EXPRESSION OF THE TYPE 1 TYROSINE KINASE GROWTH FACTOR RECEPTORS EGF RECEPTOR, c-erbB2 AND c-erbB3 IN BLADDER CANCER

JOURNAL OF PATHOLOGY, VOL. 179: 381-385 (1996)

EXPRESSION OF THE TYPE 1 TYROSINE KINASE GROWTH FACTOR RECEPTORS EGF RECEPTOR,

c-erbB2 AND c-erbB3 IN BLADDER CANCER

THANGARAJAN RAJKUMAR*, GORDON w . H. STAMP?, HARDEV s. PANDHA$, JONATHAN WAXMAN$ AND WILLIAM J . GULLICK*

*ICRF Oncology Unit, TDepartment of Histopathology, and $Department of Clinical Oncology, Royal Postgraduate Medical School, Hummersmith Hospital, London W12 ONN, U. K.

SUMMARY Expression of the c-erbB3 protein was determined in transitional cell carcinoma of the bladder by immunohistochemistry. Strong

membrane staining was observed in 10 per cent of cases (7/70) and cytoplasmic and membrane overexpression in 20 per cent (14/70). Overexpression of the epidermal growth factor (EGF) receptor (36 per cent, 25/70) and c-erbB2 proteins (9 per cent 6/70) was determined in the same series of cases. c-erbB3 overexpression was positively correlated with EGF receptor expression (P<0-025) but appeared to be inversely associated with c-erbB2 overexpression.

KEY WORDS+pidermal growth factor receptor; c-erbB2; c-erbB3; bladder cancer; oncogenes

INTRODUCTION

Bladder cancer ranks as the fourth most common cancer among men in the U.K.’ Although nearly 80 per cent are superficial, a significant proportion of these progress to invasive cancer and for those patients, the expectation for survival at 5 years is only 67 per cent. The grade and stage of the disease are currently the most important prognostic factors,2 but these lack specificity. It is therefore essential to identify additional markers in order to define prognosis more accurately.

The type 1 tyrosine kinase growth factor receptor family includes the epidermal growth factor receptor (EGFR or ~-erbBl) ,”~ ~ - e r b B 2 , ~ ~ and the more recently identified ~ - e r b B 3 ~ - ‘ ~ and c-erbB4l receptors. Among these EGFR and c-erbB2 protein expression levels have been evaluated in several malignancies including breast, stomach, ovary, head and neck, and bladder. In general, overexpression of these receptors occurs fairly frequently and is associated with a poor prognosis. In transitional cell carcinomas of the bladder, EGFR has been shown to be associated with high-grade lesions, muscle invasion, increased risk of relapse, multiplicity of lesions, and metastases.12-15 c-erbB2 overexpression has been found to be associated with high-grade tumours and poor prognosis.16 The c-erbB3 protein has been found to be overexpressed in b r e a ~ t , ’ ~ - ~ ~ g a s t r ~ i n t e s t i n a l , ~ ~ - ~ ~ cervical,24 and ovarian cancers,2s but there has been no study published to date on its expression in bladder cancer.

Lemoine ef aI.l7 found that 24 per cent of breast cancers overexpressed c-erbB3, which was weakly associated with node-positive status but not with survival. Gasparini et al. I * demonstrated c-erbB3 over-

Addressee for correspondence William J Gullick, ICRF Oncology Unit, Royal Postgraduate Medical School, Hammersmith Hospital, London W12 ONN, U K

CCC 0022-3417/96/08038 1-05 c) 1996 by John Wiley & Sons, Ltd

expression in 14 per cent of node-negative breast cancers and observed that this was associated with c-erbB2 overexpression. Quinn et aI.l9 have shown that there is a relationship between c-erbB3 overexpression in breast cancer and well-differentiated tumour grade, although this did not reach statistical significance. In ovarian c a r c i n ~ r n a s , ~ ~ however, a statistically significant association was observed between c-erbB overexpression and well-differentiated tumour grade.

In this pilot study, we present our results on the immunohistochemical analysis of 65 bladder cancers evaluated for EGFR, c-erbB2, and c-erbB3 proteins. We examine the prevalence of overexpression of c-erbB3 and the homogeneity of its distribution in tumour cells and we develop a method for scoring expression. This analysis lays the foundation for further studies on the potential of c-erbB3 as a prognostic indicator and as an indicator of response to therapy and demonstrates that the overexpressed cell surface receptor may represent a target for immunotherapy.

