Author Affiliations: Department of Hepatobiliary Surgery, West China Hospital of Sichuan University, Chengdu 610041, China (Yang Q, Zhou Y, Li FY, Mao H, Shrestha A, Ma WJ and Cheng NS); Department of Medicine, The George Washington University, Washington, District of Columbia, USA (Zhang W)

Corresponding Author: Hui Mao, MD, Department of Hepatobiliary Surgery, West China Hospital of Sichuan University, Chengdu 610041, China (Tel: +86-28-85422465; Fax: +86-28-85422468; Email: lfy_74@vip.163.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60395-2

Published online July 2, 2015.

 

 

Contributors: LFY and MH designed the research. YQ, SA and MWJ performed the histological observation and biochemistry. ZY, LFY and CNS performed the operations. ZY and MH performed Western blotting. YQ and ZY contributed equally to this paper. LFY is the guarantor.

Funding: This study was supported by grants from the National Nature Science Foundation of China (30801111 and 30972923); Science & Technology Support Project of Sichuan Province (10SZ0166, 14ZC1337 and 14ZC1335).

Ethical approval: The study was approved by the Ethical Committee of Sichuan University and informed consent was obtained from the patients.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: There is currently no effective medication to prevent stone recurrence after choledochoscopic lithotomy or to treat proliferative cholangitis (PC), which is the pathologic basis of hepatolithiasis. This study aimed to investigate whether gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, inhibited cholangio hyperplasia and lithogenesis in PC.

 

METHODS: After cholangioscopic lithotomy, indwelling catheters were placed in the diseased bile duct lumens in 94 patients with hepatolithiasis. Subsequently, 49 of the 94 patients were treated with 250 mg gefitinib solution via a catheter twice a week, and they were subjected to choledochoscopic biopsy at 6 and 12 weeks. The rest 45 hepatolithiasis patients without gefitinib treatment served as controls.

 

RESULTS: The expressions of EGFR, PCNA and procollagen I were significantly reduced in the patients treated with gefitinib in 12 weeks compared with those in the control group. Patients in the gefitinib group had a much lower degree of hyperplasia of the biliary epithelium, submucosal glands and collagen fibers compared with those in the control group. Gefitinib treatment significantly decreased mucin 3 expression and β-glucuronidase activity.

 

CONCLUSION: Postoperative gefitinib treatment could significantly inhibit PC-mediated hyperplasia and lithogenesis, which might provide a novel strategy for the prevention of biliary restenosis and stone recurrence in patients with hepatolithiasis.

 

(Hepatobiliary Pancreat Dis Int 2015;14:509-515)

 

KEY WORDS: hepatolithiasis; proliferative cholangitis; epidermal growth factor receptor blockade; recurrence; restenosis; prevention

Hepatolithiasis is recognized as an intractable disease for its high rate of recurrence and postoperative biliary restenosis. Despite the development of numerous surgical and nonsurgical procedures for hepatolithiasis, the treatment of hepatolithiasis is still intractable.^{[1, 2]} The application of choledochoscopy has partly improved the treatment of hepatolithiasis, but 33.9% of the calculi could be complicated with stenosis of secondary or even smaller subsidiary biliary ducts. These calculi are not easy to be removed completely, and have a recurrence rate of 4.2%-40.0% and a reoperation rate of 37.1%-74.4% in 4-10 years after operation.^[1-3] In fact, approximately two-thirds patients with hepatolithiasis in Asia require further surgery because of biliary stricture, stone recurrence, bile duct infection and other refractory factors. Indeed, as the frequency of stone recurrence increases, biliary stricture also turns to be more severe. This is associated with a gradual worsening of the patient's overall health and an increased risk of subsequent surgery. Such a patient is only treated by endoscopic cholelithotomy, and the recurrence of intrahepatic stone is almost inevitable.^[4] The prevention of stone recurrence is therefore very important.

 

Despite many theories concerning hepatic lithogenesis, the most common pathologic features of the stone-containing bile duct is chronic proliferative cholangitis (PC), which facilitates new stone formation via the production of muco-glycoprotein while inducing biliary stricture and cholestasis.^[5-7] Therefore, many investigations have been conducted to manage hepatolithiasis by a combined strategy including stone removal, correction of biliary tract strictures, and control of residual PC. The latter approach in particular has been traditionally ignored. The subsequent treatment of residual PC after choledochoscopic lithotomy might help to reduce stone recurrence or the rate of biliary restenosis.^[7-11] Unfortunately, there is no definitive therapy for PC at present.^{[5, 8, 12]}

 

Epidermal growth factor receptor (EGFR) is one of the receptors that are critical to cellular proliferation, differentiation and survival. Recently, gefitinib, an EGFR inhibitor, has been widely studied as a novel therapeutic agent to suppress epithelium hyperplasia, mucus accumulation and collagen deposition in inflammatory airway disease.^{[13, 14]} Since PC is also a chronic proliferative disease associated with mucin hypersecretion and fibrous tissue hyperplasia, this study was undertaken to test whether EGFR blockade with gefitinib had therapeutic effects on cholangio hyperplasia and lithogenesis associated with PC.^[15-17] The study was approved by Ethical Committee of Sichuan University and informed consent was obtained from the patients.

