Distribution of Prognostically Unfavourable Product of c-erbB-2 Oncogene and EGF-R in Carcinomas of the Breast and Utreine Cervix
A.RAY, S. L. D. NAIK AND B. K. SHARMA*
Institute of Cytology and Preventive Oncology (ICMR),
Maulana Azad Medical College Campus,
Bahadur Shah Zafir Marg,
New Delhi – 110 002.
*Corresponding Author
(Received on December 2, 2001)

Abstract: A comparative study was undertaken between cancer of the uterine cervix (n=20) and female breast cancer (n=50) with reference to the expression of c-erbB-2 oncoprotein (HER-2/neu) and that of epidermal growth factor receptor (EGF-R), both being highly homologous structurally. Expressions of EGF-R and c-erbB-2 oncoprotien were viewed in breast and cervical cancer tissues by immunochemical staining. Cervical cancer cases showed much higher expression of EGF-R, which also revealed significant association with the expression of c-erbB-2 oncoprotein and tumor grading. Among breast cancer cases, over-expression of EGF-R correlated significantly with metastasis of lymph node; and expression of c-erbB-2 oncoprotein showed a significant relationship with histological grading of the tumour. Moreover, an association was noticed between the tumour grade and the concomitant immunopositive expression of EGF-R and c-erbB-2. Our study revealed an existence of a conflicting pattern in the expression of EGF-R and c-erbB-2 oncoprotein between carcinomas of the breast and uterine cervix.

Key words:     cerbB-2 oncoprotien                EGF-R                                                 breast cancer                          cervical cancer

Introduction
Methods
Results
Discussion
References

INTRODUCTION 

Cancer of the uterine cervix and breast cancer ate the most prevalent cancers among Indian females. Cancer of the cervix is the commonest malignancy; and next to the incidence of the cervical cancer, breast cancer is the second most common cancer on women in India (1). 

Pathogenesis of cancer of the cervix is a controversial topic. The most popular theory regarding the cervical carcinogenesis is related to the viral aetiology i.e. association with human papilloma various (HPV, mainly type 16 and 18) (2). Similarly, the cause of breast cancer is not known; but epidemiological evidence points strongly towards three areas: endocrine, environmental and genetic factors. Perhaps, all these factors are interrelated (3). Surprisingly, many of the epidemiological risk factors for cancer of the uterine cervix appear to be the inverse of those for cancer of the breast (4). 

The proliferation of cells is regulated by different physiological agents of which polypeptide growth factors and corresponding growth factor receptors may play a crucial role in malignant transformation of the cells. EGF-R is found in several tumours and specific sites of this receptor possess a strong homology to the product of v-erb oncogene (avian erytroblastosis virus oncogne), suggesting its possible role in the pathogenesis of malignancy. In fact, EGF-R and c-erbB-2 and two leading members of EGF family of growth factor receptors (5). Perhaps, these two receptor proteins are also holding most prominent position amongst entire growth factor systems (including IGF, TGFβ and PDGF families). The EGF-R gene is located on the chromosome 7 (6) and encodes a170 KD transmembrane glycoprotein which possesses tyrosine kinase activity (7). On the other hand, c-erbB-2 gene is located on chromosome 17 and encodes a 185 KD transmembrane glycoprotein called p185 (c-erbB-2 oncoprotien), which also possess tyrosine kinase activity. There is 43% sequence homology in the extracellular domains and 82% sequence homology in the tyrosine kinase domains between c-erbB-2 oncoprotien and EGF-R. In the present study, an effort was made to evaluate the pattern of expression of EGF-R and c-erbB-2 oncoprotein in carcinomas of the uterine cervix and breast, whose risk factors are conflicting to each other and both are considered as common cancers among Indian women.
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METHODS

Patients: This study was conducted on the randomly selected cases who attended the associated Lok Nayak Hospital of Maulana Azad Medical College, New Delhi. A total of 50 cases of histopathologically diagnosed squamous cell carcinoma of the uterine cervix and an equal number of histopathologically proven cases of primary infiltrating duct carcinoma of the breast were selected for the study. The mean age of patients with cervical cancer was 48.6 (±10.82) years and out of these patients, 29 were postmenopausal. The grading of cervical cancer was done according to FIGO guideline (8). Well-differentiated cancer was present among 12 patients (9 were postmenopausal), 32 cases had moderately differentiated tumour (18 were postmenopausal) and the rest 6 patients belonged to poorly differentiated carcinoma. On the other hand, among 50 cases of breast cancer (mean age: 44.4 ± 10.95 years), 28 patients were premenopausal state. All breast cancer cases were graded according to the criteria of modified Bloom and Richardson grading scheme (9. 10). Patients having low grade tumor were 13 in number (5 were postmenopausal), 21 cases belonged to moderate grade (10 were postmenopausal), while 16 cases were of high grade.