MATERIALS AND METHODS

Seventy formalin-fixed, paraffin-embedded sections from 65 bladder cancer patients referred to the Medical Oncology Clinic at the Hammersmith Hospital were studied. Rabbit polyclonal antibodies 12E (against EGFR26) and 21N (against ~ - e r b B 2 ~ ~ ) were used at 4 and 3 pglml, respectively. RTJ 1 monoclonal antibody (supernatant) raised against c-erbB3 protein using a synthetic peptide2* was used at 1:20 dilution as described in ref. 22. Positive and negative controls were run with each antibody. For positive controls, a section known to be positive for imunoreactivity to the antibody was used. Peptide block (12E and 21N) or an isotype matched control monoclonal antibody (Sigma) (for RTJ 1) were used as negative controls.

Received 4 September I995 Accepted I 2 Februury 1996

382 1. RAJKUMAR ET A L

Table [----Grades of the tumours

Grade N o . of cases

I 2 (3) 2 19 (27) 3 31 (44) 4 I8 (26)

Numbers in parenthesis indicate percenlages

The sections were scored for the intensity of cytoplasmic staining (0, +, + +, + + +), the percentage of tumour cells positive for immunoreactivity (1-25, 26-50, 51-75, and >75 per cent), and whether or not there was membrane staining in the tumour cells. In the case of 12E (anti-EGFR), + + + cytoplasmic staining intensity or membrane staining was considered to be indicative of EGFR overexpression. Membrane staining alone was considered to be indicative of c-crbB2 overexpression. The following scoring system was devised. A score of 1 was given for each + intensity of’ staining (0, 1, 2, 3 for 0, +, + +, + + +, respectively) and for each of the percentage groups (1, 2, 3 , 4 for 1-25, 26-50, 51-75, >75 per cent, respectively). Membrane staining was given an additional score of 1 . ‘The scores were summed and overexpression was deemed present if the total score exceeded 5. The analysis was also performed using membrane staining only as indicative of overexpression.

The chi-squared test was used to assess the statistical significance of the results.

RESULTS

Seventy samples from 55 patients were studied for overexpression of EGFR, c-erbB2, and c-erbB3 using immunohistochemistry. Sixty-nine of the 70 were transitional cell carcinoma and one was squamous cell carcinoma. In four patients, sections were available at the time of recurrencc as well. The histological grades of the tumours arc given in Table I. There are a greater number of grade 3 and 4 tumours than of grade 1 and 2 in this series.

The normal bladder epithelium was found to give weak to moderate cytoplasmic immunoreactivity to EGFR, c-erbB2, and c-erbB3 ( Fig. 1 A) in the superficial cells. The basal cells were essentially negative.

25 (35.7 per cent) overexpressed EGFR (+ + + intensity or membrane staining). Membrane staining was observed in 6/70 (8.6 per cent) and all six cases also had + + + intensity of cytoplasmic staining. The relationship between EGFR overexpression and grade is given in Table 11. There was no significant association between EGFR overexpression and tumour grade, although all six cases positive for EGFR membrane staining were found to be high-grade tumours (grades 3 and 4) (P< 0.05).

Six (8.6 per cent) of the 70 tumours overexpressed c-erbB2 as determined by membrane staining (Fig. IB). Five of the six cases were high-grade tumours (grades 3

Fig. 1-(A) Normal bladder epithelium stained for c-erbB3 protein showing weak staining of the superficial layer. (B) Bladder cancer stained for c-erbB2 protein showing strong membrane staining. (C) Bladder cancer stained for c-erbB3 protein showing strong membrane staining

and 4), but the association did not reach statistical significance (P<0.1) (Table 11).

Fourteen (20 per cent) of the 70 tumours stained for c-erbB3 were found to have a score of 6 or more, which was considered to be indicative of overexpression (Fig. 1C); 22 (31.1 per cent) of the tumours were found to have faint to strong membrane staining in at least a few cells and 7/70 (10 per cent) of the tumours had strong

383 TYPE 1 CFR IN BLADDER CANCERS

Table II-Relationships between EGF receptor and c-erbB2 overexpression and tumour grade

EGFR c-erbB2

+ - + Grade -

112 14 7 (33) 20 1 ( 5 ) 314 31 18 (37) 44 5 (10) Total 45 25 (36) 64 6 (9)

~~

Numbers in parenthesis indicate percentages.

Table III-Relationship between c-erbB3 overexpression and tumour grade

Score Strong membrane Any membrane

+ - + Grade 6 5 >5 -

112 17 4(19) 18 3 (14) 15 6 (29) 3/4 39 10 (20) 45 4 (8) 33 16 (33) Total 56 14 (20) 63 7 (10) 58 22 (31)

Numbers in parentheses indicate percentages

Table IV-relationship between EGFR overexpression and c-erbB2 overexpression

c-erbB2

t -

EGFR 39 6 + 25 0 -

membrane staining in many of the tumour cells. The association of c-erbB3 overexpression with tumour grade is shown in Table 111. The chi-squared test did not reveal any significant association between c-erbB3 overexpression and tumour grade.