 

 

From January 2008 to May 2013, 94 patients aged from 25 to 67 years (mean 42.2) were treated for bilateral intrahepatic calculi. Among them, 45 patients had a history of biliary tract surgery (mean 3.6 times, range 2-6) (Table).

 

All the 94 patients underwent intraoperative choledochoscopic lithotomy; biopsies were performed at the intrahepatic site of PC. An indwelling catheter was placed into the bile duct lumen and threaded out of the abdomen through a T-tube. In this series, 49 patients were treated with gefitinib. Two hundred and fifty mg gefitinib (AstraZeneca, London, UK) dissolved in 250 mL of saline was administrated via a catheter twice a week for 12 weeks after operation. At 6 and 12 weeks after choledochoscopic electrohydraulic lithotripsy and lithotomy, choledochoscopic biopsy was performed at the targeted bile duct wall (2-3 samples per time) where the inflammation was most prominent. The rest 45 patients with bilateral intrahepatic calculi served as a control group. They only underwent traditional choledochoscopic lithotomy and biopsy without gefitinib therapy. There were no significant differences in age, stone distribution, and history of biliary tract surgery between the two groups. Histological sections of biopsy samples from the two groups were stained with hematoxylin and eosin (HE), Masson's trichrome, or periodic acid-Schiff and alcian blue (PAS/AB). Two pathologists who were masked to the study observed the slides.

 

Tissue sections were incubated overnight at 4 �� with the anti-EGFR or anti-Ki-67 primary antibody (Zymed Co., USA), followed by incubation with biotinylated secondary antibody for one hour at 37 ��, and then with avidin-biotin complex at 37 �� for 20 minutes, and finally diaminobenzidine was added for color development. To analyze the proliferation status, the proliferative label index of Ki-67 was determined by a computer-assisted automated image analyzer (Image Pro Plus), which examined 5 random fields per slide and calculated the ratio of the positive cell number to the total cell number.

 

Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA, USA); the cDNA was reverse transcripted using the two-step qRT-PCR kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Quantitative real-time PCR was done using an iCycler PCR system (Biorad, Munich, Germany). Fold changes were calculated after normalization to endogenous GAPDH using the comparative C_t method.

 

Using phenolphthalein-β-glucuronide (Sigma, St Louis, MO, USA) as the substrate, we used the modified Fisherman method to quantitatively measure β-glucuronidase activity, which was expressed in Fisherman units (U/mg).^[18]

 

Statistical analysis was conducted using the SPSS 10.0 (SPSS Inc., Chicago, IL, USA) software, and parametric data were compared with Student's t test. One-way analysis of variance was used for comparisons of means, and Fisher's least significant difference analysis was used for within group comparisons. A P<0.05 was considered statistically significant.

 

 

As revealed by intraoperative choledochoscopy and subsequent histology examination, all of the diseased bile ducts in hepatolithiasis patients exhibited clear signs of PC, including submucosal gland hyperplasia, papillary hyperplasia in biliary mucosa, fibrous thickening in the biliary duct wall and flocculent precipitate in the biliary lumen (Figs. 1A and 2A). Further examination using PAS/AB and Masson's staining showed a marked increase in mucoprotein expression in both the hyperplastic submucosal glands and mucosal epithelia, and the obvious proliferation of collagen fibers in the thickened duct walls. Similar histological changes were observed in the control group. In contrast, the degree of proliferation of the biliary epithelium collagen fibers and mucoprotein expression within submucosal glands were significantly reduced in the bile ducts after 6-12 weeks of gefitinib treatment compared to those in the control group. Twelve weeks after operation, the bile ducts in the gefitinib group exhibited only a mild degree of or no PC on direct visualization during choledochoscopy or microscopic examination (Figs. 1B, 1C, 2C, 2E, 3C and 3F).

 

To confirm the anti-proliferative effects of gefitinib on PC, we examined the mRNA levels or protein expressions of EGFR, Ki-67 and PCNA in the diseased bile duct. We found that the basic EGFR and PCNA mRNA expressions in the intraoperative biopsy sample of both gefitinib and control groups were significantly increased (P<0.05). After 6 weeks, the decrease of PCNA and EGFR mRNA expressions was observed in the gefitinib (G-0 vs G-6W, P<0.05) and control groups (C-0 vs C-6W, P<0.05) respectively, but there was no significant difference between the two groups. After 12 weeks, the expression of PCNA and EGFR mRNA further decreased in the gefitinib (G-6W vs G-12W, P<0.05) and control groups (C-6W vs C-12W, P<0.05), and a statistically significant reduction was observed between the two groups (G-12W vs C-12W, P<0.05) (Figs. 4 and 5). Additionally, a remarkable reduction of Ki-67 expoession was observed in the gefitinib group (Fig. 6).