Immunohistochemistry: The analysis was carried out according to the method described by Ratnakar et al (11). Paraffin embedded tissue section from tumour (5 µm thick) on poly-L-lysine coated slides were deparaffinized in xylene/toluene and then rehydrated by putting into successive lower concentration of ethanol, followed by washing in phosphate buffered saline (PBS). Tissue sections were incubated with fleshly prepared solution of methanol; and 3% hydrogen peroxide to block endogenous peroxidase activity, followed by incubation in normal mouse serum to block non-specific binding. Tissue sections were then incubated with optimally diluted primary mouse monoclonal antibody against c-erbB-2 oncoprotien (Boehringer Mannheim, Germany) and EGF-R (Sigma, USA). The sections were kept with primary antibody within a humid chamber at room temperature for 60 min. After washing with PBS, the tissue sections were incubated with secondary antibody (anti-mouse IgG, Fab specific; 1:100) for 30 min in moist chamber at room temperature. Then, the sections were washed thrice with PBS containing Tween 20, and again incubated with optimally diluted peroxidase-mouse monoclonal antiperoxidase (PAP) complex for 60 min at room temperature. After washing with PBS and Tween 20, the reaction was visualized by substrate diaminobenzidine hydrochloride (0.1% freshly prepared solution in PBS with (0.05% hydrogen peroxide). After the development of colour, the slides were washed with double distilled water and counterstained with Harris haematoxylin. The tissue sections were then dried, mounted and observed under the microscope. The sections in which more than 30% of the cells revealed membrane immunopositivily were considered positive.

Statistical analysis: The data were analysed to calculate the proportion of positivity under different variables. Moreover, chi-square test/Fisher’s exact test of significance was employed appropriately to test the differences/ association between various combinations of variable studied.
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RESULTS

Out of 50 cases of infiltrating duct carcinoma of the breast, metastasis of lymph node was present in 32 (64%) cases. Immunohistochemical staining on breast cancer tissues showed 32% (16/50) cases were positive for c-erbB-2 oncoprotein (Fig. 1) and 36% (18/50) were positive for EGF-R (Fig. 2). In both cases, the immunohistochemical procedures revealed staining in membranous portions. The immunexpressions of both c-erbB-2 oncoprotein and EGF-R showed higher proportions of positively in postmenopausal cases than premenopausal group. Out of 22 postmenopausal breast cancer cases, 12 (54.5%) and 10 (45.5%) cases were positive for c-erbB-2 oncoprotien and EGF-R respectively; whereas, 4 (14.3%) and 8 (28.6%) premenopausal breast cancer cases revealed immunopositively for c-erbB-2 and EGF-R respectively. Further, the concomitant expression of these two protein receptors in breast cancer was observed in 9 cases (18.0%), while 25 (50.0%) were negative for both receptors; 7 cases (14.0%) showed immunoexpression only for c-erbB-2 and 9 cases (18.0%) were positive only for EGF-R.

Fig.1 click for full view

Fig.2 click for full view

In our study, no significant interrelationship was observed between c-erbB-2 oncoprotein and EGF-R among breast cancer cases (P>0.05). Further, a statistically significant association was found between cases with lymph node metastasis and over-expression of EGF-R (P<0.05). However, expression of c-erbB-2 oncoprotein did not show any significant relationship with the involvement of lymph node. On the contrary, a significant relation was observed between the histological grade of the tumour and the immunoexpression of c-erbB-2 (P<0.05). Whereas, there was no statstistical association between the level expression of EGF-R and grading of breast cancer (Table I). Among premenopausal breast cancer cases (n=28), both the expressions of c-erbB-2 oncoprotein and EGF-R separately as well as concomitantly revealed a significant correlation with histological grading of the tumour (P<0.05). On the other hand, no such association was seen with cases of metastasis of lymph node (P>0.05). In premenopausal group of breast cancer, concomitant expression of these two proteins was found in 4 cases (14.3%). Among postmenopausal breast cancer cases (n=22), the over-expression of EGF-R showed a significant relationship with the involvement of lymph node (P<0.05). On the contrary, the immuno positive expression of c-erbB-2 in this group was found to have a statistically significant association with histological grading of the tumour (P<0.05). Also, Co-expression of these two proteins in postmenopausal cases exhibited a relation (P<0.05) with tumour grades. Overall, the concomitant immunopositivily of c-erbB-2 and EGF-R revealed a correlation with the histological grading (P<0.05) in breast cancer cases (irrespective of menopausal status); whereas, no significant interrelationship was detected between the co-expression of these two proteins and the lymph node metastasis (Table I).            

Table I: Shows expression of c-erbB-2 oncoprotien and EGF-R in breast cancer

              Cases with various tumor grades and involvement of lymph node.