There were interesting relationships of overexpression among the three receptor proteins. The six membrane- positive cases of c-erbB2 overexpression had no overexpression of EGFR (+ + +) or c-erbB3 (as determined by the scoring system or by the presence of strong membrane staining: Tables IV and V). EGFR overexpression, on the other hand, was found to correlate with the overexpression of c-erbB3 as determined by either the membrane staining pattern (weak + strong and strong alone) or by a c-erbB3 score of 6 or more (P<0.005; P<0.05 and P<0.025, respectively) (Table V).

In four patients, samples were evaluated at the time of relapse. One of the four patients had two relapse samples and the remaining three had one relapse sample assessed. All five samples showed an increasing trend in the c-erbB2 score; in one, the tumour converted from c-erbB2 membrane negativity to membrane positivity. Two patients were negative for EGFR overexpression in their initial and relapsed sample: in one patient, the initial presentation sample was positive for EGFR over-

Table V-Relationship between EGFR overexpression and c-erbB3 overexpression

Score Strong membrane Any membrane

+ - < 5 >5 - +

EGFR - 40 5 (11) 43 2 (4) 37 8 (18) + 16 9 (36) 20 5 (20) 11 14 (56)

P<0.025 P<0.05 P<0.005

Numbers in parentheses indicate percentages.

expression but became negative at the time of relapse: in another, an EGFR-negative tumour at presentation became positive at the time of relapse. As regards c-erbB3 status, none of the four tumour samples, either at initial presentation or a t relapse, was positive for c-erbB3 overexpression. In two of the patients, the tumours had a score of 0 in the initial and relapse samples; in the third patient, an initial score of 2 reverted to 0 at the time of second relapse; while in the fourth patient, the score increased from 3 to 5 at the time of relapse.

DISCUSSION

This is the first comparative study to evaluate EGFR, c-erbB2, and c-erbB3 receptor protein expression in bladder cancers. In normal bladder, the superficial layer of the transitional epithelium is weakly positive for EGFR28,29 and ~-erbB2~~,'O and moderately positive for c-erbB3 immunoreactivity.29

EGFR has been reported to be relatively overexpressed in 25-61 per cent of transitional cell bladder carcinomas, using paraffin12'31 and frozen sections.15J2 With the exception of the study by Nguyen et aL3I most have shown EGFR overexpression to be associated with high grade, invasiveness, and increased risk of recurrence and progression. 12.' 532,33 c-erbB2 overexpression has been reported in several s t ~ d i e s . ' ~ , ' ~ , ~ ~ ' ~ All of these except that by Zhau et al.37 used membrane immunoreactivity to define overexpression in bladder cancers. c-erbB2 overexpression has been associated with high grade and poor prognosis,I6 but in a multivariate analysis it had no independent prognostic value. 38 Others have shown an association with tumour recurrence39 and invasive cancers with overexpression of mutant p53 ~ r 0 t e i n . l ~ It is notable, however, that the range of positive values reported varies between 2 and 74 per cent. This may be due to differences in techniques of staining such as the use of immunofluorescence,37 the composition of the cases in individual series, and some ambiguity as to the strictness of the criteria used to define expression.

The relatively lower incidence of grade 1 tumours in the present study is because patients referred to the Medical Oncology Unit have advanced disease. The present study shows that EGFR is overexpressed in 35.7 per cent, c-erbB2 in 8.6 per cent, and c-erbB3 in 20 per

3 84 T. RAJKUMAR ET AL.

cent of the bladder cancers. Interestingly, EGFR overexpression and c-erbB3 overexpression were related irre- spective of which criteria were used to define c-erbB3 overexpression. An inverse relationship was present between c-erbB2 overexpression and the other two receptor proteins, with none of the six membrane- positive c-erbB2 cases having overexpression of EGFR or c-erbB3, as determined by either scoring system. The c-erbB3 protein has been reported to heterodimerize with EGFR40 and with ~-erbB2.~I Heterodimerization of c-erbB3 with EGFR has been found to activate phosphatidylinositol 3-kinase (P13-kinase),") which is not activated appreciably either by homodimerization of EGFR or c-erbB2, or by heterodimerization between EGFR and c-erbB2. The opportunity for more selective utilization of the EGFR-c-erbB3 heterodimerization indicates that there may be a preferential utilization of the P13-kinase pathway in these bladder cancers. How- ever, with only relatively few cases evaluated, it is appropriate to be cautious in interpreting these results and it will be necessary to study a larger number of cases before any concrete conclusions can be reached.

