Xanthogranulomatous cholecystitis: a premalignant condition?

New Delhi, India

Xanthogranulomatous cholecystitis: a premalignant condition?

Mila Ghosh, Puja Sakhuja and Anil K Agarwal

New Delhi, India

BACKGROUND: Xanthogranulomatous cholecystitis (XGC) is an uncommon variant of chronic cholecystitis, characterized by marked thickening of the gallbladder wall and dense local adhesions. It often mimics a gallbladder carcinoma (GBC), and may coexist with GBC, leading to a diagnostic dilemma. Furthermore, the premalignant nature of this entity is not known. This study was undertaken to assess the p53, PCNA and beta-catenin expression in XGC in comparison to GBC and chronic inflammation.

METHODS: Sections from paraffin-embedded blocks of surgically resected specimens of GBC (69 cases), XGC (65), chronic cholecystitis (18) and control gallbladder (10) were stained with the monoclonal antibodies to p53 and PCNA, and a polyclonal antibody to beta-catenin. p53 expression was scored as the percentage of nuclei stained. PCNA expression was scored as the product of the percentage of nuclei stained and the intensity of the staining (1-3). A cutoff value of 80 for this score was taken as a positive result. Beta-catenin expression was scored as type of expressionmembranous, cytoplasmic or nuclear staining.

RESULTS: p53 mutation was positive in 52% of GBC cases and 3% of XGC, but was not expressed in chronic cholecystitis and control gallbladders. p53 expression was lower in XGC than in GBC (P＜0.0001). PCNA expression was seen in 65% of GBC cases and 11% of XGC, but not in chronic cholecystitis and control gallbladders. PCNA expression was higher in GBC than XGC (P=0.0001), but there was no significant difference between the XGC, chronic cholecystitis and control gallbladder groups. Beta-catenin expression was positive in the GBC, XGC, chronic cholecystitis and control gallbladder groups. But the expression pattern in XGC, chronic cholecystitis and control gallbladders was homogenously membranous, whereas in GBC the membranous expression pattern was altered to cytoplasmic and nuclear.

CONCLUSION: The expression of p53, PCNA and beta-catenin in XGC was significantly different from GBC and similar to chronic cholecystitis, thus indicating the inflammatory nature of XGC and may not support a premalignant nature of the lesion.

(Hepatobiliary Pancreat Dis Int 2011; 10: 179-184)

xanthogranulomatous cholecystitis; gallbladder cancer; p53; PCNA; beta-catenin

Introduction

Xanthogranulomatous cholecystitis (XGC) is a rare presentation of chronic cholecystitis, characterized by xanthogranuloma, severe fibrosis and foam cells. The imaging of a mass-forming XGC usually mimics gallbladder cancer (GBC) and can pose a diagnostic challenge/dilemma. Patients with XGC are frequently misdiagnosed intraoperatively as having GBC and are treated with extensive excision.[1-3]It can mimic stage IVA GBC,[4]in the form of a mass lesion, originating from the gallbladder and extending up to the liver parenchyma, with associated enlargement of nodes in the hepatoduodenal ligament. Akin to GBC, the mass may involve the liver hilum, right lobe of the liver, right colonic flexure, and duodenum.[5]Cases may present with high CA19-9 levels and radiological findings suggestive of metastatic gallbladder carcinoma; however, the elevated CA19-9 returns to normal postoperatively.[6]Definite diagnosis depends exclusively on pathological examination. Often pathologists are also in dilemma due to severe inflammatory atypia. Not only does XGC occasionally present as a mass formation with adjacent organ invasion like a malignant neoplasm, it is also infrequently associated with GBC.[7-10]As XGC may occasionally coexist with GBC,[11]the association with cancer is a controversial issue.[12]Many studies have demonstrated an association of gallstones with GBC.[13]But with more inflammation and mass formation, the role of XGC in GBC has not been much studied.

