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Transforming growth factor alpha

immunoreactivity. A study in hepatocellular

carcinoma and in non-neoplastic liver tissue ANNALS OF HEPATOLOGY, MEXICO, v. 11, n. 4, supl., Part 3, pp. 495-499, JUL-AUG, 2012http://www.producao.usp.br/handle/BDPI/37538

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European Journal of Surgical Oncology 1998; 24:38~,2

Transforming growth factor alpha immunoreactivity in human gallbladder and extrahepatic biliary tract tumours

C. Soon Lee

Department of Pathology, The University of Melbourne, Australia

Aims. Transforming growth factor alpha (TGF-alpha), a protein structurally similar to epidermal growth factor (EGF), is implicated in the development of many human tumours. This study examines the expression of TGF-alpha in gallbladder and extrahepatic biliary tract tumours in which EGFR expression has been previously shown to be important. Methods. A monoclonal antibody to the TGF-alpha protein was used to investigate the immunohistochemical expression of TGF-alpha in carcinoma of the gallbladder (11 = 13), common bile duct (CBD) (n = 6) and ampulla of Vater (n = 8). Tissues from cases of chronic cholecystitis (n = 11), gallbladder dysplasia 01 = 3) and adenoma 01 = I), and ampullary carcinoma hi situ (CIS) (n=3) were used as non-malignant controls. These cases were previously studied for EGFR expression. Results. TGF-alpha overexpression, defined as intense immunoreactivity in more than two-thirds of cells immunostained for TGF-alpha, was present in most gallbladder carcinomas (n= 10; 77%) but with no significant differences in expression between different tumour grades. None of the cases of gallbladder dysplasia or chronic cholecystitis had strong TGF-alpha expression and this was significantly different from the carcinomas (P=0.013 and P=0.0001, respectively; Z" test), although a few cases of chronic cholecystitis showed weak (n =4), moderate (n=6) or no (n= I) immunoreactivity. A few ampullary carcinomas (n=2; 25%) and CIS 01= 1; 33%), and half of the CBD carcinomas (50%) had strong TGF-alpha immunoreactivity. There was correlation between TGF-alpha and EGFR immunoreactivity in the tumour cases ( r= 0.70, ia= 0.49, P = 0.0001; simple regression analysis), although the rate of EGFR immunoreactivity in CBD and ampullary carcinomas was somewhat higher than that of TGF- alpha. However, no statistically significant correlation between TGF-alpha expression with patient survival or tumour recurrence ( r = 0 . 1 1 , / = 0 . 0 1 2 , P=0.65; simple regression analysis) was found. Conclusions. Increased TGF-alpha expression occurs more frequently in gallbladder carcinoma than in gallbladder dysplasia, chronic cholecystitis, CBD or ampullary tumour, with no specific relationship to tumour grade, suggesting that TGF-alpha overexpression occurs early in the development of gallbladder cancers, and that biliary tract cancers have a different molecular origin. Correlation was found between TGF-alpha and EFGR expression in gallbladder and biliary tract tumours.

Key words: TGF-alpha; EGF receptor; gallbladder neoplasms; biliary tract neoplasms; immunohistochemistry; tumour markers; tyrosine kinase.

Introduction

Cancers of the gallbladder and extrahepatic bile ducts are relatively uncommon with 135 new cases diagnosed in 1992 in the state of New South Wales, Australia. These tumours account for about 1.2°/,, of all cancer-related deaths and have a peak incidence during the seventh decade of life) Males and females are affected with almost equal frequency. Both carcinoma of the gallbladder and extrahepatic bile ducts have a poor prognosis with 5-year survival rates of 5 and 3.6%, respectively. 24

Correspondence to: A/Professor C. S. Lee, Department of Anatomical Pathology, John Hunter Hospital, Locked Bag No. I, Hunter Region Mail Centre, Newcastle, New South Wales, Australia 2310.

Increased expression of transforming growth factor-alpha (TGF-alpha) is found in several types of tumour, s ~6 TGF- alpha is structurally and functionally similar to epidermal growth factor (EGF) and both growth factors bind to the epidermal growth factor (EGFR)) 7 20 The role of TGF- alpha in the development of human cancers may be related to its responsiveness and mitogenic effects in many human tissues that possess EGFRs. "j

Overexpression of the EGFR has been previously reported as important in gallbladder and biliary tract cancers-'-' and another histogenetically related tumour, pancreatic carcinoma." This study attempts to demonstrate and examine the expression of TGF-alpha in gallbladder and biliary tract cancers, and assess its prognostic value, using cases of chronic cholecystitis as the non-neoplastic control group.

