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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2012  |  Volume : 55  |  Issue : 1  |  Page : 75-79
Role of C-erbB2 expression in gallbladder cancer


1 Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Surgical Gastroenterology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

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Date of Web Publication11-Apr-2012
 

   Abstract 

Background: Gallbladder cancer (GBC) is a lethal malignancy presenting at an advanced stage. The pathogenesis is not well categorized, and surgery is the only treatment available at the early stage of the disease. There have been few reports on role of growth factor receptors in GBC. C-erbB2 is one such receptor whose over-expression is being explored in GBC as one of the factors involved in carcinogenesis and possible target for therapy. Materials and Methods: One hundred and four consecutive cases of GBC were retrospectively studied with regard to clinical features, histological type, grade and stage of tumor. Immunohistochemistry for C-erbB2 was done and expression was correlated with different clinic-pathological parameters and survival. Results: C-erbB2 overexpression was seen in 9.4% cases with complete staining and both complete and incomplete staining (2+ and 3+) was seen in 13.4% cases. Eighty percent of the C-erbB2 over-expressed cases were well differentiated and in stage II to stage IV disease. Dysplasia adjacent to carcinoma did not show any expression. No correlation was found with tumor grade, stage, gall stones, and patient survival. Xanthogranulomatous inflammation was inversely correlated with C-erbB2 over-expression. Median survival was 30 months in C-erbB2 over-expressed cases, and 12 months in C-erbB2 negative cases. Conclusion: We found complete membranous staining of C-erbB2 in 9.4% of GBC which was frequent in well differentiated and stage II to stage IV tumors. C-erbB2 tumors had longer median survival than C-erbB2 negative tumors. C-erbB2 is not involved early in the carcinogenetic process as none of the dysplasia showed expression. C-erbB2 over-expression may be considered as target for therapy in advanced stage of GBC.

Keywords: C-erbB2, dysplasia, gallbladder cancer

How to cite this article:
Kumari N, Kapoor V K, Krishnani N, Kumar K, Baitha DK. Role of C-erbB2 expression in gallbladder cancer. Indian J Pathol Microbiol 2012;55:75-9

How to cite this URL:
Kumari N, Kapoor V K, Krishnani N, Kumar K, Baitha DK. Role of C-erbB2 expression in gallbladder cancer. Indian J Pathol Microbiol [serial online] 2012 [cited 2019 Dec 7];55:75-9. Available from: http://www.ijpmonline.org/text.asp?2012/55/1/75/94862



   Introduction Top


Gallbladder cancer (GBC) is an uncommon neoplasm demonstrating considerable geographic and gender variation. It is a highly malignant neoplasm presenting at advanced stages in majority of the cases. GBC is an aggressive malignancy where surgical excision is possible in only one fifth of the patients. Rest of the patients with GBC is not even suitable for surgery because of the advanced stage of disease at presentation. North India is one of the regions having highest incidence of GBC. The high-incidence areas are reported in Latin America, Eastern Europe, and Japan, apart from North India [1],[2],[3],[4],[5],[6] GBC, similar to other epithelial tumors, precedes by pre-invasive lesions like intestinal metaplasia and dysplasia. [7] Epithelial tumorigenesis is a multi-step process resulting from sequential genetic alterations at different stages of evolution. Although development of early molecular markers for detection, prediction to response of adjuvant therapies, and development of molecules for target therapies would be beneficial; however, there is limited information available about the genetic changes occurring in GBC.

C-erbB2, a cell surface growth factor receptor, is gaining popularity as a candidate for targeted therapy in different cancers. Although started as Herceptin, the drug against C-erbB2 for breast cancer is now being explored in gastrointestinal cancers. There is an increasing evidence that over-expression of tyrosine kinase growth factor receptors such as C-erbB2 may play an important role in the development of biliary tract carcinomas. [8] C-erbB2, a proto-oncogene, also known as Her-2/neu, is a trans-membrane glycoprotein having tyrosine kinase activity. Over-expression of C-erbB2 has been studied in various tumors like breast, gastric, colorectal, pulmonary, and gallbladder, and has become a valid indication for targeted therapy by trastuzumab in breast cancer and is being studied in gastric and esophageal cancers. [8],[9],[10] Although not yet validated, but similar targeted therapy for GBC in future might become a target for therapy which would be superior to conventional cytotoxic therapies used at present.

Our aim was to evaluate the overexpression of C-erbB2 in GBC in this high prevalence area of north India and to see their relation with the clinic-pathologic parameters and survival.


