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Value of Epidermal Growth Factor Receptor, HER2, p53, and Steroid Receptors in Predicting the Efficacy of Tamoxifen in High-Risk Postmenopausal Breast Cancer Patients

From the Oncological Research Center, Odense University Hospital, Odense, and Roskilde County Hospital, Roskilde, Denmark; and Gray Laboratory Cancer Research Trust, Northwood, United Kingdom.

Address reprint requests to Ann S. Knoop, MD, PhD, Oncological Research Center, Odense University Hospital, DK-5000 Odense C, Denmark; email: knoop{at}dadlnet.dk

	ABSTRACT

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PURPOSE: Few studies have examined the possible importance of biologic prognostic factors in breast cancer connected with differentiation and growth in predicting response to a specific adjuvant treatment. HER2, epidermal growth factor receptor (EGFR), and p53 have all been suggested as possible markers of tamoxifen resistance. The aim of this study was to investigate interactions between adjuvant treatment with tamoxifen and the content of EGFR, HER2, and p53 in steroid receptor–positive patients.

PATIENTS AND METHODS: A total of 1,716 high-risk postmenopausal breast cancer patients were randomly assigned to treatment with tamoxifen (868 women) or to observation (848 women) in a prospective trial (Danish Breast Cancer Cooperative Group’s 77c protocol). The content of the steroid receptors and expression of p53, EGFR, and HER2 were determined by immunohistochemical analysis of paraffin-embedded tissue. The length of follow-up was 10 years. The end point for this analysis was disease-free survival.

RESULTS: Multivariate analysis demonstrated no increased risk of recurrence after treatment with tamoxifen for HER2-, EGFR-, and p53-positive, high-risk, steroid receptor–positive patients. Patients with steroid receptor–positive tumors and positive immunohistochemical staining for HER2, EGFR or p53 benefited from treatment with tamoxifen for 1 year, although the latter variable contained independent prognostic information by itself.

CONCLUSION: With the statistical power of the present randomized study, we did not find support for the hypothesis that HER2/EGFR or p53 status predicts benefit from tamoxifen treatment in estrogen receptor–positive patients with early-stage breast cancer. Thus, neither HER2, EGFR, nor p53 overexpression/accumulation should be used as a contraindication for giving tamoxifen.

	INTRODUCTION

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ADJUVANT ANTIESTROGEN therapy with tamoxifen has been established as treatment for postmenopausal women with primary breast cancer. Tamoxifen increases overall survival and recurrence-free survival, especially in steroid receptor–positive patients.¹ Furthermore, 5 years of adjuvant treatment is superior to 1 or 2 years,^2-4 and no particular group of steroid receptor–positive patients with resistance to tamoxifen treatment has been identified.

The expression of two members of the epidermal growth receptor family, epidermal growth factor receptor (EGFR) and HER2, in breast cancer cells is regulated by multiple factors and hormones, which modulate growth and differentiation of all cells. Studies have shown that estrogens specifically inhibit HER2 expression and that mammary cells respond to estrogen by modifying the receptor array on their surface, thereby setting their own sensitivity to the different autocrine and paracrine factors. The value of EGFR and HER2 as prognostic factors has been investigated in several studies, but the combined results from these studies remain inconclusive. Few studies have examined the possible importance of factors connected with differentiation and growth, such as EGFR, the related HER2, and the tumor suppressor protein p53, in predicting the efficacy of a specific treatment. The overall pattern is that EGFR,^5-7 HER2,^8-11 and perhaps p53^12,13 may be candidates, either alone or in combination with estrogen receptor (ER), for predicting a reduced response to endocrine treatment, but none of these studies had prediction toward treatment as its primary end point. The present study aimed to investigate interactions between treatment with tamoxifen and the content of EGFR, HER2, and p53 in steroid receptor–positive patients in a retrospective analysis of the Danish Breast Cancer Cooperative Group (DBCG) 77c protocol. In the DBCG 77c study, patients were randomized between observation and systemic treatment with tamoxifen for 1 year.

	PATIENTS AND METHODS

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Patients and Treatment
Patients in the present study (N = 1,716) were postmenopausal high-risk patients enrolled onto the DBCG 77c protocol between August 1977 and November 1982. A woman was defined as postmenopausal when amenorrhoea had persisted for at least 5 years. Patients were designated as being at high risk for recurrent disease when positive axillary lymph nodes were identified, when the tumor was more than 5 cm in diameter, or when the tumor invaded skin or deep fascia. To enter the study, the patients had to have no evidence of advanced disease by physical examination, radiography of the chest and bone, or bone scintigraphy. Furthermore, they were required to have no previous or concomitant malignant disease and to give their verbal informed consent.¹⁴ After total mastectomy and lower axillary dissection, all received radiotherapy and were randomly allocated either to tamoxifen 10 mg three times per day (n = 868) or to observation (n = 848).

