Qin Yang, Yong Zhou, Fu-Yu Li, Hui Mao, Anuj Shrestha, Wen-Jie Ma, Nan-Sheng Cheng and Wei Zhang

Chengdu, China

Effects of epidermal growth factor receptor inhibitor on proliferative cholangitis in hepatolithiasis

Qin Yang, Yong Zhou, Fu-Yu Li, Hui Mao, Anuj Shrestha, Wen-Jie Ma, Nan-Sheng Cheng and Wei Zhang

Chengdu, China

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)

hepatolithiasis;

proliferative cholangitis;

epidermal growth factor receptor blockade;

recurrence;

restenosis;

prevention

Introduction

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 stonecontaining bile duct is chronic proliferative cholangitis (PC), which facilitates new stone formation via the production of muco-glycoprotein while inducing biliarystricture 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.

Methods

Patients and surgical procedure

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.

Table. Patients' demographics

Immunohistochemistry staining of EGFR and Ki-67

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.

Real-time PCR detection of EGFR, PCNA, mucin 3 and collagen I

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 Ctmethod.

Biochemical determination of endogenous β-glucuronidase

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

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.

Results

Choledochoscopic and histological examination

Fig. 1. Choledochoscope before treatment (A), 6 weeks (B) and 12 weeks (C) after gefitinib treatment. Chronic proliferative cholangitis with a lot of flocculent precipitate in the ductal lumen (A), which was obviously cleared after the 6-12 weeks of gefitinib treatment (B and C).

Fig. 2. HE staining of the biliary duct (original magnification ×200). The histology before treatment: gefitinib group (A) and control group (B); C and D were from the gefitinib group and control group at 6 weeks post surgery; E and F were from the gefitinib group and control group at 12 weeks post surgery.

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).

EGFR, Ki-67 and PCNA expression

Fig. 3. Gefitinib decreased hyperplasia of biliary epithelium and collagen fiber and mucoprotein accumulation within submucosal gland (original magnification ×400). A-C: PAS/AB staining; D-F: Masson's staining. A and D: before treatment; B and E: the control group at 12 weeks post surgery; C and F: the gefitinib group at 12 weeks post surgery.

Fig. 4. Immunohistochemistry (original magnification ×400) and RT-PCR analysis of EGFR. A: before gefitinib treatment; B: the gefitinib group at 6 weeks post surgery; C: the gefitinib group at 12 weeks post surgery; D: RT-PCR analysis. C: control; G: gefitinib. C-0 and G-0: before surgery; C-6W and G-6W: 6 weeks after surgery; C-12W and G12W: 12 weeks after surgery. *: P＜0.05, compared with baseline; #: P＜0.05, compared with the control group.

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).

Mucin 3 and procollagen I expression

Fig. 5. RT-PCR analysis of PCNA (A), mucin 3 (B) and procollagen I (C) expression in biliary duct wall. C-0 and G-0: before treatment; C-6W and G-6W: 6 weeks post surgery; C-12W and G-12W: 12 weeks post surgery. *: P＜0.05, versus gefitinib group or compared with baseline; #: P＜0.05, compared with the control group.

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 controlgroup (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).

Fig. 6. Immunohistochemistry analysis of Ki-67 (original magnification ×400). A: before gefitinib treatment; B: the control group at 12 weeks post surgery; C: the gefitinib group at 12 weeks post surgery.

Fig. 7. Endogenous β-glucuronidase activity in the bile duct wall. *: P＜0.05, versus the gefitinib group or control group before treatment.

Endogenous β-glucuronidase activity determination

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).

Discussion

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 antiproliferative 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, longterm follow-up is required to confirm this finding.

Hyperplasia of collagen fibers in PC and the hypersecretion 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 PCand 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 hyperproliferation 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.

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.

References

1 Al-Sukhni W, Gallinger S, Pratzer A, Wei A, Ho CS, Kortan P, et al. Recurrent pyogenic cholangitis with hepatolithiasis--the role of surgical therapy in North America. J Gastrointest Surg 2008;12:496-503.

2 Uchiyama K, Onishi H, Tani M, Kinoshita H, Ueno M, Yamaue H. Indication and procedure for treatment of hepatolithiasis. Arch Surg 2002;137:149-153.

3 Li FY, Cheng NS, Mao H, Jiang LS, Cheng JQ, Li QS, et al. Significance of controlling chronic proliferative cholangitis in the treatment of hepatolithiasis. World J Surg 2009;33:2155-2160.

4 Chen XG, Liu JQ, Peng MH, Wang WG, Yang DH, Hu DH, et al. Clinical epidemiological study on intrahepatic cholelithiasis: analysis of 8585 cases. Hepatobiliary Pancreat Dis Int 2003;2:281-284.

5 Li FY, Jiang LS, de Jong MC, Cheng NS, Cheng JQ, Li N, et al. Clinical prospect of applying the chemical bile duct embolization to achieve a chemical hepatectomy in the treatment of highly selected hepatolithiasis. Surg Laparosc Endosc Percutan Tech 2009;19:183-189.

6 Terada T, Nakanuma Y. Pathologic observations of intrahepatic peribiliary glands in 1000 consecutive autopsy livers: IV. Hyperplasia of intramural and extramural glands. Hum Pathol 1992;23:483-490.

7 Lu S, Yan L, Rao L, Xia T, Gou J, Zhang S, et al. Down stream involvement of the bile duct in hepatolithiasis. Chin Med J (Engl) 2002;115:62-64.

8 Park SM, Choi JW, Kim ST, Cho MC, Sung RH, Jang LC, et al. Suppression of proliferative cholangitis in a rat model by local delivery of paclitaxel. J Hepatobiliary Pancreat Surg 2003;10: 176-182.