0748-7983198/010038 + 05 $12.00•0 © 1998 W.B. Saunders Company Limited

TGF-alpha expression & gallbladder and bil&ry tract cancers 39

M a t e r i a l s a n d m e t h o d s

Tissue

Paraffin-embedded, archival tissues from patients with gallbladder adenocarcinoma (n = 13), dysplasia (n = 3) and adenoma (n= I), chronic cholescystitis (n= 11), common bile duct carcinoma (n=6), together with ampullary carcinoma (n=6) and carcinoma in situ (C1S) (n=3) were studied. The gallbladder adenocarcinomas were further classified as moderate-to-poorly differentiated (n=4) or poorly differentiated (n=9). The moderate-to-poorly differentiated adenocarcinomas were identified as those with areas of both moderate glandular differentiation and anaplasia. The poorly differentiated tumours consisted predominantly of solid sheets of tumour cells with little or no evidence of glandular differentiation.

Table 1. Summary of TGF-alpha immunostaining results in gallbladder and biliary tract neoplasia, and chronic cholecystitis

lmmunoreactivity

Diagnosis n Negative Weak Moderate Strong

Gallbladder adenocarcinoma Moderately differentiated 4 - - - - I 3 Poorly differentiated 9 - - 1 1 7

Gallbladder adenoma I - - - - - - I Gallbladder dysplasia 3 - - 1 2 - - Chronic cholecystitis I I I 4 6 - - Common bile duct

carcinoma 6 - - - - 3 3 Ampullary carcinoma 8 1 1 4 2 . Amupullary carcinoma

in situ 3 - - 2 - - I

Abbreviations: n: number of cases. TGF-alpha: transforming growth factor alpha.

hnnmnohis tochemis try

lmmunohistochemistry was performed on sections which had been cut at 41am, mounted on 5-aminopropyl- triethoxysilane (AAS)-coated slides and allowed to dry overnight. Two adjacent sections were used for the study, one for immunostaining and the other as negative control. After dewaxing in xylene, blocking of endogenous peroxidase, washing in phosphate-buffered saline (PBS), and blocking of non-specific binding of secondary antibody with normal swine serum, routine streptavidin-biotin- peroxidase immunostaining with diaminobenzidine was applied to the sections incubated overnight with a monoclonal antibody to TGF-alpha (Oncogene Science lnc, Manhasset, NY; clone GSI0) (1:100, dilution). Immunohistochemistry for EGFR (Serotec; distributed by Australian Laboratory Science, Rockdale, NSW, Australia; clone MCA-571) (1:1500, dilution) was performed on the same cases as previously described, z-' The primary antibody was substituted with PBS in sections used as negative controls. The sections were counterstained with Harris Haematoxylin.

lmmunohistochemical staining of cells was assessed according to both the intensity and proportion of cells stained. The intensity was scored on a semiquantitative 4-point scale as follows: 0 = n o staining; i=weak ; 2 = moderate; and 3=in tense staining. The proport ion of cells staining was also scored on a 4-point scale: 0 = n o cells staining; l = l e s s than one-third of cells staining; 2=be tween one-third and two-thirds of cells staining; 3 = m o r e than two-thirds of cells staining. An immunostaining score, ranging from 0 to 6, was derived by adding the above two scores, lmmunohistological expression of TGF-a lpha was then classified according to the immunostaining score as follows: immunostaining score of 0 = n o expression (N); l - 2 = w e a k (W); 3--4= moderate (M); >4 = strong (S) expression. Cases classified as having strong TGF-a lpha immunoreactivity are those that demonstrate staining stronger than that observed in the normal gallbladder epithelium in cases of chronic cholecystitis.

Stat is t ical analysis

Categorical variables were analysed with the chi-squared (;(2) contingency test. Only P-values of less than 0.05 were considered significant.

R e s u l t s

Overexpression of TGF-alpha was defined as strong cytoplasmic immunostaining of cells and this was found in most of the cases of gallbladder adenocarcinoma (n= I0; 77%) (Table 1; Fig. 1). The majority of the poorly differentiated (n=7; 78%) and moderately differentiated gallbladder carcinomas (n=3; 75%) showed TGF-alpha overexpression. However, the differences in TGF-alpha expression between the different grades of gallbladder carcinomas were not statistically significant (P=0.91, ;(2 test).

In contrast, none of the cases of gallbladder dysplasia demonstrated strong expression of TGF-alpha and this is statistically significantly different to the carcinoma cases (P=0.013; ~ test). Similarly, the cases of chronic cholecystitis used as non-neoplastic controls showed weak (n =4), moderate (n =6) or no (n = 1) immunoreactivity and none had evidence of TGF-alpha overexpression. The difference in TGF-alpha immunoreactivity between gallbladder cancers and chronic cholecystitis was significant (P=0.0001; ;(2 test).

Only a minority of the ampullary carcinomas (n =2; 25%) and CIS ( n = l ; 33%), and half of the CBD carcinomas (50%) (Fig. 2) showed strong immunoreactivity to the TGF- alpha protein.

