) | 15 (18.8%) | 0.001 |
| Gallbladder adenomyomatosis, n (%) | 55 (11.0%) | 14 (17.5%) | 0.098 |
| Bile amylase, U/L | 10 (0–20) | 480 (218–1630) | < 0.001 |
| Age, years | 44 (35–57) | 52 (44–61) | < 0.001 |
| Male, n (%) | 186 (37.3%) | 32 (40.0%) | 0.650 |
| Height, m | 1.64 (1.60–1.70) | 1.64 (1.59–1.72) | 0.571 |
| Weight, kg | 65 (57–73) | 62 (56–72) | 0.413 |
| BMI, kg*m−2 | 23.83 (21.65–26.00) | 23.08 (21.54–24.83) | 0.174 |
| Abdominal pain, n (%) | 243 (48.8%) | 40 (50.0%) | 0.841 |
| Hypertension, n (%) | 95 (19.1%) | 18 (22.5%) | 0.474 |
| Diabetes, n (%) | 39 (7.8%) | 8 (10.0%) | 0.510 |
| Diameter of CBD > 8 mm, n (%) | 26 (5.2%) | 3 (3.8%) | 0.777 |
| GWT > 3 mm, n (%) | 199 (40.0%) | 40 (50.0%) | 0.091 |
| Hyper-bilirubinemia, n (%) | 24 (4.8%) | 2 (2.5%) | 0.523 |
| Hyper-γ-GT, n (%) | 52 (10.4%) | 11 (13.8%) | 0.378 |
| Hyper-ALP, n (%) | 19 (3.8%) | 5 (6.3%) | 0.477 |
Data are represented as the median (IQR) or percentage
Abbreviations: OPBR occult pancreaticobiliary reflux, BMI body mass index, CBD common bile duct, GWT gallbladder wall thickness, Hyper-γ-GT hyper-γ-glutamyltransferase, Hyper-ALP hyper-alkaline phosphatasemia
Significant p-values are highlighted in bold
aA total of 728 benign gallbladder disease diagnoses were recorded in 578 patients, as some individuals presented with more than one condition
Table 4.
Predictors of OPBR in patients with benign gallbladder disease using logistic regression
| Variables | Univariable analysis | Multivariable analysis | ||
|---|---|---|---|---|
| OR (95% CI) | P-value | OR (95% CI) | P-value | |
| Group | P trend | 0.007 | P trend | 0.028 |
| A | 5.09 (1.74–14.88) | 0.003 | 4.05 (1.26–13.99) | 0.019 |
| B | 3.99 (1.55–10.25) | 0.004 | 3.59 (1.38–9.35) | 0.009 |
| C | 1.94 (0.59–6.37) | 0.272 | 1.82 (0.55–6.04) | 0.329 |
| D | 1.00 (reference) | - | 1.00 (reference) | - |
| Age > 50 years | 2.03 (1.26–3.27) | 0.004 | 1.96 (1.20–3.19) | 0.007 |
| Male | 1.12 (0.69–1.81) | 0.650 | ||
| BMI ≥ 24 kg*m−2 | 0.65 (0.40–1.05) | 0.077 | 0.60 (0.37–0.99) | 0.047 |
| Abdominal pain | 1.05 (0.65–1.68) | 0.841 | ||
| Hypertension | 1.23 (0.70–2.18) | 0.474 | ||
| Diabetes | 1.31 (0.59–2.91) | 0.511 | ||
| Diameter of CBD > 8 mm | 0.71 (0.21–2.39) | 0.578 | ||
| GTW > 3 mm | 1.50 (0.94–2.41) | 0.092 | 1.16 (0.67–2.01) | 0.593 |
| Hyperbilirubinemia | 0.51 (0.12–2.19) | 0.362 | ||
| Hyper-γ-GT | 1.37 (0.68–2.75) | 0.380 | ||
| Hyper-ALP | 1.68 (0.61–4.64) | 0.316 | ||
Abbreviations: OPBR occult pancreaticobiliary reflux, OR odds ratio, CI confidence interval, BMI body mass index, CBD common bile duct, GWT gallbladder wall thickness, Hyper-γ-GT hyper-γ-glutamyltransferase, Hyper-ALP hyper-alkaline phosphatasemia
Patients grouping: Group A (those with GA), Group B (those with GS alone), Group C (those with GS and GP) and Group D (those with GP alone)
Significant p-values are highlighted in bold
In Group A, a higher percentage of OPBR patients presented with hyper-ALP, and multivariate regression analysis pinpointed gallbladder sludge and hyper-γ-GT as significant independent risk factors for OPBR, each with an OR of 9.56 (95% CI: 1.38–66.43, p = 0.022) (Table 5 and Fig. 3A). In group B, patients with OPBR were generally older and more often had gallbladder sludge (Table 5). Notably, regression analysis for Group B indicated that gallbladder sludge (OR: 5.29, 95% CI: 1.91–14.67, p = 0.001) and age over 50 (OR: 2.49, 95% CI: 1.32–4.67, p = 0.005) are strong predictors of OPBR. Additional factors include male sex (OR: 1.98, 95% CI: 1.03–3.81, p = 0.041), while BMI ≥ 24 kg/m² (OR: 0.51, 95% CI: 0.27–0.98, p = 0.043) and abdominal pain (OR: 0.49, 95% CI: 0.26–0.92, p = 0.027) were inversely associated (Fig. 3B). For Groups C and D, no clinical characteristics were notably associated with OPBR (Table 5).