   Materials and Methods Top


All cases of GBC within a period of five years from January 2001 to December 2005 who had available follow-up data were included in this study. The clinical, imaging, and operative details of the specimens were retrieved from the records of hospital information system. Hematoxylin and eosin stained slides of these cases were reviewed and the following histological features - type of malignancy (whether adenocarcinoma, squamous cell carcinoma, or any other type), grade (well, moderate, and poor) of malignancy, depth of invasion, pre-neoplastic changes in adjacent mucosal epithelium like metaplasia and dysplasia and presence of associated xanthogranulomatous inflammation. Representative sections from formalin fixed paraffin embedded tissue blocks were selected for IHC. Polyclonal antibody for C-erbB2 (Dako; dilution 1:300) was used. Five micron thick sections were cut, dewaxed in xylene, and rehydrated in graded alcohol. Endogenous peroxidase blocking was done by using 3% H 2 O 2 . Antigen retrieval was done using Tris EDTA at pH. 8.4. After adding primary antibody, the slides were incubated for three hours at room temperature. Biotinylated secondary antibody was put for 30 minutes (Dako, LSAB) followed by streptavidin peroxidase for 30 minutes. Diaminobenzidine was used as chromogen and counterstaining was done by Harris hematoxylin. Carcinoma breast tissue was used as positive control for C-erbB2.

C-erbB2 was considered overexpressed when complete membranous staining of cell cytoplasm was seen in at least 10% of tumor cells. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) version 15. The significance of correlation between grade and stage of tumor with C-erbB2 expression was tested using Spearman's correlation coefficient. The survival analysis was performed by Kaplan-Meier method and a probability of <0.05 was considered statistically significant.


   Results Top


The age range of the patients with GBC was 31-82 years with a mean of 55.68 years. There were 27 males and 77 females with M:F ratio of 1:2.8. Gallstones were found in 70 patients (67.3%).

Histopathology

There were 97 cases adenocarcinoma, 6 adenosquamous carcinoma, and 1 squamous cell carcinoma . The different variants of adenocarcinoma are given in [Table 1]. Evaluable mucosa was present in 68 cases and dysplasia was seen in 60 of the 68 cases (21 low grades and 39 high grades). Mucosa was normal in 8 cases. Associated xanthogranulomatous inflammation was found in 21 cases.
Table 1: Histological distribution of gallbladder carcinoma

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Immunohistochemistry

Ten (9.8%) cases of GBC showed complete membranous (3+) positivity for C-erbB2 [Figure 1]. Eight of these (80%) were well differentiated carcinoma and two (20%) were moderately differentiated carcinoma. Forty percent of the C-erbB2 positive GBC were in stage II, followed by thirty percent in stage IV [Table 2]. Four cases had incomplete membranous (2+) positivity with two of them being in stage II disease and a well differentiated morphology [Figure 2] and [Table 3]. Considering both 3+ and 2+ staining as overexpression, C-erbB2 overexpression was seen in 13.4% cases of GBC. None of the cases with C-erbB2 expression had associated xanthogranulomatous inflammation; however, 10 of 14 cases (3+ staining in seven cases and 2+ staining in three cases) showed presence of gallstones. C-erbB2 expression was not seen in the dysplastic epithelium adjacent to carcinoma. Survival was available for 0-60 months. Kaplein Meir survival analysis showed a median survival of 12 months, irrespective of grade and stage of the disease. Patients with 3+ staining of C-erbB2 had survival ranging from 0.5 to 37 months and those with 2+ staining had 1 to 45 months. Statistical analysis using Chi square did not show any significant correlation of C-erbB2 3+ staining with tumor stage, tumor grade, presence of xanthogranulomatous inflammation, and gallstone. When both 2+ and 3+ staining were combined there was a significant inverse relationship between associated xanthogranulomatous inflammation and C-erbB2 overexpression (P value=0.04). Kaplein Meir log rank survival analysis showed median survival of 30 months in cases with C-erbB2 3+ over-expression, whereas it was 12 months in C-erbB2 negative cases; however, the difference was not statistically significant (P value=0.15) [Figure 3]a and b. Associated xanthogranulomatous inflammation or presence of gallstones did not have any effect on survival.
Figure 1: C-erbB2 expression in gallbladder carcinoma; 3+ positivity (IHC, ×40)

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Figure 2: C-erbB2 expression in gallbladder carcinoma; 2+ positivity (IHC, ×40)

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Figure 3: (a) Survival analysis in C-erbB2 over-expressed and C-erbB2 negative patients. Survival difference in complete membranous positivity of C-erbB2 and C-erbB2 negative tumors, (b) Survival analysis in C-erbB2 over-expressed and C-erbB2 negative patients. Survival difference in both complete and incomplete membranous positivity of C-erbB2 and C-erbB2 negative tumors

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Table 2: Relation of grade of tumor with C-erbB2 expression and loss of FHIT expression

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Table 3: Relation of tumor stage with C-erbB2 expression