Mod = moderate, Pos=Positive, Neg=Negative, L.node=Lymph node

*Significant

Amongst 50 cases of squamous cell carcinoma of the uterine cervix, 5 cases (10%)m were positive for the involvement of lymph node. The immunostaining for c-erbB-2 oncoprotein (Fig. 3). On tissue sections of cervical cancer revealed positivity in 26% (13/50) cases, of which 7 (out of 29, 24.1%) were postmenopausal and 6 (out of 21, 28.6%) were premenopausal. on the other hand 50% (25/50) cervical cancer cases showed immunopositivity of EGF-R (Fig. 4). Out of 29 postmenopausal cases, 13 (44.8%) were positive of EGF-R: whereas, 12 premenopausal cases (57.1%) showed over-expression of EGF-R. Overall, in cervical cancer tissues, the concomitant immunopositive expression of c-erbB-2 and EGF-R was found in 10 (20%) cases; 44% (cases were negative for both these receptors; whereas, 3 (6%) were positive for c-erbB-2 oncoprotein only and 15 (13%) cases solely revealed over-expression of EGF-R.

Fig.3 click for full view

Fig.4 click for full view

A Significant association (P<0.05) was observed between the levels of immunopositive expression of EGF-R and c-erbB-2 to oncoprotien in cancer of the uterine cervix but, neither the immunohistochemical expression of these two receptors disunitedly nor their co-expression did show any statistically significant interrelationship with the involvement of lymph node (P<0.05). However, there was a fewer number of cervical cancer cases with metastases to lymph node compared with identical cases of breast cancer (i.e. involvement of lymph node). Nevertheless, a significant (P<0.05) relationship was observed between the over-expression of EGF-R and cervical tumour grading. No statistical association was seen between c-erbB-2 immunopositivity and histological grade which also did not exhibit any significant relation with concomitant expression of c-erbB-2 oncoprotein and EGF-R. However, when the cervical cancer cases were divided according to menopausal status, a statistical correlation (P<0.05) was noticed only between over-expression of EGF-R and histological differentiation (grade) among premenopausal group (Table II).

WD = well differentiated, MD = Moderately differentiated, PD = Poorly differentiated
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DISCUSSION

 The role of oncogenes in the development and prognosis of various cancers is a subject of intense investigation. The development of cancer is a multifactorial process that includes the sequential activation of oncogenes and other genetic derangement. Currently, one of the oncogenes, which is being studied most extensively, is c-erbB-2. The c-erbB-2 oncogene codes for a putative transmembrane receptor protein similar in structure to the EGF-R those tumours which are positive for EGF-R or similar receptors may be responsive to circulating EGF-and autocrine growth factors for growth stimulation (12).

Many investigators have demonstrated that EGF-R is expressed in dysplastic and malignant cervical epithelium. Maruo et al (13) found that EGF-R was absent in normal cervical epithelium but was detectable in approximately 75% of dysplastic and 50% of malignant squamous lesions. Hale et al (14) observed immunostaining for EGF-R in approximately 50% of early stage invasive squamous cervical cancers and the presence of staining was associated with poor prognosis. In the present study, we also noticed a similar type of over-expression of EGF-R in cancer of the uterine cervix. Interestingly, in the present study, a significant relation was observed between over-expression of EGF-R and cervical tumor grading. This relation was predominatingly present among premenopausal cases of cervical cancer. Further, a significant interrelationship was found between the expression of EGF-R and c-erbB-2 in cervical cancer. Ozanne et al (15) reported that in some but not all cases of squamous cell carcinomas, over-expression of EGF-R has been associated with c-erbB-2 gene amplification. Lakshmi et al (16) observed that the overall expression of EGF-R and c-erbB-2 was similar (88.6% and 90.5%, respectively) in cervical squamous lesions. They also observed a highly significant correlation between the expression of the two proteins and the histological grade of the lesions.

In our study, 26% of cervical cancer cases showed c-erbB-2 oncoprotein expression. Identical frequency of expression was also observed by Bhadauria et al (17). On the other hand, Brumm et al (18) reported a high rate (75%) of expression of c-erbB-2 protein in squamous cell carcinoma of the cervix. On the contrary, Berchuck et al (19) found that only 4% if cervical squamous cell malignancy were positive for c-erbB-2 immunostaining. So, it appears that the expression of c-erbB-2 oncoprotein varies widely among different studies. However, several studies (20-22) have demonstrated that c-erbB-2 oncoprotein expression showed significantly poorer prognosis in patients with squamous cell carcinoma of the cervix. The c-erbB-2 protein might be associated with aggressive biological behavior (19) and radiation resistance (22) of the tumor. Nevertheless, Hale et al (20) and Nakano et al (22) reported a positivity rate of 42%; whereas, Oka et al (21) observed that 19% of cervical cancers were positive for c-erbB-2 oncoprotein expression.