Most studies have stressed the role of oncogene activation and tumor suppressor gene inactivation in promoting aberrant tumor-cell proliferation. p53 has an independent role in GBC. Beta-catenin is a key regulator of the cadherin-mediated cell adhesion system. Altered expression of these adhesion molecules is involved in the progression and metastasis of GBCs. Proliferative activity is an important indicator of the growth and behavior of various human tumors. Immunostaining of tissue sections with proliferating cell nuclear antigen (PCNA) antibodies appears to be reliable in the assessment of tumor cell proliferation.

To understand the malignant potential of XGC if any, we studied the immunohistochemical expression of p53, PCNA and beta-catenin in surgically resected specimens of GBC, XGC, chronic cholecystitis and control gallbladders.

Methods

Tissue specimens

We retrospectively examined sections from surgically resected specimens from 69 cases of GBC, 102 of XGC (37 discarded as no viable epithelium was identified for further immunostaining), 18 of chronic cholecystitis with gallstone disease and 10 control gallbladder specimens removed as part of another surgical procedure with no gallbladder pathology, such as in Whipple's resection in cancer of the head of the pancreas or periampullary carcinoma. This study was conducted in GB Pant Hospital, a tertiary referral hospital, Maulana Azad Medical College, New Delhi, India.

Antibodies

Immunohistochemical staining was performed with monoclonal antibody p53 (DakoCytomation), monoclonal antibody PCNA (Diagnostic Biosystem) and beta-catenin (Santa Cruz Biotechnology) in all cases.

Immunohistochemistry

Sections 4 μm thick were cut from paraffin-embedded tissues from cases of GBC, chronic cholecystitis and XGC, and from control gallbladders. The sections were placed on polyL-lysine-coated slides and deparaffinized, using the standard protocol. Antigen retrieval was performed in citrate buffer at pH 6 in a microwave for 2 minutes at half power and 2 minutes at full power for both p53 and PCNA antibody, while for beta-catenin it was done at full power for 15 minutes. After washing with Tris buffer, primary antibodies were applied against p53 and PCNA from a ready-to-use solution. Primary antibody of betacatenin was diluted at 1∶200. A negative control was made by replacing primary antibody with Tris buffer while sections from breast and colonic cancer were used as positive controls. After the application of primary antibodies, slides were incubated overnight in a humid chamber and subsequently washed in three changes of Tris buffer for 30 minutes. Biotinylated secondary antibodies were applied for 1 hour in the same chamber at room temperature followed by washing with Tris buffer. Next, streptavidin peroxidase reagent was applied and incubated in the same chamber for 1 hour at room temperature. Finally, the reaction product was visualized by developing color using diaminobenzidine tetrahydrochloride. Sections were counterstained with hematoxylin, mounted in Crystal Mount, and coverslipped.

Evaluation of p53, PCNA and beta-catenin expression

The expression of p53, PCNA and beta-catenin in the epithelium was examined under a light microscope. In each case of XGC, chronic cholecystitis and control gallbladder, two sections were taken from areas with intact surface epithelium and high levels of inflammation. In GBC cases, one section from intact epithelium and one from an area of deeper invasion were used. Immunohistochemical scoring was performed in the areas of highest expression.

p53 expression was scored as the percentage of nuclei stained. p53 expression was judged positive when the nuclei of ＞5% cells were stained. The stained cell fraction was evaluated as grade I when 5%-30% of epithelial nuclei in one section were stained, grade II when 30%-60% of epithelial nuclei were stained, and grade III when ＞60% epithelial nuclei were stained. The results were scored semi-quantitatively and statistical analysis was performed.

To score PCNA expression, we devised a Quick score which was the product of the percentage of nuclei stained and the intensity of staining (1-3). Thus a maximum Quick score of 300 was obtainable. Since the highest PCNA score in our chronic cholecystitis cases was 80, a cut-off value of ＞80 for the Quick score was taken as a positive result.[14]

Beta-catenin expression was judged positive when＞5% of cells were stained. Staining was evaluated as membranous, cytoplasmic or nuclear.