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   Discussion Top


Gallbladder cancer is an aggressive disease and its pathogenesis is not well known. Various genetic and molecular markers studied have been studied which include p53, CEA, CDNK1, FHIT, MUC1, growth factors and their receptors and mismatch repair genes. However, till now, there is no clear understanding of GBC pathogenesis and no candidate molecule has been explored for targeted therapy. GBC is one of the commonest causes of cancer related mortality in women, in the northern and north eastern states of India. [11] According to Indian Council of Medical Research, cancer registry 2001, in North India, the incidence of gallbladder cancer was 2.5-8.9/1,00,000 female population. [12] To the best of our knowledge, there is only one report from India on expression of C-erbB2 in GBC. [10],[13]

Ten (9.6%) cases showed over-expression of C-erbB2 based on complete membranous (3+) staining [Table 3]. Chaube et al. studied 40 cases of GBC and showed over-expression of C-erbB2 in 25% cases based on IHC. [10] Nakazawa et al. [8] showed over-expression of C-erbB2 in 16% of their 89 cases by combining both immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH). Kamel D et al. [9] studied 30 cases of GBC and showed over-expression of C-erbB2 in 10% of cases using IHC. Expression of C-erbB2 has varied between 10% and 46.5% in gallbladder carcinomas and its expression was correlated with increasing stage. [14] Nakazawa et al.,[8] in their study, showed over-expression of C-erbB2 in 21% of cases by considering both 2+ (cytoplasmic and incomplete membranous staining) and 3+ staining (complete membranous staining) on IHC. However, after gene amplification by FISH, they found only 16% cases to show over-expression as 33% of their 2+ positive cases on IHC did not show gene amplification. Considering 3+ complete membranous staining as positive they had only 8% C-erbB2 expression in their study. If we include our GBC with both 2+ and 3+ (incomplete and complete membranous stainingthe expression rate of C-erbB2 will rise to 13.4%. FISH should be reserved for GBC with 2+ staining for C-erbB2 as they are equivocal for C-erbB2 over-expression. C-erbB2 over-expression was higher in the study by Chaube et al.; [8] however, they studied less number of cases and considered both cytoplasmic and membranous staining as positive staining on IHC. Our results are comparable to Kamel et al. [9] where complete membranous staining was considered as positive for C-erbB2 over-expression similar to our study. The results of CerbB2 over-expression in gastric and esophageal cancers are also similar to gallbladder cancers. [15],[16]

Eight of the ten cases that were positive for C-erbB2, were well differentiated tumors. In the study by Chaube et al. [8] 50% of well differentiated, 30% of moderately differentiated, and 20% of poorly differentiated tumors showed C-erbB2 positivity. [10] The present study had 80% of C-erbB2 positive tumors in well differentiated category and 20% in moderately differentiated category.

None of the dysplasia or intestinal metaplasia showed C-erbB2 positive staining in our study as reported by Kamel et al. [9] As dysplasia did not show positive staining and C-erbB2 expression was not seen in poorly differentiated tumors, it may be possible that C-erbB2 is not involved at an early stage in gallbladder carcinogenesis and the expression decreases with loss of tumor differentiation. In a study by Kawamoto et al., C-erbB2 over-expression (2+ and 3+ staining) was identified in 31.2% of gallbladder cancers, 31.3% of extrahepatic bile duct cancers, and 33.3% of intrahepatic bile duct cancers, and gene amplification was found in 20.9%, 21.4%, and 0%, respectively. [17] As over-expression of C-erbB2 has been shown to indicate worse prognosis in tumors of various organs and is used as an important marker to identify lesions having a poorer prognosis, it appears likely that it may be associated with neoplastic progression and its expression may be a marker for worse prognosis in GBC, as it has been shown in some studies that C-erbB2 positivity increases with stage progression. [18] In the present study also, C-erbB2 expression was more frequent from stage II disease onwards. The C-erbB2 overexpressed tumors had median survival of 30 months compared to C-erbB2 negative tumors which had survival of 12 months although the difference in survival was not statistically significant (P value=0.15). Therefore, expression of C-erbB2 in higher stage tumors indicates a worse prognosis, but a longer survival although not statistically significant. This difference in survival needs to be studied in larger number of cases and with longer follow-up periods to see whether C-erbB2 over-expressed cases really survive longer.

As more and more targeted therapeutic approaches are being developed against various molecules involved in cell cycle regulation, over-expression of C-erbB2 in GBC may need to be evaluated as candidate for targeted therapy similar to that in breast cancer and would be helpful in higher stage tumors.