The following variables were prospectively recorded: age at operation, histologic type (ductal carcinoma not otherwise specified, lobular carcinomas, or other type), histologic grade according to Bloom and Richardson,¹⁵ pathologically estimated size (largest diameter of the invasive component), surgically estimated size (the largest diameter of the palpable mass before surgery), and numbers of removed and positive axillary lymph nodes. The 27 departments of pathology participating in the DBCG trials performed the histopathologic examinations.

A total of 1,515 paraffin-embedded blocks from the primary breast lesions were obtained from pathology departments, meaning that immunohistochemical information was obtained from 88% of the patients in the study. (Forty-three blocks were lost because of routine destruction of archived tissue after 10 years, four were used in other studies, and 120 were lost for unknown reasons during the long period. In 21 cases, the remaining tumor tissue was insufficient for immunohistochemical handling, and in 12 cases the invasive component could not be identified in the remaining blocks). Slide stained with hematoxylin and eosin, prepared from each block, were used for pathologic confirmation of breast cancer. There was no difference in either disease-free (P = .86) or overall survival (P = .9) between the groups with and without available blocks. The incidence of classical prognostic variables (size, histologic grade, and proportion of positive nodes) was equally distributed between the two treatment arms (data not shown).

Methods
Five-micrometer consecutive serial sections were cut from each paraffin-embedded tumor. The sections were mounted on capillary gap Plus slides (ChemMate kit; Dako, Glostrup, Denmark), dried for 24 to 72 hours at room temperature, and stored in black boxes at 4°C until use. Before the staining procedure, the sections were dried for 60 minutes at 58°C to 60°C, the paraffin was removed with xylene, and the sections were hydrated with graded ethanol and washed in water for 5 minutes. Two sections from each case, the first and last slide, were stained with hematoxylin and eosin by the conventional technique.

Immunohistochemical Staining
When a microwave oven was used to demask antigens, slides were boiled with citrate buffer (pH 6.0) or Tris 10 mmol/L with ethyleneglycoltetra-acetic acid 0.5 mmol/L (pH 9) (T-EG buffer) for 25 minutes at 600 W, depending on the antigen. The slides were cooled in the solution for 15 minutes and rinsed for 5 minutes in water. Before EGFR was demasked, the slides were rinsed with distilled water for 5 minutes. Demasking of EGFR with pepsin was done at 35°C to 37°C for 20 minutes; afterward, slides were also washed for 5 minutes in water. For details about antibodies, dilution, and incubation times, see Table 1.

Interpretation of Staining Results
Only nuclear or distinct membranous immunostaining, depending on the epitope, was interpreted as a positive result. Cytoplasmic reaction, if any, was ignored. As a positive control, a multitissue block with known positive reactions in different tissue, including breast carcinomas, was used. As a negative control, tumor specimens, pretreated in T-EG buffer and microwave oven, were stained under the same conditions but without the primary antibody. Reactions were negative in all cases.

In order to estimate the distribution and thereby the median value of the immunohistochemical variables, some of the specimens (n = 500) were estimated into 10% steps, from 10% to 100%. Ten percent was used as the cut point for distinguishing between positive and negative specimens for EGFR, ER, PgR, and p53. For HER2, it was decided before the start of the study that only tumors with known amplification (examined by Southern blot on cell lines) were to be considered positive.

All of the specimens were semiquantitatively divided into two (EGFR and PgR) or three groups (HER2, p53, and ER) and blinded with respect to the patient outcome. When more than two groups were considered, the positive specimens were divided according to the median value into low and high positive groups. The median value for HER2 was 50%; for p53, 33%; and for ER, 75%. Intra- and interobserver kappa statistics were between 0.72 and 0.83 (data not shown).

Statistical Analysis
The association between the classical prognostic variables and ER, PgR, HER2, EGFR, and p53 (Table 2) was analyzed using the ² test. Univariate analysis was used to screen for putative prognostic factors and predictors, and P values were adjusted for multiple comparisons with Bonferroni statistic (Tables 3 and 4).