Results

A total of 69 cases of GBC adenocarcinoma were included in the study. Twenty-seven cases were well differentiated, 32 moderately, and 10 poorly differentiated. Tumorinvasion was assessed by TNM staging--30 cases were in the T3 category where the tumor involves the adjacent liver, 28 were T2, where the tumor invades perimuscular connective tissue, but no extension beyond the serosa or into the liver, and 11 were T1, where the tumor is limited to the lamina propria or muscle layer.[15]XGC epithelium is often ulcerated and we had to search for areas with intact mucosal lining. The epithelium in XGC was predominantly flattened. No papillary pattern of the epithelium was present in XGC, whereas in chronic cholecystitis, simple papillary hyperplasia of the epithelium was often present. Four cases of GBC were associated with areas of XGC. Those cases were included with GBC. The results of immunohistochemistry are summarized in Table.

p53 expression

p53 staining was nuclear with increased expression in GBC (Fig. 1A). Its expression was positive in 52% (36/69) GBC cases. In XGC, expression was positive in 3% (2/65) (Fig. 1B). It was not found in chronic cholecystitis (0/18) (Fig. 1C) or in control gallbladders (0/10) (Fig. 1D). p53 expression occurred in fewer XGC cases than in GBC (P＜0.0001). In XGC, both cases were grade 1, but in GBC, 33% of positive cases were grade 1, 28% grade 2 and 39% grade 3.

PCNA expression

PCNA expression was nuclear with high Quick scores in GBC. Its expression in GBC was positive in 65% (45/69) (Fig. 2A). In 4 cases PCNA expression was greater in invading areas but very low to negative in the surface epithelium. In XGC, expression was positive in 11% (7/65) (Fig. 2B). In both chronic cholecystitis (Fig. 2C) and control gallbladders (Fig. 2D), PCNA expression was negative (Quick score ＜80). The median Quick score for PCNA expression in GBC was 180, and the median score was 60 in XGC and 27.5 in chronic cholecystitis.

PCNA expression was higher in GBC than XGC (P=0.0001) but there was no significant difference in PCNA expression among XGC, chronic cholecystitis and control gallbladders (Fig. 3).

Table. p53 and PCNA expression in gallbladder tissues

Fig. 1. Microphotographs of diseased and control gallbladder stained with immunoreactive p53 antibody. A: GBC with high p53 expression with nuclear staining (original magnification ×200); B: XGC with high p53 expression with no nuclear staining (original magnification ×200); C: Chronic cholecystitis with no p53 expression (original magnification ×200); D: Control gallbladder with no p53 expression (original magnification ×200).

Fig. 2. Microphotographs of diseased and control gallbladder stained with immunoreactive PCNA antibody (original magnification ×200). A: GBC with high PCNA expression by nuclear staining; B: XGC with focal PCNA expression with nuclear staining; C: Chronic cholecystitis with no PCNA expression with nuclear staining; D: Control gallbladder with no PCNA expression.

Fig. 4. Microphotographs of diseased and control gallbladder stained with immunoreactive beta-catenin antibody (original magnification ×200). A: GBC with high cytoplasmic and focal nuclear beta-catenin expression; B: XGC with focal beta catenin membranous and cytoplasmic expression; C: Chronic cholecystitis with focal beta-catenin membranous expression; D: Control gallbladder with focal beta catenin membranous expression-low intensity.