Table 5.
Comparison of clinicopathologic characteristics between control and OPBR patients across four groups
| Variables | Group A (n = 69) | Group B (n = 325) | Group C (n = 79) | Group D group (n = 105) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control (n = 55) |
OPBR (n = 14) |
P-value | Control (n = 271) |
OPBR (n = 54) |
P-value | Control (n = 72) |
OPBR (n = 7) |
P-value | Control (n = 100) |
OPBR (n = 5) |
P-value | |
| With gallstone, n (%) | 35 (63.6%) | 12 (85.7%) | 0.207 | - | - | - | - | - | - | - | - | - |
| Multiple stones, n (%) | 25 (45.5%) | 8 (57.1%) | 0.434 | 189 (69.7%) | 35 (64.8%) | 0.475 | 53 (73.6%) | 5 (71.4%) | 1.000 | - | - | - |
| Gallbladder sludge, n (%) | 2 (3.6%) | 3 (21.4%) | 0.053 | 10 (3.7%) | 9 (16.7%) | 0.001 | 1 (1.4%) | 0 (0.0%) | 1.000 | - | - | - |
| With gallbladder polyp, n (%) | 21 (38.2%) | 3 (21.4%) | 0.389 | - | - | - | - | - | - | - | - | - |
| Multiple polyps, n (%) | 17 (30.9%) | 2 (14.3%) | 0.364 | - | - | - | 50 (69.4%) | 7 (100.0%) | 0.200 | 74 (74.0%) | 3 (60.0%) | 0.863 |
| Max size of polyps ≥ 10 mm, n (%) | 3 (5.5%) | 0 (0.0%) | 1.000 | - | - | - | 10 (13.9%) | 1 (14.3%) | 1.000 | 50 (50.0%) | 2 (40.0%)- | 1.000 |
| Bile amylase, U/L | 10 (0–20) | 595 (160–1630) | < 0.001 | 10 (0–20) | 465 (203–1593) | < 0.001 | 10 (0–20) | 470 (390–1595) | < 0.001 | 10 (0–20) | 410 (310–1255) | < 0.001 |
| Age, years | 49 (39–60) | 48 (35–54) | 0.467 | 45 (35–57) | 56 (48–63) | < 0.001 | 44 (36–59) | 55 (49–59) | 0.184 | 39 (32–52) | 44 (33–56) | 0.583 |
| Male, n (%) | 23 (41.8%) | 5 (35.7%) | 0.678 | 78 (28.8%) | 22 (40.7%) | 0.082 | 36 (50.0%) | 3 (42.9%) | 1.000 | 49 (49.0%) | 2 (40.0%) | 1.000 |
| BMI, kg*m−2 | 23.03 (20.94–24.22) | 222.93 (21.60–24.44) | 0.682 | 24.05 (21.92–26.31) | 23.14 (20.75–25.62) | 0.087 | 24.22 (22.04–26.17) |
23.03 (22.41–24) |
0.529 | 23.44 (21.48–25.64) | 24.80 (21.79–29.03) | 0.220 |
| Abdominal pain, n (%) | 16 (29.1%) | 7 (50.0%) | 0.244 | 175 (64.6%) | 28 (51.9%) | 0.078 | 36 (50.0%) | 5 (71.4%) | 0.492 | 16 (16.0%) | 0 (0.0%) | 1.000 |
| Hypertension, n (%) | 7 (12.7%) | 4 (28.6%) | 0.300 | 55 (20.3%) | 13 (24.1%) | 0.533 | 18 (25.0%) | 1 (14.3%) | 0.865 | 15 (15.0%) | 0 (0.0%) | 1.000 |
| Diabetes, n (%) | 2 (3.6%) | 1 (7.1%) | 0.499 | 23 (8.5%) | 6 (11.1%) | 0.722 | 6 (8.3%) | 0 (0.0%) | 1.000 | 8 (8.0%) | 1 (20.0%) | 0.367 |
| Diameter of CBD > 8 mm, n (%) | 2 (3.6%) | 2 (14.3%) | 0.181 | 21 (7.7%) | 1 (1.9%) | 0.201 | 2 (2.8%) | 0 (0.0%) | 1.000 | 1 (1.0%) | 0 (0.0%) | 1.000 |
| GWT > 3 mm, n (%) | 55 (100%) | 14 (100%) | - | 100 (36.9%) | 21 (38.9%) | 0.783 | 26 (36.1%) | 4 (57.1%) | 0.492 | 18 (18.0%) | 1 (20.0%) | 1.000 |
| Hyper-bilirubinemia, n (%) | 7 (12.7%) | 0 (0.0%) | 0.362 | 11 (4.1%) | 2 (3.7%) | 1.000 | 2 (2.8%) | 0 (0.0%) | 1.000 | 4 (4.0%) | 0 (0.0%) | 1.000 |
| Hyper-γ-GT, n (%) | 2 (3.6%) | 3 (21.4%) | 0.086 | 42 (15.5%) | 7 (13.0%) | 0.634 | 4 (5.6%) | 0 (0.0%) | 1.000 | 4 (4.0%) | 1 (20.0%) | 0.220 |
| Hyper-ALP, n (%) | 0 (0.0%) | 2 (14.3%) | 0.039 | 14 (5.2%) | 3 (5.6%) | 1.000 | 2 (2.8%) | 0 (0.0%) | 1.000 | 3 (3.0%) | 0 (0.0%) | 1.000 |
Data are represented as the median (IQR) or percentage
Abbreviations: OPBR occult pancreaticobiliary reflux, BMI body mass index, CBD common bile duct, GWT gallbladder wall thickness, Hyper-γ-GT hyper-γ-glutamyltransferase, Hyper-ALP hyper-alkaline phosphatasemia
