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Journal of Clinical Oncology, Vol 25, No 28 (October 1), 2007: pp. 4405-4413 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.09.8822 Prognostic Significance of Overexpression and Phosphorylation of Epidermal Growth Factor Receptor (EGFR) and the Presence of Truncated EGFRvIII in Locoregionally Advanced Breast Cancer
From the Department of Stem Cell Transplantation and Cellular Therapy, The University of Texas M.D. Anderson Cancer Center, Houston, TX; and the Department of Pathology, the University of Colorado, Denver, CO Address reprint requests to Yago Nieto, MD, Department of Stem Cell Transplantation and Cellular Therapy, Unit 423, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: ynieto{at}mdanderson.org
Purpose The prognostic value of the epidermal growth factor receptor (EGFR) in breast cancer and more specifically, in patients with locoregionally advanced disease, is still undefined. We hypothesized that EGFR status plays a major prognostic role in this setting, through expression, activation, or the presence of its mutated variant EGFRvIII.
Patients and Methods We reviewed tumor samples of 225 patients treated uniformly in prospective trials of high-dose chemotherapy for four to nine positive axillary nodes, Results EGFR expression, phospho-EGFR, and mutated EGFRvIII were detected in 43%, 54%, and 4% of the patients, respectively. EGFR expression correlated with negative hormone receptor status, and was associated with significantly worse relapse-free survival (59% v 79%; P < .001) and overall survival (61% v 81%; P = .001) than no expression. There was no association of phospho-EGFR or EGFRvIII with outcome. Multivariate models confirmed the prognostic effect of EGFR independent of other known prognostic variables in this population. The prognostic value of EGFR was most prominent in the human epidermal growth factor receptor 2 (HER-2) –positive and the estrogen receptor/progesterone receptor–negative subgroups. Conclusion EGFR expression, but not phospho-EGFR or EGFRvIII expression, is an independent adverse prognostic factor in patients with high-risk primary breast cancer, particularly when it is coexpressed with HER-2. Our results suggest the potential benefit of dual EGFR/HER-2 receptor targeting in this setting.
Locoregionally advanced breast cancer is associated with a 50% or greater risk of distant recurrence despite modern multimodal management. Identification of relevant new biologic targets in this population may allow for improvements in outcome. The epidermal growth factor receptor (EGFR) is a new therapeutic target in solid tumors. EGFR is part of the ErbB receptor family, along with human epidermal growth factor receptor 2 (HER-2), HER-3, and HER-4. EGFR is a transmembrane protein with an extracellular EGF-binding domain, a transmembrane region, and an intracellular domain with ligand-activated tyrosine kinase (TK) activity.1 On ligand binding, the erbB receptors form homodimers or heterodimers, which causes phosphorylation of their cytoplasmic TK domain, triggering intracellular signaling that results in increased proliferation, survival, angiogenesis and metastatic potential. Overexpression of HER-2 is an adverse prognostic factor in breast cancer,2,3 as well as in the particular population with high-risk primary tumors.4,5 In contrast, the relationship between EGFR and outcome in breast cancer remains unclear.6,7 Several authors have observed a significant association,8-10 whereas others have not.11-13 Methodology differences, with biochemical ligand-binding assays in early studies and immunohistochemistry (IHC) in more recent studies, absence of multivariate analyses in many reports, and patient and treatment heterogeneity preclude firm conclusions. In addition, it is unclear whether an effect of EGFR would stem from the wild-type receptor or from EGFRvIII, a constitutively activated truncated variant first described in gliomas, which has been shown to increase breast cancer tumorigenicity in vitro.14 Furthermore, little is known about the prognostic effect of its activated phosphorylated form (phospho-EGFR). We hypothesized that EGFR status plays an important prognostic role in high-risk primary breast cancer (HRPBC). To test this hypothesis and to determine the effects of the wild-type, activated, and mutant receptors, we performed retrospective IHC in tumor samples from patients with HRPBC treated uniformly within prospective trials of high-dose chemotherapy (HDC) and subjected to long-term follow-up.