Beta-catenin expression

Beta-catenin expression was membranous, cytoplasmic or nuclear, with a nuclear pattern of staining only in GBC. Its expression was seen in all 69 cases of GBC. Expression was heterogeneous and strong in the tumor area; the expression pattern, though predominantly membranous, was also seen in the cytoplasm in 56/69 cases (81%), but it was relatively focal and paler, and showed a nuclear pattern in 65% (45/69) (Fig. 4A). In XGC, beta-catenin expression was seen in all 65 cases; the expression pattern was homogeneous and predominantly membranous with focal cytoplasmic expression (Fig. 4B). In 18 chronic cholecystitis (Fig. 4C) and 10 control gallbladder specimens (Fig. 4D), betacatenin expression was seen in all cases but expression pattern is homogeneous and membranous. No nuclear expression of beta-catenin was observed in XGC, chronic cholecystitis and control gallbladders.

Immunohistochemistry of XGC foci in GBC

The foci of XGC seen in four cases of GBC did not show p53 expression, the PCNA Quick score was ＜80 and beta-catenin expression was membranous. The carcinoma areas however showed grade 3 p53 expression in 2 cases, the PCNA Quick score was ＞80, and betacatenin was expressed in the nucleus in 3 cases.

Discussion

The malignant potential of XGC is controversial and highly disputed. Immunohistochemical study using an oncogenic protein, a tumor suppressant and a proliferation marker was done to assess this potential. The staining intensities and patterns of p53, PCNA and beta-catenin were studied.

An association of XGC with GBC has been reported.[7-10]In our study, 4/65 cases of XGC were associated with cancer, but the expression of p53, PCNA and beta-catenin in the areas showing XGC-like morphology were the same as in other cases of XGC. In the cancer area it was the same. In this study, p53 expression was increased in GBC compared to XGC. Many others have already reported p53 expression in GBC,[16-18]however there are only a few reports of p53 expression in XGC. Takada et al[12]reported results similar to ours with no p53 expression in XGC cases. In this study, p53 expression in XGC was noted in only 2 cases. The PCNA Quick scores in these cases were 0 and 180. Beta-catenin expression was only membranous in both. The p53 and PCNA overexpression in rare cases may be due to severe inflammation. Often, inflammatory conditions like ulcerative colitis also show increased p53 expression.[19]

PCNA expression was also not high in most XGC cases. PCNA expression with a Quick score ＞80 in 7 cases of XGC may be due to a high degree of inflammatory reaction and regenerative proliferation of epithelium. Expression of PCNA in GBC (median score 180) was significantly higher than in XGC (median score 60) but there was no difference in PCNA expression among XGC, chronic cholecystitis (27.5) and control gallbladders. These findings may not support the premalignant nature of XGC.

The expression of beta-catenin in XGC was predominantly membranous and thus similar to that in chronic cholecystitis and control gallbladders. No nuclear beta-catenin expression was noted in XGC, chronic cholecystitis or control gallbladders, unlike in GBC. This further supports the inflammatory nature of the lesion and may not support a neoplastic nature of XGC.

Our results indicate that XGC is an inflammatory condition. It is generally agreed that rupture and intramural extravasation of the inspissated bile and mucin from the occlusion of Rokitansky-Aschoff sinuses are the main causes of a xanthogranulomatous reaction in the gallbladder wall.[20,21]Another possible primary cause of XGC is chronic infection and calculi associated with bile stasis recurrent inflammation, and calculi provoke degeneration and necrosis of the gallbladder wall, with subsequent intramural abscess formation.[22]Regardless of the primary cause of XGC, the intramural abscesses are replaced by xanthogranulomas with foamy histiocytes and foreign body giant cells.

Severe XGC often mimics a GBC.[1-3]Guzman-Valdivia reported that a malignant lesion was suspected in 30% of cases and cancer was found in 3%.[23]Another report shows that 12% of cases had coexisting XGC and GBC.[24]Howard et al[25]reported 40% cases suspected as cancer. In some cases, the normal levels of tumor markers (like CA19-9 and DUPAN II) in all clinical courses might be a reason to consider XGC instead of GBC.[4]But cases are reported with high levels of tumor markers in XGC where the markers decreased after surgery.[26]More studies are required to find the reason for this.