Patients grouping: Group A (those with GA), Group B (those with GS alone), Group C (those with GS and GP) and Group D (those with GP alone)
Significant p-values are highlighted in bold
Fig. 3.
Multivariate Logistic Regression Analysis of Risk Factors for OPBR in Groups A and B. A Risk Factors for OPBR in Group A. B Risk Factors for OPBR in Group B
Discussion
Gallbladder cancer stands as the predominant malignancy within the biliary tract [12]. Its typically silent progression results in late diagnosis, which significantly compromises patient outcomes. As such, early identification is paramount, particularly since surgical intervention can offer curative possibilities [4]. Notably, benign gallbladder diseases (GS, GP, and GA) and PBR (PBM and OPBR) are all considered risk factors for gallbladder cancer [3, 6, 11–13]. For individuals predisposed to gallbladder cancer, cholecystectomy can effectively mitigate tumor genesis, albeit with its inherent surgical risks [1, 9]. While modalities like abdominal ultrasound efficiently diagnose GS, GP, and GA; PBM—with its congenital anomalies—presents identifiable markers discernible through ERCP, PTC, and MRCP [1, 2]. Conversely, OPBR, characterized by its NPBJ, eludes traditional imaging techniques and lacks distinct biomarkers. This poses a diagnostic challenge, thereby elevating the risk for gallbladder cancer development [4, 5]. To address this, our study incorporated 578 patients with NPBJ and benign gallbladder disease. The objective was to delineate the clinical characteristics of OPBR, facilitating its early identification and reducing gallbladder cancer risks.
Our analysis indicates a clear association between OPBR and both GS and GA, but not with GP. Specifically, the incidence of OPBR was more pronounced in patients with GA (Group A) and those with GS alone (Group B). In contrast, those with GP, either combined with GS (Group C) or alone (Group D), showed a reduced prevalence of OPBR at 8.9% and 4.8% respectively, aligning closely with the 6.1% observed in healthy adults [16]. In addition, current literature doesn’t establish a correlation between OPBR and GP, suggesting that OPBR may not influence GP formation. Further examination of the clinical features across the four OPBR patient groups revealed a singular distinction: Group D patients had a decreased occurrence of gallbladder wall thickening compared to the other cohorts (p < 0.01). This observation dovetails with the variance observed across the entirety of the patient group. Chronic thickening of the gallbladder wall is recognized as an indicator of sustained gallbladder inflammation, which escalates gallbladder cancer risk [11, 17]. This inflammatory environment, driven by OPBR and GS, sets off a cycle of injury and repair in the gallbladder mucosa. This results in genetic mutations, such as in P53 and K-ras, along with DNA damage, cumulatively steering the tissue towards malignancy [3, 6, 11, 17]. Nonetheless, it’s pivotal to underscore that the malignant metamorphosis of GP is hypothesized to traverse a polyp-adenoma-adenocarcinoma continuum, distinctly segregating it from the inflammation-induced carcinogenesis of the gallbladder mucosa [12, 18]. Thus, it’s likely that OPBR isn’t pivotal in GP’s malignant progression.