   Conclusion Top


This study demonstrates the role of C-erbB2 in the pathogenesis of GBC as a late event in north Indian population which is a high prevalence region for GBC. We found complete membranous staining of C-erbB2 in 9.6% cases and both complete and incomplete membranous staining in 13.4% cases of GBC. The expression was frequent in well differentiated and high stage tumors i.e. stage II onward tumors. Associated xanthogranulomatous inflammation was inversely correlated with C-erbB2 over-expression. Median survival was better in C-erbB2 over-expressed tumors as compared to C-erbB2 negative tumors, although not statistically significant. C-erbB2 may become a candidate for therapy in GBC as is being used in breast cancer and being explored in gastric cancer.

 
   References Top

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2.Lazcano-Ponce EC, Miquel JF, Muñoz N, Herrero R, Ferrecio C, Wistuba II, et al. Epidemiology and molecular pathology of gallbladder cancer. CA Cancer J Clin 2001;51:349-64.  Back to cited text no. 2
    
3.Misra S, Chaturvedi A, Misra NC, Sharma ID. Carcinoma of the gallbladder. Lancet Oncol 2003;4:167-76.  Back to cited text no. 3
    
4.Randi G, Franceschi S, La Vecchia C. Gallbladder cancer worldwide: Geographical distribution and risk factors. Int J Cancer 2006;118:1591-602.  Back to cited text no. 4
    
5.Pandey M. Risk factors for gallbladder cancer: A reappraisal. Eur J Cancer Prev 2003;12:15-24.  Back to cited text no. 5
    
6.Kapoor VK, McMichael AJ. Gallbladder cancer: An 'Indian' disease. Natl Med J India 2003;16:209-13.  Back to cited text no. 6
    
7.Albores-Saavedra J, Henson DE. Tumors of gallbladder and extrahepatic bile ducts. Fascicle 23, Ed. 3. Washington DC: Armed Forces Institute of Pathology; 2000.  Back to cited text no. 7
    
8.Nakazawa K, Dobashi Y, Suzuki S, Fujii H, Takeda Y, Ooi A. Amplification and overexpression of C-erbB-2, epidermal growth factor receptor, and c-met in biliary tract cancers. J Pathol 2005;206:356-65.  Back to cited text no. 8
    
9.Kamel D, Pääkkö P, Nuorva K, Vähäkangas K, Soini Y. p53 and C-erbB-2 protein expression in adenocarcinomas and epithelial dysplasia of the gall bladder. J Pathol 1993;170:67-72.  Back to cited text no. 9
    
10.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 10;6:126. Available from: Http://www.biomedcentral.com/1471-2407/6/126. [Last accessed on 2010 Sep 14].  Back to cited text no. 10
    
11.Nandakumar A, Gupta PC, Gangadharan P, Visweswara RN, Parkin DM. Geographic pathology revisited: Development of an atlas of cancer in India. Int J Cancer 2005;116:740-54.  Back to cited text no. 11
    
12.Indian Council of Medical Research. National cancer registry programme. Consolidated report of the population based cancer registries 1990-1996. Incidence and Distribution of cancer. New Delhi: Indian Council of Medical Research; 2001. p. 53.  Back to cited text no. 12
    
13.Priya TP, Kapoor VK, Krishnani N, Agrawal V, Agarwal S. Fragile histidine triad (FHIT) gene and its association with p53 protein expression in the progression of gallbladder cancer. Cancer Invest 2009;27:764-73.  Back to cited text no. 13
    
14.Kim YW, Huh SH, Park YK, Yoon TY, Lee SM, Hong SH. Expression of the c-erb-B2 and p53 protein in gallbladder carcinomas. Oncol Rep 2001;8;1127-32.  Back to cited text no. 14
    
15.Reichelt U, Duesedau P, Tsourlakis MC, Tsourlakis MC, Quaas A, Link BC, et al. Frequent homogeneous HER-2 amplification in primary and metastatic adenocarcinoma of the esophagus. Mod Pathol 2007;20:120-9.  Back to cited text no. 15
    
16.Marx AH, Tharun L, Muth J, Dancau AM, Simon R, Yekebas E, et al. HER- 2 amplification is highly homogenous in gastric cancer. Hum Pathol 2009;40:769-77.  Back to cited text no. 16
    
17.Kawamoto T, Krishnamurthy S, Tarco E, Trivedi S, Wistuba II, Li D, et al. HER Receptor Family: Novel Candidate for Targeted Therapy for Gallbladder and Extrahepatic Bile Duct Cancer. Gastrointest Cancer Res 2007;1:221-7.  Back to cited text no. 17
    
18.Goldin RD, Roa JC. Gallbladder cancer: A morphological and molecular update. Histopathology 2009;55:218-29.  Back to cited text no. 18
    

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Correspondence Address:
Niraj Kumari
Department of Pathology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.94862

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    Tables

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