Patient Population Between 1990 and 2001, 264 patients with four to nine positive axillary lymph nodes (n = 93),15,16  10 positive lymph nodes (n = 120),17,18 or inflammatory breast cancer (IBC; n = 51)19 were accrued to phase II and III trials of HDC for HRPBC at the University of Colorado (Denver, CO). The retrospective study of their tumor blocks was approved by the Institutional Review Board, and written informed consent was obtained. Fourteen patients who died from HDC-related complications were excluded from this analysis. Tumor samples from 25 eligible patients could not be obtained. The remaining 225 patients comprised our study group. The organ function and staging tests for the HDC trials were described previously.15-19 Patients received four cycles of doxorubicin-containing standard-dose chemotherapy (SDC) and HDC within 6 months of primary surgery. Patients with IBC received preoperative chemotherapy followed by surgery and HDC.19 Absence of relapse during pretransplantation SDC was required. After collection of hematopoietic progenitor cells, patients received high-dose cyclophosphamide/cisplatin/carmustine.15-19 Post-transplantation treatment included locoregional radiotherapy on platelet recovery and tamoxifen for 5 years in patients with estrogen receptor (ER) or progesterone receptor (PR) –positive disease.
Immunohistochemical Analyses Tissue blocks were sectioned to 5 µm and mounted onto charged glass slides, deparaffinized with xylene, rehydrated through graded alcohols, and incubated in protease-1 for 10 minutes in a 40°C humidity chamber. Endogenous peroxidase was blocked by 3% H2O2 for 15 minutes. Slides were incubated overnight at 4°C with antibodies to EGFR (1:100 dilution), phospho-EGFR (1:100), and EGFRvIII (1:50). Subsequently, sections were developed using 3,3'-diaminobenzidine (DAKO, Carpentaria, CA), counterstained with Harris hematoxylin, dehydrated in graded alcohols, and covered with a cover slip. As negative controls, all sections were processed using corresponding concentrations of a subclass-matched antimouse immunoglobulin generated against unrelated antigens. Paraffin-embedded sections of breast and lung cancer with known EGFR and phospho-EGFR positivity and EGFRvIII-positive glioblastoma multiforme served as positive controls.
Staining intensity was evaluated as 1+ (weak), 2+ (moderate), or 3+ (strong). Samples were graded based on the overall proportion of cells stained with moderate or strong intensity: 0% cells, 0; 1% to 33%, 1+; 34% to 66%, 2+; and 67% to 100%, 3+. Samples with
Statistical Methods
Correlations between categoric and continuous variables were assessed using the Multivariate analyses of RFS and OS used proportional hazards Cox regression models,24 including variables previously identified as independent prognostic factors in our population: nodal ratio (ie, number of involved axillary nodes/number of dissected nodes), combined ER/PR, the pathologic tumor size, and HER-2 status.4,25 Other variables lacking an independent prognostic effect in our patients were excluded from the multivariate analyses (eg, age, menopausal status, family history, histologic grade, ploidy, S phase fraction, multifocality, vascular or lymphatic invasion, extensive intraductal component, or p53 status).25 Likewise, the nodal ratio was the only axillary node–related variable independently associated with outcome, in contrast to the absolute number of involved nodes, involved nodes more than 2 cm, or extracapsular extension.
In the Cox models, tumor size, nodal ratio, and ER/PR were analyzed both separately and as their joint score25: score = (nodal ratio x 3.05) + (tumor size x 0.15) – (ER/PR x 1.19). In this formula, tumor size is entered in centimeters and ER/PR is assigned a value of 1 if positive (ie, if ER and/or PR are positive) and zero if negative (ie, if both are negative). Pretreatment scores The significance of the multivariate models was evaluated with the likelihood ratio test. Individual coefficients were tested using Wald's statistic. The proportionality assumption for the variables was assessed with Kaplan-Meier curves. All P values are two tailed. All statistical analyses used Statview 5.01 software (SAS Inc, Cary, NC).