9 Li F, Zhou Y, Cheng N, Mao H, Jiang L, Li N, et al. Epidermal growth factor receptor as a target for anti-proliferative treatment of proliferative cholangitis in hepatolithiasis. J Surg Res 2011;166:87-94.

10 Terao R, Honda K, Hatano E, Uehara T, Yamamoto M, Yamaoka Y. Suppression of proliferative cholangitis in a rat model with direct adenovirus-mediated retinoblastoma gene transfer to the biliary tract. Hepatology 1998;28:605-612.

11 Yoshida M, Yamamoto N, Nitta T, Uehara T, Terao R, Hatano E, et al. Suppression of proliferative cholangitis by E2F decoy oligodeoxynucleotide. J Surg Res 2002;102:95-101.

12 Hwang JH, Yoon YB, Kim YT, Cheon JH, Jeong JB. Risk factors for recurrent cholangitis after initial hepatolithiasis treatment. J Clin Gastroenterol 2004;38:364-367.

13 Deshmukh HS, Case LM, Wesselkamper SC, Borchers MT, Martin LD, Shertzer HG, et al. Metalloproteinases mediate mucin 5AC expression by epidermal growth factor receptor activation. Am J Respir Crit Care Med 2005;171:305-314.

14 Ishii Y, Fujimoto S, Fukuda T. Gefitinib prevents bleomycininduced lung fibrosis in mice. Am J Respir Crit Care Med 2006;174:550-556.

15 Hur GY, Lee SY, Lee SH, Kim SJ, Lee KJ, Jung JY, et al. Potential use of an anticancer drug gefinitib, an EGFR inhibitor, on allergic airway inflammation. Exp Mol Med 2007;39:367-375.

16 Sato Y, Harada K, Furubo S, Kizawa K, Sanzen T, Yasoshima M, et al. Inhibition of intrahepatic bile duct dilation of the polycystic kidney rat with a novel tyrosine kinase inhibitor gefitinib. Am J Pathol 2006;169:1238-1250.

17 Yamamoto K. Intrahepatic periductal glands and their significance in primary intrahepatic lithiasis. Jpn J Surg 1982;12:163-170.

18 Li N, Xiao LJ, Chen SW, Li L, Xiao BL, Chen WB, et al. Mucus histochemical study of bilirubin cholangiolithiasis in rabbit model. Hua Xi Yi Ke Da Xue Xue Bao 1989;20:417-420.

19 Ishii M, Vroman B, LaRusso NF. Morphologic demonstration of receptor-mediated endocytosis of epidermal growth factor by isolated bile duct epithelial cells. Gastroenterology 1990;98: 1284-1291.

20 Miyata H, Sasaki T, Kuwahara K, Serikawa M, Chayama K. The effects of ZD1839 (Iressa), a highly selective EGFR tyrosine kinase inhibitor, as a radiosensitiser in bile duct carcinoma cell lines. Int J Oncol 2006;28:915-921.

21 Burgel PR, Nadel JA. Roles of epidermal growth factor receptor activation in epithelial cell repair and mucin production in airway epithelium. Thorax 2004;59:992-996.

22 Tamaoka M, Hassan M, McGovern T, Ramos-Barbón D, Jo T, Yoshizawa Y, et al. The epidermal growth factor receptor mediates allergic airway remodelling in the rat. Eur Respir J 2008;32:1213-1223.

23 Takeyama K, Tamaoki J, Kondo M, Isono K, Nagai A. Role of epidermal growth factor receptor in maintaining airway goblet cell hyperplasia in rats sensitized to allergen. Clin Exp Allergy 2008;38:857-865.

24 Fran?ois H, Placier S, Flamant M, Tharaux PL, Chansel D, Dussaule JC, et al. Prevention of renal vascular and glomerular fibrosis by epidermal growth factor receptor inhibition. FASEB J 2004;18:926-928.

25 Purdom S, Chen QM. Epidermal growth factor receptor-dependent and -independent pathways in hydrogen peroxide-induced mitogen-activated protein kinase activation in cardiomyocytes and heart fibroblasts. J Pharmacol Exp Ther 2005;312: 1179-1186.

26 Song JS, Hyun SW, Lillihoj E, Kim BT. Mucin secretion in the rat tracheal epithelial cells by epidermal growth factor and Pseudomonas aeruginosa extracts. Korean J Intern Med 2001;16:167-172.

27 Hardie WD, Davidson C, Ikegami M, Leikauf GD, Le Cras TD, Prestridge A, et al. EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 2008;294: L1217-1225.

28 Flamant M, Tharaux PL, Placier S, Henrion D, Coffman T, Chatziantoniou C, et al. Epidermal growth factor receptor trans-activation mediates the tonic and fibrogenic effects of endothelin in the aortic wall of transgenic mice. FASEB J 2003; 17:327-329.

29 Chen CY, Shiesh SC, Tsao HC, Lin XZ. Human biliary betaglucuronidase activity before and after relief of bile duct obstruction: is it the major role in the formation of pigment gallstones? J Gastroenterol Hepatol 2000;15:1071-1075.

30 Boonyapisit ST, Trotman BW, Ostrow JD. Unconjugated bilirubin, and the hydrolysis of conjugated bilirubin, in gallbladder bile of patients with cholelithiasis. Gastroenterology 1978;74: 70-74.

31 Kato T, Nishio K. Clinical aspects of epidermal growth factor receptor inhibitors: benefit and risk. Respirology 2006;11:693-698.

Accepted after revision March 22, 2015

March 13, 2014