Several inflammatory conditions have neoplastic potential on long-term follow-up. Ulcerative colitis has shown evidence of such neoplastic potential; foci of dysplasia and malignancy arising in this background are well documented. Such evidence is not available for XGC.

Currently, a correct pre-operative diagnosis is often not made. With increased awareness and a high index of suspicion, radiological diagnosis is possible. On histologic examination too, diagnostic difficulty can arise due to presence of deep Rockitansky Aschoff sinuses and inflammatory atypia in the epithelium. In such cases, combined immunohistochemical staining with p53, PCNA and beta-catenin can be of some help. In patients with no nuclear beta-catenin staining, no or low p53 positivity and a low PCNA index score may help clear the ambiguity in diagnosis of XGC cases. This can prevent a patient from extensive surgery.

In conclusion, this study supports the inflammatory nature of XGC but does not show any evidence of a premalignant condition. A panel of immunohistochemical staining with p53, PCNA and beta-catenin in XGC may be useful in diagnosis of ambiguous cases in future.

Funding: This study is done with financial support from the Department of Science and Technology, Ministry of Science & Technology, Government of India, Fast Track Scheme.

Ethical approval: Not needed.

Contributors: GM, SP and AAK proposed the study. GM wrote the first draft. All authors contributed to the design and interpretation of the study and to further drafts. AAK is the guarantor.

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.

1 Baykara M, Karahan OI. Xanthogranulomatous cholecystitis mimicking carcinoma of gallbladder. Tani Girisim Radyol 2004;10:56-58.

2 Pinocy J, Lange A, K?nig C, Kaiserling E, Becker HD, Kr?ber SM. Xanthogranulomatous cholecystitis resembling carcinoma with extensive tumorous infiltration of the liver and colon. Langenbecks Arch Surg 2003;388:48-51.

3 Goldar-Najafi A, Khettry U. Xanthogranulomatous choledochitis: a previously undescribed mass lesion of the hepatobiliary and ampullary region. Semin Liver Dis 2003;23:101-106.

4 Enomoto T, Todoroki T, Koike N, Kawamoto T, Matsumoto H. Xanthogranulomatous cholecystitis mimicking stage IV gallbladder cancer. Hepatogastroenterology 2003;50:1255-1258.

5 Spinelli A, Schumacher G, Pascher A, Lopez-Hanninen E, Al-Abadi H, Benckert C, et al. Extended surgical resection for xanthogranulomatous cholecystitis mimicking advanced gallbladder carcinoma: A case report and review of literature. World J Gastroenterol 2006;12:2293-2296.

6 Clarke T, Matsuoka L, Jabbour N, Mateo R, Genyk Y, Selby R, et al. Gallbladder mass with a carbohydrate antigen 19-9 level in the thousands: malignant or benign pathology? Report of a case. Surg Today 2007;37:342-344.

7 Rao RV, Kumar A, Sikora SS, Saxena R, Kapoor VK. Xanthogranulomatous cholecystitis: differentiation from associated gall bladder carcinoma. Trop Gastroenterol 2005; 26:31-33.

8 Lee HS, Joo KR, Kim DH, Park NH, Jeong YK, Suh JH, et al. A case of simultaneous xanthogranulomatous cholecystitis and carcinoma of the gallbladder. Korean J Intern Med 2003;18: 53-56.

9 Krishnani N, Shukla S, Jain M, Pandey R, Gupta RK. Fine needle aspiration cytology in xanthogranulomatous cholecystitis, gallbladder adenocarcinoma and coexistent lesions. Acta Cytol 2000;44:508-514.

10 Kwon AH, Sakaida N. Simultaneous presence of xanthogranulomatous cholecystitis and gallbladder cancer. J Gastroenterol 2007;42:703-704.

11 Zou S, Zhang L. Relative risk factors analysis of 3922 cases of gallbladder cancer. Zhonghua Wai Ke Za Zhi 2000;38:805-808.