EGFR status was determined in 225 patients (85% of the eligible population; Table 1). Of them, 96 (42.6%) had positive IHC for EGFR (Figs 1A and 1B), and 43 of these (45%) coexpressed HER-2. EGFR was graded as 1+ in 15 patients, 2+ in 21 patients, and 3+ in 60 patients. The prevalence of EGFR expression was higher in patients with ER/PR-negative disease than in those with ER/PR-positive disease (63% v 28.5%; P < 10–6). EGFR-positive tumors were slightly larger than EGFR-negative tumors (median, 3.8 v 3 cm; P = .02). In contrast, EGFR expression was not associated significantly with age (P = .2), menopausal status (P = .6), HER-2 overexpression (P = .4), p53 expression (P = .15), IBC (P = .9), the absolute number of positive nodes (P = .3), or the nodal ratio (P = .6).
EGFR expression was associated with significantly worse RFS (59% v 79%; P < .001; Fig 2A) and OS (61% v 81%; P = .001) than no expression. No differences in RFS or OS were detected among the different grades of EGFR staining (P = .4 and P = .3, respectively).
Multivariate models included EGFR expression along with HER-2 status, nodal ratio, tumor size, and ER/PR status. In the first model, these last three variables were combined as the scoring system, as described previously. The second model analyzed all five variables separately. Both models showed the independent prognostic effect of EGFR on RFS and OS (Table 2).
EGFR expression had a substantial impact on the subset of patients with HER-2–positive tumors (n = 93). Patients with concurrent EGFR and HER-2 tumor expression had substantially worse RFS (75% v 45%; P = .001; Fig 3A) and OS (78% v 51%; P = .001) than those with HER-2–positive and EGFR-negative tumors. In contrast, differences between patients with EGFR-negative and EGFR-positive tumors within the HER-2–negative group (n = 126) were smaller and did not reach significance for RFS (81% v 72%; P = .18; Fig 3B) or OS (84% v 74%; P = .14). The combined analysis of EGFR and HER-2 allowed for the identification of three risk groups: expression of neither receptor (83% RFS), expression of one of the two receptors (72% RFS), and expression of both receptors (44% RFS; Fig 4A and 4B).
Likewise, EGFR expression had a different effect on the ER/PR-negative and ER/PR-positive subsets. In the former group (n = 91), patients with EGFR-negative tumors had better RFS than those with EGFR-positive disease (77% v 54%; P = .01). The RFS difference between EGFR-negative and EGFR-positive subgroups was less prominent in the ER/PR-positive group (n = 129; 79% v 65%; P = .1). Within this hormone receptor–positive group, the RFS differences according to EGFR status were not significant within the subgroups with PR-positive (n = 102; P = .17) or PR-negative (n = 27; P = .4) tumors. Finally, within the low-score group (n = 164), patients with EGFR-negative tumors had better RFS (83% v 66%; P = .01) and OS (86% v 70%; P = .02) than those with EGFR-positive tumors. In the smaller high-score group (n = 61), the differences between EGFR-negative and EGFR-positive subgroups were of similar magnitude, but did not achieve statistical significance in RFS (61% v 47%; P = .2) or OS (65% v 50%; P = .1). Phospho-EGFR was present in 54% of the samples (7% membranous staining; 90% cytoplasmic; Fig 1C). Its detection was not associated with EGFR expression (P = .8), but was more frequent among HER-2–positive tumors than HER-2–negative tumors (62% v 48%; P = .01). There was no association of phospho-EGFR with RFS (P = .4; Fig 2B) or OS (P = .5). We observed no differences between patients with membranous and cytoplasmic phospho-EGFR staining in RFS (P = .7) or OS (P = .9). We detected the presence of EGFRvIII in only nine (4%) of our patients (Fig 1D). Its presence was not associated with expression of either EGFR or HER-2. These few patients did not present different RFS from the larger EGFRvIII-negative group (P = .6).