View larger version (25K): [in this window] [in a new window]   Fig 1. Numbers of patients: (A) tamoxifen (TAM)-treated and HER2+/EGFR+, n = 51; TAM-treated and HER2-/EGFR-, n = 491; observed (Obs) only and HER2+/EGFR+, n = 58; Obs and HER2-/EGFR-, n = 459); (B) TAM-treated and p53+, n = 51; TAM-treated and p53-, n = 492; Obs and p53+, n = 42; Obs and p53-, n = 476.

Twenty percent of the pathologically estimated tumor sizes were missing, compared with 0.6% of the surgically estimated tumor sizes, probably because the latter was used as an inclusion criterion for the DBCG 77c trial. Also, surgical tumor size had a stronger association with outcome in the multivariate analysis. We therefore chose the surgically estimated tumor size as a prognostic factor in order to avoid loss of information in the final model. The prognostic or predictive value of a given characteristic was quantified by the ratio of hazards rates or relative risk (RR) of failure to control the disease in patients with or without this characteristic. In analyses of the impact of treatment with tamoxifen, DFS was used as end point. Recurrence was a more precise measurement than death because patients often had been treated after their first relapse and the effect of treatment might have confounded the true impact of the variable, DFS was defined as the period from enrollment to relapse or death without a relapse within the first 10 years after surgery.

	RESULTS

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Content and Correlation Between Clinicopathologic and Immunohistochemical Variables
Overexpression of HER2, EGFR, and p53 was seen in 18%, 13%, and 16%, respectively (Table 2). Patients whose tumors had 10% or more of their ER or PgR retained constituted 66% and 43% of the population, respectively. Positive contents of the immunohistochemical variables HER2, EGFR, and p53 were correlated with increasing histologic grade, steroid receptor negativity, and younger age, although not significantly for EGFR. None of the variables was correlated with increasing proportion of positive nodes, and only positive p53 status was significantly correlated with increasing tumor size. The three immunohistochemical variables, HER2, EGFR, and p53, were all closely related (P < .0001; data not shown).

Patients who received adjuvant treatment with tamoxifen were equally distributed with respect to the variables listed in Table 2 (² test, P > .05), except for HER2. Only 8% of the HER2-positive patients received tamoxifen, compared with 14% of HER2-positive patients who received radiotherapy alone (P = .001).

Univariate Analysis
Prognosis. In the univariate analysis (Table 3), all five immunohistochemical variables were able to predict DFS. Patients with steroid receptor–positive tumors had significantly better DFS, but the subdivision of the ER-positive tumors (10% to 75% v > 75%) did not provide further prognostic information. The subdivision of p53-positive tumors provided only little additional information. Positive EGFR and p53 status and high level of HER2 were associated with a significantly reduced 5- and 10-year DFS compared with negative tumors (5-year DFS, 36% v 47%, 30% v 48%, and 38% v 47%, respectively, although the latter difference became insignificant after multiple comparisons were adjusted for). Patients with HER2-negative tumors had the same DFS as patients with low-positive tumors.

The classical variables showed decreased DFS with increasing tumor size, proportion of positive nodes, and histologic grade. Histologic types other than ductal carcinoma not otherwise specified had a 5- and 10-year DFS between those of grade 1 and grade 2 carcinomas. No significant difference in DFS according to age or histologic type of tumor was found (data not shown).

Prediction. Overall, the patients benefited from 1 year of treatment with tamoxifen (P = .02; data not shown). However, in this univariate analysis, when only the steroid receptor–positive patients were included, the benefit was not uniformly distributed among the different patient and tumor characteristics (Table 4). The increased DFS probability was mainly found in patients with HER2-, EGFR-, and p53-negative tumors, whereas no treatment advantages were found in the positive tumors, after the P values were adjusted. The interaction between tamoxifen and positive HER2, EGFR, and p53 status, in the steroid receptor–positive group of patients, is illustrated in the survival curves (Fig 1, A and B). Patients positive for one of the two growth factor receptors were here added together, because the number of patients positive for one of the factors in question (HER2 or EGFR) was too small to yield a survival curve with any useful precision. Despite this combining of the two factors, the number of patients who were positive for HER2 or EGFR and who were treated with tamoxifen was only 51 (5%). Patients with positive HER2/EGFR tumors (n = 109) did not achieve an increased DFS when treated (RR, 0.92; 95% confidence interval [CI], 0.73 to 1.16; log-rank test, P = .5). HER2/EGFR-negative patients (P = .002), p53-positive patients (P = .047), and p53-negative patients (P = .0003) all had significantly increased DFS after treatment with tamoxifen.