In Groups A and B, both characterized by an elevated incidence of OPBR, we delineated the clinical disparities between patients diagnosed with OPBR and their respective controls. A multivariate regression analysis was subsequently performed on clinical variables that yielded a p-value < 0.1 during the univariate assessments. Within Group A, there was a pronounced inclination towards elevated ALP among individuals with OPBR. The multivariate regression data further elucidated that hyper-γ-GT and gallbladder sludge emerged as independent risk factors for OPBR in the patients with GA (Group A). Elevations in serum ALP and γ-GT levels are well-recognized indicators of bile duct obstruction and inflammation [19, 20]. Intriguingly, GA itself does not inherently cause biliary stasis or inflammation. However, the emergence of OPBR correlates with poor outflow from the pancreaticobiliary common channel and persistent irritative effects of PBR might instigate biliary epithelial inflammation, subsequently elevating serum ALP and γ-GT levels [5, 21, 22]. Although the precise pathophysiological mechanisms underpinning GA remain unclear, PBR has been hypothesized as a potential initiator in GA pathogenesis [13, 23, 24]. Historically, literature has primarily addressed the relationship between PBM and GA [13, 23, 24]. However, our findings extend this association to OPBR. Previously, we analyzed OPBR incidence across different GA subtypes, revealing a lower prevalence in fundal GA (10.4%, 5/48) compared with segmental (28.6%, 8/28) and diffuse GA (2/5), while also identifying OPBR as a significant risk factor specifically for segmental and diffuse forms of GA [25]. Based on these findings, we propose several plausible mechanisms by which OPBR may contribute to gallbladder adenomyomatosis. Impaired pancreaticobiliary outflow characteristic of OPBR may lead to biliary stasis and elevated gallbladder intraluminal pressure. Concurrently, sustained irritation from PBR, especially when accompanied by gallstones, can induce progressive gallbladder mucosal epithelial atrophy and muscular layer hypertrophy. Such pathological remodeling promotes mucosal invagination into the gallbladder wall, resulting in the formation of Rokitansky-Aschoff sinuses, ultimately facilitating the development of GA [13, 23].
In Group B, it was discerned that patients with OPBR were typically of a more advanced age and exhibited a notable inclination towards the presence of gallbladder sludge. Multivariate regression analyses revealed that the presence of gallbladder sludge and an age > 50 years were robust independent determinants for OPBR in subjects diagnosed with GS alone. Noteworthily, pathologies such as stenosing papillitis and periampullary diverticulum, known inducers of OPBR, are predominantly observed in middle-aged demographics, offering a plausible rationale for the heightened OPBR susceptibility post the age of 50 [5, 26–28]. Advancing age, progressive weakening of intestinal smooth muscle, and increased intraduodenal pressure may encourage outpouching of the duodenum at sites of these defects, leading to the formation of diverticula [26]. Serendipitously, both gallbladder malignancies and GS prevalence demonstrate an age-correlated ascent [11, 12]. OPBR could act as a catalyst in the formation of GS by initiating inflammatory cascades within the gallbladder and disrupting the molecular balance that maintains cholesterol solubility in bile [14, 29]. Additionally, although definitive evidence for GS as a direct cause of gallbladder cancer is lacking, GS have traditionally been regarded as an etiological factor based on findings from case–control and autopsy studies, which suggest that recurrent cycles of cell death and epithelial regeneration induce a constant inflammatory state [12, 17, 30, 31]. Gallbladder carcinogenesis in patients with PBR follows a multistage progression through a metaplasia–dysplasia–carcinoma sequence driven by chronic inflammation, similar to the pathway observed in gallbladder cancer related to GS [3, 4, 6, 17]. Therefore, the combined stimulation from PBR and GS may further facilitate gallbladder cancer development.