There was statistically significant correlation between TGF-alpha expression and EGFR immunoreactivity obtained in a previous study 22 of the same tumour cases (r=0.70, r2=0.49, P=0.0001; simple regression analysis). In gallbladder carcinomas the proportion of increased TGF- alpha immunoreactivity (77%) was comparable to that of EGFR (100%). However, EGFR immunoreactivity in CBO

40 C S. Lee

Fig. 1. Strong TGF-alpha immunoreactivity in the cytoplasm of neoplastic cells in a gallbladder adenocarcinoma (Immunoperoxidase, x 200).

Fig. 2. Strong cytoplasm TGF-alpha immunostaining of neoplastic cells in a common bile duct carcinoma (lmmunoperoxidase, x 200).

TGF-alpha expression in gallbladder and biliary tract cancers 41

(86%), ampullary (67%) carcinomas, appears to be somewhat higher than that of TGF-alpha. 22

However, there was no statistically significant correlation between TGF-alpha expression with patient survival or tumour recurrence (r=0.11, r2=0.012, P=0.65; simple regression analysis).

Discussion

Overexpression of TGF-alpha, a polypeptide with significant homology to EGF, ~8 is associated with cell proliferation shown experimentally in cell lines which possess EGFR. 2~ Neoplastic transformation of cells by TGF- alpha involves autocrine stimulation of cells 23 in which cells with surface EGFR are stimulated to grow when either TGF-alpha or EGF binds to the receptors. 23'24 TGF-alpha has been shown to be important in the development of many human cancers. 5 ,,.2s .~J In some tumours, TGF-alpha is implicated in their progression to a less differentiated state. 7 Increased levels of serum TGF-alpha are noted in several types of gastrointestinal cancers such as gastric, pancreatic and colorectal carcinomas/3

The current study has demonstrated a statistically significant rate of increase in TGF-alpha immunoreactivity in gallbladder carcinomas than in gallbladder dysplasia or in cases of chronic cholecystitis. However, there was no significant difference in the expression of TGF-alpha between the different grades of gallbladder carcinomas. These findings suggest that increased expression of TGF- alpha may be important in the early development rather than the progression of gallbladder carcinoma. On the other hand, TGF-alpha does not appear to be important in ampullary or CBD carcinoma with only a minority of these tumours displaying strong immunoreactivity to the TGF- alpha protein.

in common with the current study, increased expression of TGF-alpha is observed to be an early event in gastric carcinogenesis) 2 Similarly, immunohistochemical detection of TGF-alpha is present in 95% of pancreatic cancers, a tumour that is histogenetically related to gallbladder and biliary tract cancers." The immunostaining is cytoplasmic with membrane accentuation." Although TGF-alpha is also implicated in the development of pancreatic carcinoma, there is no report to indicate that TGF-alpha is important in its progression." Of interest, TGF-alpha immuno- reactivity in squamous cell carcinoma of the larynx is found mostly in the better differentiated areas. ~~

However, no statistically significant correlation between TGF-alpha expression with patient survival or tumour recurrence ( r= 0.11, r 2 = 0.012, P = 0.65; simple regression analysis) is found. This may be related to the small number of cases with adequate clinical follow-up examined in the current study. Further studies with longer follow-up in a larger series may be required to elucidate the prognostic significance of TGF-alpha expression in these tumour types.

The correlation between TGF-alpha expression and EGFR immunoreactivity in the gallbladder and biliary tract carcinomas is consistent with the current concept of cancer development in which there is a sequential process of multiple genetic events with activation of several growth

factors and inactivation of tumour suppressor genes. ~'~5 However, the higher rate of TGF-alpha overexpression in gallbladder carcinomas than in CBD (50%), ampullary (25%) carcinomas and yet a similar rate of EGFR expression in all of these tumour types indicate that the role of TGF- alpha and EGFR in these tumours is complex. This may be explained by the possible activation of EGFRs by an autocrine mechanism involving EGF, EGFR and TGF- alpha that has been shown in previous studies of other types of tumours. 23"24 Moreover, the current findings also support the possibility that the molecular and genetic events leading to the development of gallbladder cancers may be different from those in extrahepatic biliary tract tumours.

In summary, this study has demonstrated increased TGF- alpha expression in the majority of gallbladder carcinomas with no specific relationship to tumour grade suggesting that although it may be important in the development of gallbladder carcinomas, it may not be important in their progression, lmmunohistochemical detection of TGF-alpha does not appear to be of prognostic value in gallbladder and biliary tract carcinomas. There is significant correlation between TGF-alpha and EGFR expression in gallbladder and biliary tract carcinomas although TGF-alph'a overexpression is lower in the latter tumours, suggesting that EGFR may be activated by another mechanism in those tumours.

Acknowledgements

The author is grateful to Anne Pirdas for technical support and to Dr David Ellis, then Director of Anatomical Pathology at St. Vincent's Hospital, for allowing access to the case material and laboratory facilities.

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Accepted for publication 1 December 1997