EGFR expression had a powerful adverse prognostic effect in our analysis of 225 HRPBC patients. The effect of EGFR status was independent of other known predictors, such as the nodal ratio, tumor size, or hormone receptor or HER-2 status. In contrast, detection of phospho-EGFR was not associated with outcome. Expression of EGFRvIII was rarely present and lacked a prognostic impact. Because current treatment standards for patients with HRPBC include anthracyclines, which have been shown to decrease the prognostic effect of other c-erbB family members,26 our results are important in placing the effect of EGFR in a modern setting. Similar to our data, Buchholz et al27 have reported an adverse effect of EGFR expression in 82 patients with locally advanced tumors treated with anthracyclines. The detrimental effect of EGFR was more pronounced among patients with HER-2–positive tumors. It is well established that overexpression of HER-2 can potentiate EGFR signaling and can contribute to EGFR-mediated transformation and tumor progression.28 Concurrent expression of EGFR and HER-2 in breast tumors may indicate heterodimerization. Preclinical studies have shown a stronger proliferative effect from c-erbB heterodimers than their corresponding homodimers.29,30 Furthermore, HER-2/EGFR heterodimers show ineffective endocytosis and destruction of ligand-bound EGFR, in contrast to EGFR homodimers.31 Our observations confirm prior reports by Suo et al32 and DiGiovanna et al33 in heterogeneous populations with node-negative and node-positive tumors who received various types of postoperative treatment or no postoperative treatments; the worst outcome was noted among those patients with concurrent EGFR/HER-2 expression. A similarly detrimental prognostic effect of their dual detection has also been described in non–small-cell lung cancer.34-36 In keeping with previous reports,10,37 we observed an inverse correlation of EGFR and hormone receptor expression. Furthermore, the prognostic effect of EGFR was more prominent in patients with ER/PR-negative tumors than in those with positive receptors. These findings suggest a mechanistic link between EGFR upregulation and estrogen independence, or incompatibility between the EGFR and ER signaling pathways. The variations in expression of EGFR seem to reside in the transcription-enhancing element located in the first intron of the EGFR gene. This element is stimulated selectively in ER-negative breast cancer cells.38,39 We did not observe a higher expression of EGFR among IBC patients, consistent with previous gene expression array40 and tissue microarray reports,41,42 which suggests that EGFR is unlikely to be primarily responsible for the high proliferation rate that characterizes IBC. Importantly, there was no prognostic effect of EGFR activation in our patients. As reported previously in other tumors,43-46 detection of phospho-EGFR was not associated with EGFR expression. Furthermore, the prevalence of positive IHC for phospho-EGFR in cytoplasm and membrane was higher than for membranous EGFR. The most likely explanation is the internalization and downregulation that membrane EGFR undergoes on ligand binding and phosphorylation,47,48 a process regulated by downstream signaling proteins such as p38 mitogen-activated protein kinase.49 Experiments using the same antiphospho-EGFR mAb (Tyr1086) as in our study have shown that after phosphorylation EGFR is internalized in clathrin-coated pits.50 In contrast to the lack of association of phospho-EGFR and EGFR expression, we noted a direct association between detection of phospho-EGFR and HER-2 overexpression. In a prior report of 46 patients with HER-2–overexpressing metastatic disease, a higher response rate to trastuzumab was noted among patients with detectable phospho-EGFR.51 We studied EGFR activation status determined by phosphorylation of its Tyr1086 residue, which activates signal transducer and activator of transcription 3 (STAT 3)52 and serves as docking site for Src homology–2 or phosphotyrosine binding domains of multiple proteins involved in intracellular signaling, such as the mitogen-activated protein kinase or phosphatidylinositol 3–kinase pathways.1,53 Given that EGFR contains several autophosphorylation sites, it is possible that activation at other sites may have prognostic importance. To our knowledge, there have been no prior prognostic reports of the activation status of EGFR in breast cancer. Our results suggest that the adverse effect of EGFR expression may not be mediated by its own activation and triggering of its intracellular signaling, but rather through heterodimerization and enhancement of HER-2 activation or via cross talk with other pathways. Observations from phase II trials of EGFR TK inhibitors in breast cancer are consistent with this hypothesis. Treatment with erlotinib54 or gefitinib55-57 causes a decrease in tumor EGFR phosphorylation and downstream signaling, which does not