Multivariate Model in Steroid Receptor–Positive Patients
Table 6 shows the RR reduction from tamoxifen use in the steroid receptor–positive group when the covariates indicated in Table 7 were accounted for. Tamoxifen alone gave an RR reduction of 27%. Increasing proportion of positive nodes and grade influenced the risk of recurrence, and p53 positivity was the only immunohistochemical variable that retained prognostic information by itself in this analysis, with an RR of 1.54 (95% CI, 1.19 to 1.99). There was no evidence of an interaction between either HER2, EGFR, or p53 positivity and tamoxifen.

	DISCUSSION

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In the daily clinic, relapse and death are usually predicted by the well-established prognostic factors tumor size, lymph node involvement, and histologic grade, and the value of these factors is confirmed by our study. Postmenopausal patients with tumors that are positive for ERs are known to benefit from tamoxifen treatment,² as confirmed in this study, in which tamoxifen treatment for just 1 year was sufficient to show a significant difference in DFS.

In this large-scale randomized study, we found no evidence of an independent ability of HER2, EGFR, or p53 to predict tamoxifen resistance, nor a detrimental effect of tamoxifen use on the risk of recurrence, in postmenopausal, steroid receptor–positive, high-risk patients treated with tamoxifen for 1 year.

As primary end point, no studies have addressed the role of HER2/EGFR as a resistance marker for endocrine therapy in breast cancer. A few studies investigated this issue as a secondary end point in univariate or multivariate analyses. Leitzel et al,¹⁶ Wright et al,¹⁷ Berns et al,⁹ Giai et al,⁸ and Yamauchi et al¹⁸ found, in metastatic disease, that HER2 predicted resistance to hormonal therapy. In the adjuvant settings, this was confirmed by studies by Borg et al,¹⁰ Carlomagno et al,¹⁹ and Bianco et al.²⁰ Studies showing HER2 to have no predictive value, eg, those by Archer et al²¹ and Soubeyran et al,²² have also been published. Elledge et al²³ included only ER-positive patients and found no predictive value of HER2. In a more recent nonrandomized adjuvant study by Berry et al²⁴ of 651 ER-positive patients, tamoxifen treatment improved DFS and overall survival irrespective of HER2 status. These patients received an anthracycline-containing regimen that has been shown to overcome HER2-induced resistance, but the authors found no evidence of a three-way interaction. In the studies of the prognostic impact of EGFR, the proportion of patients who were given endocrine treatment or chemotherapy varied between 19% and 93%.^25,26 In two studies, in which the authors had adjusted for the effect of adjuvant chemotherapy or hormone therapy, EGFR had prognostic significance only in the group that did not receive adjuvant therapy.^27,28 In metastatic disease, EGFR overexpression has predicted tamoxifen resistance, especially in ER-positive tumors.^6,7,29 An overall conclusion regarding steroid receptor–positive patients cannot be drawn from the data published so far. HER2 and EGFR correlate with ER and PgR negativity and degree of anaplasia and are more frequently expressed in younger patients. These parameters are seldom, if ever, comparable in the different predictive studies. Furthermore, as often mentioned, there is no consensus on the best method to use or cutoff value. With these inborn limitations of the applicability of the joined results, we cannot rule out that resistance to tamoxifen or even an increased risk of recurrence might be present in a small group of ER-positive patients.

As already mentioned, strong precautions have to be taken when the predictive value of HER2 is analyzed in ER-positive patients because of the inverse correlation between HER2 and ER. For example, in order to find a significant RR of 1.4 (for a two-sided 5% significance level and 90% statistical power) for the EGFR x tamoxifen variable in the steroid receptor–positive patients with the same study design, more than 3,000 patients with approximately 2,000 events would have to be included in the analysis. A prospective study in which half of the steroid receptor–positive patients remained untreated would of course, with our present knowledge, never see daylight. In the literature, the proportion of ER-positive patients among the HER2-positive patients is approximately 10% (in our study, only 6%). With the lower number of events in the ER-positive group, an enormous group of patients would be required to give enough statistical strength for any clinical conclusion regarding prediction. So far, none of the published studies, including ours, has the required strength.