It is an interesting finding of the present study that expression of c-erbB-2 was higher in breast cancer cases (32%) than cervical cancer (26%); while over-expression of EGF-R was more in cases of cervical cancer (50%) compared to breast cancer (36%). Amongst breast cancer cases, immuno positive expressions of both these receptor-proteins were higher in postmenopausal group (c-erbB-2 oncoprotien: 54.5% and EGF-R: 45.5%) than premenopausal women (c-erbB-2 oncoprotein: 14.3% and EGF-R: 28.6%). On the contrary, increased rate of c-erbB-2 as well as EGF-R immunoexpressions was observed among premenopausal cervical cancer cases (c-erbB-2 oncoprotein: 28.6% and EGF-R: 57.1%) than corresponding postmenopausal cases (c-erbB-2 oncoprotein: 24.1% and EGF-R: 44.8%). Thus, like conflicting nature of epidemiological risk factors among breast cancer and cervical cancer, the present study clearly revealed a conflicting pattern of expression of EGF-R and c-erbB-2 oncoprotein between these two cancers. However, the overall (irrespective of menopausal status) concomitant   expressions of these two proteins were almost same in both carcinomas of the breast (18%) and the uterine cervix (20%).

Usually, one third to half of breast cancer cases have shown positivity for EGF-R in various studies (11, 23, 24). In the present study, a significant relationship was observed between over-expression of EGF-R and clinically palpable lymph node.  Similar findings were obtained in some other studies also (25, 26). Furthermore, in premenopausal breast cancer cases of our study, an association was found between histological grade of the tumour and over-expression of EGF-R. According to Sainsbury et al (27), EGF-R expression is a marked of poor differentiation, and therefore, it has a correlation with higher Bloom and Richardson (9) scores for ductal carcinomas. In one study, Sharma et al (28) observed an identical frequency of expression of EGF-R and c-erbB-2 oncoprotein (33% for each protein), both of which showed a significant correlation with high histological grade of breast cancer; whereas Bezwoda (29) found positive c-erbB-2 immunostaining in 26% cases of metastatic breast cancer. On the other hand, Bhatavdekar et al (30) noticed c-erbB-2 positivity in 68% breast cancer cases. The expression of c-erbB-2 oncoprotein always showed a significant relation with histological grades in our study. Similar to the results of the present study, Lipponen et al (31) concluded that the expression of c-erbB-2 positively correlated with high tumour grade. Like the over-expression of EGF-R, usually c-erbB-2 oncoprotein has been expressed more in the histologically severe grades. It is notable that the c-erbB-2 gene has been reported to be amplified in 10-30% of most types of human adenocarcinomas, including breast cancer (32, 33). Further, in the present study, concomitant immunoexpression of c-erbB-2 and EGF-R revealed a significant correlation with tumour grade. This association was observed in both premenopausal and postmenopausal cases of breast cancer. However, our study clearly reflects the difference in the behaviour of expression of EGF-R and c-erbB-2 between carcinomas of the breast and uterine cervix, whose epidemiological risk factors are conflicting to each other. Therefore, it is necessary to conduct several studies on different cancers for understanding the nature of involvement of the tyrosine kinase receptor in the disease process.

The c-erbB-2 oncoprotein is an important member of the tyrosine kinase receptors family. Its expression has been detected in several human cancers and it is believed to be associated with poor prognosis, aggressive biological behaviour of the tumor and metastatic potentiality. There have been reports in the literatures that c-erbB-2 over-expression is correlated with reduced benefit of adjuvant therapy and c-erbB-2 over-expressing patients may be resistant to therapy with tamoxifen (34, 35). Many studies have convincingly shown that repression of c-erbB-2 suppresses the malignant phenotypes of c-erbB-2 over-expressing cancer cells. These findings strongly suggest that c-erbB-2 may serve as an excellent target for developing anticancer agents specific for c-erbB-2-over-expressing tumour (36). Different approaches to target the c-erbB-2 oncoprotein have been devised (36-39). It has been demonstrated that targeting growth factor receptors using specific antibodies such as trastuzumab (a recombinant humanized monoclonal antibody directed against c-erbB-2 oncoprotein) can induce regression in some human cancers. Similarly, antibodies directed against the EGF-R directly inhibit the growth of receptor-bearing tumors in vitro and in vivo and clearly synergize with a number of antineoplastic agents, such as cisplatin and doxorubicin (40). Such antibodies modulate receptor-associated tyrosine kinase activity. The study on different cancers among Indian population will enrich our knowledge concerning the role of EGF-R and c-erbB-2 in pathophysiology of malignancy, response to treatment and promise of monoclonal antibody therapy as well as other approaches in cancer. 

ACKNOWLEDGEMENTS 

Authors wish to express thanks to Mr. Bishan Singh Negi for his laboratory support and Mr. Mohanan T for his secretarial help.                                               
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