Significantly, gallbladder sludge emerged as a consistent independent risk factor for PBR in both Group A and B, with patients possessing gallbladder sludge presenting a striking 48.0% likelihood of developing OPBR. Gallbladder sludge, defined as a mixture of particulate debris suspended in bile, sometimes contains larger, distinct particles known as microlithiasis (1–3 mm) [32]. The onset of gallbladder sludge is influenced by an array of factors, including total parenteral nutrition, rapid weight loss, pregnancy, organ transplantation, and use of ceftriaxone or octreotide [32, 33]. Furthermore, PBR can exacerbate gallbladder sludge formation by inducing chronic inflammatory damage to the gallbladder mucosa, thus accelerating cholesterol crystallization [14]. This suggests that gallbladder sludge could be the precursor stage of GS spurred by OPBR. For gallbladder sludge formation, we believe the combined effects of the following mechanisms occur: pancreatic juice reflux into the gallbladder hydrolyzes bile phospholipids, disrupting the balance among bile salts, cholesterol, and phospholipids [3, 14]. This disruption reduces lipid solubility and results in cholesterol supersaturation. Additionally, pancreatic juice and its hydrolysis products, such as lysophosphatidylcholine, free fatty acids, and various inflammatory factors, cause mucosal inflammation and damage, impairing the gallbladder epithelium’s lipid absorption capacity [14, 21, 34]. This ongoing inflammatory damage can induce gallbladder hypomotility, promote mucin production, and enhance secretion, thus facilitating cholesterol crystal nucleation and growth, ultimately forming sludge and stones [14, 34].
Limitations
Several limitations of our study should be acknowledged. First, as a single-center investigation of cholecystectomy patients with benign gallbladder disease, referral and selection biases may limit the generalizability of our findings. Second, because the incidence of gallbladder cancer at our center is low, we excluded cancer cases and were therefore unable to compare OPBR incidence or bile amylase levels between benign and malignant conditions. Third, OPBR diagnosis depended on a single preoperative MRCP and one-time bile amylase measurement, which cannot capture temporal fluctuations in reflux or sphincter of Oddi function. Finally, follow-up data on long-term outcomes—such as biliary malignancy development or sludge recurrence—were not yet available. Future multicenter, prospective studies with inclusion of gallbladder cancer cases and extended serial assessments will be essential to validate and expand upon these results.
Conclusion
In summary, our findings indicate no association between GP and OPBR, regardless of the presence of GS. However, there’s a marked need to be vigilant about the potential onset of OPBR in GA patients who manifest elevations in serum ALP and γ-GT, as well as in patients with GS alone who are aged above 50 years. Significantly, our study reveals gallbladder sludge as a predominant and consistent risk factor for OPBR in both GS-alone and GA patients, with OPBR observed in 48.0% of individuals with gallbladder sludge.
Acknowledgements
We are grateful to the laboratory technicians for their participation and contributions.
Abbreviations
- ALP
Alkaline phosphatase
- BMI
Body mass index
- CI
Confidence interval
- ERCP
Endoscopic retrograde cholangiopancreatography
- GA
Gallbladder adenomyomatosis
- GP
Gallbladder polyp
- GS
Gallbladder stone
- γ-GT
Gamma-glutamyl transpeptidase
- IQR
Interquartile range
- MRCP
Magnetic resonance cholangiopancreatography
- NPBJ
Normal pancreaticobiliary junction
- OR
Odds ratio
- OPBR
Occult pancreaticobiliary reflux
- PBM
Pancreaticobiliary maljunction
- PBR
Pancreaticobiliary reflux
- PTC
Percutaneous transhepatic cholangiography
- SOD
Sphincter of Oddi dysfunction
Authors’ contributions
Yukai Xiang, Cheng Zhang, and Yulong Yang contributed to the conception and design of the study, prepared figures and tables, and drafted the manuscript. Chen Qiu, Hai Hu, Jingli Cai, Gang Zhao, Anhua Huang, Anan Xu, and Chuanqi He performed data collection and sample analysis. Zhaoyan Jiang critically revised and finalized the manuscript. All authors reviewed and approved the final version of the manuscript.
Funding
This study was funded by Key Specialty Construction Project of Shanghai Pudong New Area Health Commission (Grant No. PWZzk2022-17); Shanghai East Hospital Clinical Research Project (Grant No. DFLC2022019); the Featured Clinical Discipline Project of Shanghai Pudong District (Grant No. PWYts2021-06); Shanghai East Hospital Research Initiation Fund (Grant No. DFRC2018014).
Data availability
The datasets used and/or analyzed during the current study are not publicly available due to patient privacy and institutional data protection policies, but are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki, and approved by Shanghai East Hospital Ethics Committee (approval No. 2019tjdx46). Informed consent was obtained from all subjects involved in the study.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Contributor Information
Yulong Yang, Email: yyl516@tongji.edu.cn.
Zhaoyan Jiang, Email: jiangzhaoyan@tongji.edu.cn.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are not publicly available due to patient privacy and institutional data protection policies, but are available from the corresponding author upon reasonable request.