result in clinical responses. Taken together, our observations and these clinical studies suggest a lack of dependence of breast tumors on EGFR signaling, and suggest that the greatest benefit from anti-EGFR interventions in breast cancer might derive from therapeutics that target its extracellular domain or interfere with its heterodimerization and enhancement of the signaling pathways of other receptors. In this regard, recent studies have shown substantial activity of the new dual EGFR/HER-2 TK inhibitor lapatinib, even after failure of prior trastuzumab, in patients with advanced tumors that co-overexpressed both receptors,58,59 as well as a more prolonged time to progression when added to capecitabine in patients with metastatic disease.60 Randomized trials of lapatinib in nonmetastatic populations, such as those with HRPBC, are planned or underway. In contrast to our observations of an absence of prognostic effect of phospho-EGFR and its lack of association with EGFR expression, prior reports on phosphorylated HER-2 indicate that it is only detected in patients with HER-2–overexpressing tumors,61,62 and that it carries poor prognosis.33,64 In addition, HER-2 does not undergo endocytosis and downregulation on phosphorylation,29,31 which constitutes another difference from EGFR. We did not detect the variant EGFRvIII receptor in most of our patients. This mutant receptor is a truncated form that lacks exons 2 to 7, resulting in its constitutive activation. It was originally proposed as a potential tumor-specific growth receptor, in contrast to the wild-type form, which is expressed in many normal tissues.14,64 Our IHC observations are consistent with recently reported reverse transcriptase polymerase chain reaction results in breast cancer cell lines and archival clinical samples in which EGFRvIII mRNA was not detected.65 Taken together, these results argue strongly against a meaningful effect of EGFRvIII in breast cancer. Our study has several potential limitations. One of them is the inherent bias of all retrospective archival studies (perhaps lessened in ours by the high sample collection rate and the fact that all samples were assessable for IHC). Another limitation is that, although treatment was uniform, it contained HDC, which is not currently part of the standard management of HRPBC. Although some randomized trials have shown significant superiority of HDC over SDC,66,67 several others have not shown any appreciable outcome differences.18,68-70 In addition, our analysis excluded those patients who experienced relapse during pretransplantation SDC, which could represent a selection bias. However, this subgroup with very poor prognosis represented only 2% of the accrual.18 These limitations notwithstanding, we believe our results can be extrapolated to other HRPBC patients treated with standard multimodal therapy. In conclusion, we found that EGFR expression is an important, independent adverse prognostic factor in patients with HRPBC, with a particularly detrimental effect when coexpressed with HER-2. In contrast, the presence of phospho-EGFR or EGFRvIII is not associated with worse outcome in this population.
The author(s) indicated no potential conflicts of interest.
Conception and design: Yago Nieto Financial support: Yago Nieto Administrative support: Yago Nieto Provision of study materials or patients: Yago Nieto, Roy B. Jones, Elizabeth J. Shpall, Samia Nawaz Collection and assembly of data: Yago Nieto, Fatima Nawaz, Samia Nawaz Data analysis and interpretation: Yago Nieto, Samia Nawaz Manuscript writing: Yago Nieto Final approval of manuscript: Yago Nieto, Fatima Nawaz, Roy B. Jones, Elizabeth J. Shpall, Samia Nawaz
We thank the referring institutions for submitting the tissue sample blocks; Rima Saliba, PhD, for statistical assistance; Wilbur Franklin, MD, James Ho, Marc Lippman, MD, and Ming Tsao, MD, for assistance; and Francisco J. Esteva, MD, for his comments on the manuscript.
Supported by Grant No. 1 R21 CA095762-01 (to Y.N.) from the National Cancer Institute, National Institutes of Health, Bethesda, MD. Presented in part at the 39th Annual Meeting of the American Society of Clinical Oncology, May 31-June 3, 2003, Chicago, IL. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
less than .05, our analyses had 77% statistical power to detect a 15% absolute RFS difference between the high- and low-risk groups, 92% power to detect a 20% RFS difference, and more than 99% power to detect a 30% RFS difference. Thus, our sample was deemed sufficiently large to detect a relevant prognostic effect of EGFR, if present. 
2 or Fisher's exact test, and t test, respectively. Median follow-up times were estimated among surviving patients. RFS was defined as the time from HDC to documented relapse or death without relapse. Overall survival (OS) was defined as the time from HDC to death as a result of any cause. All survival times were analyzed using the Kaplan-Meier method.