One of the problems associated with the scoring of tumours for c-erbB3 protein expression is the heterogeneity of the immunoreactivity seen, both with regard to the number of tumour cells positive and with regard to their intensity of staining. Most of the studies evaluating c-erbB3 expression have used different scoring systems. Lemoine et 171. , I 7 in assessing c-erbB3 expression in breast tumours, compared the immunoreactivity seen in the tumours with that seen in the normal breast tissue. They considered the normal breast ductal tissue to express moderate levels of c-erbB3 protein and hence tumours that were scored as having any area with greater immunoreactivity than the normal tissue were deemed to overexpress c-erbB3 protein. Interestingly, the study reported only 11195 cases as exhibiting membrane immunoreactivity. In a series of node-negative breast carcinomas, membrane staining was used as the criterion for overexpression of c-erbB2 and c-erbB3 protein. l 8 However, the membrane-positive cases were divided into three subsets based on the proportion of tumour cells which were positive. The authors also reported that there was heterogeneous immunoreactivity in the tumours. Their study reported a higher prevalence of cases showing any membrane staining (65 per cent) or strong generalized membrane staining (1 3 per cent) than Lemoine et u1.I' This could be due in part to the use of different antibodies, the former using the RTJ1 monoclonal antibody and the latter a rabbit polyclonal antibody raised against the same peptide.

In contrast to breast carcinoma, 35 per cent of pancreatic carcinomas were found to show membrane and cytoplasmic immunoreactivity and it was reported that the staining was usually homogeneous throughout an individual tumour."' This study scored c-erbB3 immunoreactivity on a semi-quantitative scale: - , negative; + , weakly positive; + +, moderately positive; and + + + , strongly positive with membrane staining. Sanidas ct reporting on the pattern of expression of c-erbB3 protein in gastric carcinomas, used a scoring system similar to the one described in this paper, evaluating

the proportion of tumour cells stained (1 to 4); the distribution of staining evaluated as focal, patchy, or extensive; the cellular localization of staining, whether cytoplasmic, membrane, or both; and the intensity of staining (1 to 4). Their study, however, differed from the present report in that they evaluated tumours sequen- tially with each of the above parameters, rather than summing the total aggregate scores. Their study also reported that the c-erbB3 expression was heterogeneous, particularly in diffuse-type gastric tumours.

The most striking feature of the four reports discussed above is that each of the studies has used a different scoring system to evaluate the expression of c-erbB3 protein in tumours; three of the studies indicate that the c-erbB3 expression within an individual tumour is heterogeneous, while only one of' them, on pancreas, states that the expression is homogeneous. Unlike c-erbB2 overexpression in breast carcinomas, where membrane staining has been associated with gene ampli- f i ~ a t i o n ~ ~ and has been shown to have an adverse effect on distant metastasis-free s~rv iva l ,~ ' there have been no data to date to show evidence f'or c-erbB3 gene amplification or for membrane staining influencing survival. Under these circumstances, it is not ideal to rely on a single parameter, such as membrane staining, for scoring c-erbB3 expression in tumours, as this may ignore useful information.

Cytoplasmic immunoreactivity had been considered to be non-specific in the case of c-erbB2 e x p r e s s i ~ n . ' ~ . ~ ~ However, this has been questioned by Kumar r i a/.,45 who showed, using biochemical methods, that even in a cell line expressing high levels of c-erbB2 due to gene amplification, only about 19 per cent of the receptor protein was expressed on the cell membrane, the remain- der being found in the other intracellular fractions. In addition, Coombes et ( 1 1 . ~ ~ have shown that the cytoplasmic staining for c-erbB2 in bladder cancer can be blocked by the immunizing peptide, suggesting that this represents expression of the c-erbB2 protein. These authors have also advocated a scoring system for c-erbB2 based on the percentage of cells expressing the c-erbB2 product and the amount of product in each

The scoring system used in this study was devised to take into account the intensity of staining, the proportion of tumour cells staining positive, and whether or not there is membranc immunoreactivity. Thc cut-of' values have been set to establish a clean definition of overexpression in tumours. Similar scoring systems have been used in the past, even for c-erbB2 protein expres s io~ i .~~ .~*

It is possible that in the future, overexpression of these receptors on bladder cancer cells may be exploited either intravenously or, in superficial tumours, intravesically. We have recently isolated a monoclonal antibody which specifically recognizes the extracellular domain of the c-erbB protein.49 This is being developed in several recombinant forms as a potential therapeutic reagent.

4 1 . 4 6

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Documents

EXPRESSION OF THE TYPE 1 TYROSINE KINASE GROWTH FACTOR RECEPTORS EGF RECEPTOR, c-erbB2 AND c-erbB3 IN BLADDER CANCER