12 Takada M, Horita Y, Okuda S, Okumoto S, Samizo M,Wada T, et al. Genetic analysis of xanthogranulomatous cholecystitis: precancerous lesion of gallbladder cancer? Hepatogastroenterology 2002;49:935-937.

13 Mlinaric-Vrbica S, Vrbica Z. Correlation between cholelithiasis and gallbladder carcinoma in surgical and autopsy specimens. Coll Antropol 2009;33:533-537.

14 Ang KK, Berkey BA, Tu X, Zhang HZ, Katz R, Hammond EH, et al. Impact of epidermal growth factor receptor expression on survival and pattern of relapse in patients with advanced head and neck carcinoma. Cancer Res 2002;62:7350-7356.

15 Fleming ID, Cooper JS, Henson DE, Hutter RVP, Kennedy BJ, Murphy GP, et al. AJCC Cancer staging Manual. In, Fifth edition. Philadelphia-New York: Lippincot-Raven;1997.

16 Arévalo F, Arias Stella C, Monge E. Immunoexpression of p53 and cyclin D1 in adenomas of the gallbladder. Rev Esp Enferm Dig 2007;99:694-697.

17 Chaube A, Tewari M, Garbyal RS, Singh U, Shukla HS. Preliminary study of p53 and c-erbB-2 expression in gallbladder cancer in Indian patients. BMC Cancer 2006;6: 126.

18 Wang SN, Chung SC, Tsai KB, Chai CY, Chang WT, Kuo KK, et al. Aberrant p53 expression and the development of gallbladder carcinoma and adenoma. Kaohsiung J Med Sci 2006;22:53-59.

19 Sipos F, Molnár B, Zágoni T, Berczi L, Tulassay Z. Growth in epithelial cell proliferation and apoptosis correlates specifically to the inflammation activity of inflammatory bowel diseases: ulcerative colitis shows specific p53- and EGFR expression alterations. Dis Colon Rectum 2005;48:775-786.

20 Goodman ZD, Ishak KG. Xanthogranulomatous cholecystitis. Am J Surg Pathol 1981;5:653-659.

21 Dao AH, Wong SW, Adkins RB Jr. Xanthogranulomatous cholecystitis. A clinical and pathologic study of twelve cases. Am Surg 1989;55:32-35.

22 Ladefoged C, Lorentzen M. Xanthogranulomatous cholecystitis. A clinicopathological study of 20 cases and review of the literature. APMIS 1993;101:869-875.

23 Guzmán-Valdivia G. Xanthogranulomatous cholecystitis: 15 years' experience. World J Surg 2004;28:254-257.

24 Krishnani N, Dhingra S, Kapoor S, Pandey R. Cytopathologic diagnosis of xanthogranulomatous cholecystitis and coexistent lesions. A prospective study of 31 cases. Acta Cytol 2007;51:37-41.

25 Howard TJ, Bennion RS, Thompson JE Jr. Xanthogranulomatous cholecystitis: a chronic inflammatory pseudotumor of the gallbladder. Am Surg 1991;57:821-824.

26 Uwagawa T, Misawa T, Nojiri T, Kitajima K, Kawakami M, Yanaga K. Xanthogranulomatous cholecystitis expressing high levels of DUPAN-II. J Gastroenterol 2007;42:593-596.

Received July 20, 2010

Accepted after revision December 11, 2010

Author Affiliations: Department of Gastrointestinal Surgery (Ghosh M and Agarwal AK) and Department of Pathology (Sakhuja P), GB Pant Hospital and Maulana Azad Medical College, New Delhi 110002, India

Anil K Agarwal, Professor, Department of Gastrointestinal Surgery, GB Pant Hospital & Maulana Azad Medical College, Delhi University, New Delhi 110002, India (Email: aka.hpb@gmail.com)

? 2011, Hepatobiliary Pancreat Dis Int. All rights reserved.