Second, an increasing number of experimental studies are finding that antiestrogen enhances survival of ER-positive, HER2-overexpressing breast cancer cells, possible by many different mechanisms.^30-34 In HER2-positive tumors, tamoxifen may act by resolving the inhibition of the HER2 amplification controlled by estrogen.^35-37 Experimental in vitro studies have shown a dramatic decrease in HER2 mRNA and protein expression in ER-positive breast cancer cell lines treated with 17-estradiol,³⁸ suggesting that estrogen acts on or by an estrogen-dependent part of the HER2 promoter. Furthermore, an upregulation of HER2 has been seen in ER-positive cells treated with tamoxifen.^37,39 ER-positive breast cancer cells transfected with HER2 cDNA remain sensitive to estrogens, but they are not growth-inhibited by tamoxifen in vitro or in nude mice.⁴⁰

A main concern when trying to interpret results from a retrospective study, especially when using archived tumor specimens, is whether the results are reproducible. The samples used in this study were consecutive and taken from a randomized, nationwide protocol with a long follow-up including many events. Only a small number of the specimens (12%) were lost for analysis. Violation of the criteria set by Simon and Altman⁴¹ for a proper prognostic study was minimized because no attempt was made to search for optimal cut points.⁴²

In the literature, the range of patients found to be positive with an immunohistochemical method was 14% to 56% for EGFR,^43,44 9% to 44% for HER2,^45,46 and 10% to 50% for p53.^47,48 The proportion of EGFR-, HER2-, and p53-positive tumors in this old archived material was 13%, 18%, and 16%, respectively. Although these proportions are at the lower end of the above ranges, the results are acceptable. HER2, p53, and, to some degree, EGFR were correlated with younger age in these specimens. Restricting the analysis to 40- to 60-year-old patients increased the proportion of positive tumors to 17%, 26%, and 21% for EGFR, HER2, and p53, respectively. This is more comparable with the figures obtained with specimens from groups that included pre- and postmenopausal patients. Whether more quantitative analysis of the EGFR and HER2, eg, by fluorescence in situ hybridization, which allows visualization and quantitation of specific genes on a cell-by-cell basis, gives more specific prognostic/predictive information is an important but as yet unsolved question.

PgR and ER were positive in 43% and 66% of the tumors, respectively. As an indicator of the disease stage at surgery, we know from the DBCG database that the proportion of patients with very large tumors (> 50 mm), estimated by the pathologist from 1977 to 1982, was 17%, compared with 8% from 1990 to 1997. The PgR content of only 43% in our specimens can therefore be explained by a higher disease stage at the time of surgery during that period.

The ability of p53 to predict patient outcome independently has been well established by immunohistochemical studies,^13,49-56 although a few studies did not find that p53 was connected to prognosis.^57-60 Few studies have examined the interaction between p53 and tamoxifen. Gasparini et al^61,62 found no benefit from adjuvant tamoxifen in highly angiogenic tumors, irrespective of steroid receptor status, and they hypothesized that genetic changes in p53 could result in enhanced angiogenesis and thus lead potentially to hormone-independent growth. Elledge et al⁶³ found that p53 was not associated with response to tamoxifen in metastatic breast cancer, which again was confirmed by Berry et al²⁴ in an adjuvant study. Bergh et al¹² found that tamoxifen with radiotherapy was without benefit for patients with a p53 mutation, and Berns et al⁶⁴ found that in 401 patients with metastatic disease, p53 was a predictor of tamoxifen resistance.

In a study similar to ours, Silvestrini et al¹³ examined 240 postmenopausal, node-positive, tamoxifen-treated women. Irrespective of steroid receptor status, they found an RR of 2.1 (95% CI, 1.3 to 3.4) for metastasis-free survival and 1.7 (95% CI, 1.1 to 2.7) for recurrence-free survival for patients with p53-positive tumors. This result is comparable to the findings in the present study. Although the univariate analysis hinted that aggressiveness induced by p53 positivity was associated to some degree with tamoxifen resistance, the multivariate analysis in the steroid receptor–positive patients showed that it was more a question of an inherent biologic aggressiveness given by the altered p53 protein.

This large analysis of patients entered onto a randomized study did not support that HER2, EGFR, or p53 status was of importance in steroid receptor–positive patients with early-stage breast cancer as a predictor of benefit from tamoxifen treatment. Unfortunately, this large study does not have enough statistical power to finally confirm or disconfirm whether a small group of steroid receptor–positive patients is resistant to or have a detrimental response to tamoxifen treatment.

	ACKNOWLEDGMENTS

 
Supported by grants from the Clinical Institute, Odense University; grant committee of the Consultancy Council, Odense University Hospital; Clinical Oncological Research Unit, Odense University Hospital; Foundation of Medical Research, County of Funen; Danish Medical Association; Gluds Foundation; and Hjorteberg’s Foundation.

We are grateful to the board of the DBCG for their permission to perform this study. We thank the DBCG secretariat and the pathology institutes throughout Denmark for their assistance, and Ole Nielsen and Lene Nielsen for the meticulous technical supervision and work.

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Submitted November 8, 1999; accepted April 11, 2001.

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