 

To determine the effect of gefitinib on mucin production and collagen fiber proliferation in PC, we examined mucin 3 and procollagen I mRNA levels in the diseased bile duct, and found a significantly increased expression of both genes in the intraoperative biopsy samples in the gefitinib and control groups. However, after 6 weeks of gefitinib treatment, the expressions of mucin 3 and procollagen I mRNA were significantly decreased in the gefitinib group compared with those in the control group (C-0 vs C-6W, P<0.05; G-0 vs G-6W, P<0.05). The differences reached to a significant level at 12 weeks after gefitinib treatment (G-12W vs C-12W, P<0.05) (Fig. 5).

 

To evaluate the influence of gefitinib on the lithogenesis in PC, we examined the activity of endogenous β-glucuronidase in the diseased bile duct. The intraoperative biopsy samples of the bile duct displayed very high levels of β-glucuronidase activity, which decreased significantly after 6-12 weeks of gefitinib treatment. In addition, the β-glucuronidase activity in the diseased bile duct after 6-12 weeks of gefitinib treatment tended to be slightly lower than that in the control group, but the difference was not statistically significant (P>0.05) (Fig. 7).

 

 

High postoperative recurrence rate of hepatolithiasis is mainly due to the absence of an effective method to prevent stone recurrence after choledochoscopic lithotomy or to treat PC.^[2-6] We investigated whether gefitinib could inhibit hyperplasia and lithogenesis in PC and found that postoperative gefitinib treatment significantly inhibited hyperplasia and lithogenesis in PC, suggesting a novel therapeutic strategy for the prevention of biliary restenosis and stone recurrence in patients with hepatolithiasis. We also found that gefitinib could efficiently inhibit the mRNA or protein expression of genes related to proliferation, including EGFR and PCNA. As a result, the proliferative degree of biliary epithelium, submucosal gland, and collagen fiber were much lower in the bile ducts after 6-12 weeks of gefitinib treatment than those in the control group. In addition, some bile ducts in the gefitinib group had almost no histopathological evidence of PC. There were two pathways for gefitinib: the anti-proliferative pathway and the pro-apoptotic pathway.^[19,20] Initial biopsy was performed at the site where PC was the most prominent. Therefore, this site should be the best spot for the investigation of the anti-proliferative effects of gefitinib on PC. In our study, postoperative long-term gefitinib treatment controlled PC postoperatively. The inhibition of hyperplasia of the ductal epithelium in patients with PC would possibly reduce the incidence of biliary restenosis. However, long-term follow-up is required to confirm this finding.

 

Hyperplasia of collagen fibers in PC and the hyper-secretion of mucin from PC play an important role in the pathogenesis of biliary strictures and intrahepatic stones, but there are no effective therapies at present. EGFR regulates mucin and collagen productions, while the blockage of EGFR signaling pathways by gefitinib has also been used as a novel therapeutic strategy for suppressing mucin synthesis in hypersecretory diseases such as asthma as well as the development of pulmonary fibrosis, renal fibrosis and vascular stricture by inhibiting abnormal formation of extracellular matrix through a MAPK-mediated mechanism.^{[14-16, 21-27]} These findings suggested that gefitinib inhibits the production of mucin and collagen in PC. We found that gefitinib significantly inhibited the EGFR expression and production of mucin 3 and procollagen I in the bile duct wall. Gefinitb therefore produces anti-fibrotic effects via the inhibition of collagen fiber proliferation. These inhibitory effects would also possibly reduce the incidence of stone recurrence and biliary restenosis secondary to PC.

 

In our study, the expressions of EGFR, PCNA, mucin 3 and procollagen I reduced in some degree in the control group. This reduction may be due to stone removal and biliary drainage, which relieved the infection of the biliary tract. However, clinical experience has shown that T-tube drainage alone can, to some extent, improve the degree of biliary tract infection but cannot eradicate PC and prevent PC-mediated stone recurrence. In our study, however, postoperative gefitinib treatment significantly inhibited PC, especially after 12 weeks. In some cases, gefitinib cured PC and, therefore, prevented stone recurrence.

 

Considering the dominant role of β-glucuronidase in the formation of pigment stones, we further evaluated the effect of gefitinib on the lithogenesis of PC by assessing endogenous β-glucuronidase activity.^[16,28] The chronic inflammatory stimuli or cellular hyper-proliferation in PC resulted in high-level endogenous β-glucuronidase activity. After 6-12 weeks of gefitinib treatment, the endogenous β-glucuronidase activity decreased by 50%. This was due to the inhibitory effect of gefitinib on the biliary epithelium and submucosal gland proliferation, which decreased the amount of endogenous β-glucuronidase secreted from the diseased bile ducts. This effect of gefitinib on β-glucuronidase activity may also help to prevent recurrent stones.^{[3, 4, 29-31]}

 

In conclusion, PC-mediated hyperplasia and lithogenesis can be effectively inhibited by gefitinib as an EGFR inhibitor. Our results suggest the clinical application of gefitinib is feasible. Since cholangioscopic lithotomy or resection of the diseased hepatic segment is still the first treatment of choice, gefitinib might be valuable in clinical practice. However, well-controlled clinical studies and long-term follow-up are needed to validate its long-term efficacy, related complications and dosage before clinical application.

 

 

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