Introduction

Triple-negative breast cancer is a subtype of breast cancer characterized by the absence of expression of estrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2), accounting for 10–20% of all breast cancers.1, 2, 3 Histopathologically, the majority of triple-negative breast cancers are high-grade invasive carcinomas of no special type, metaplastic carcinomas, and medullary carcinomas.3 Triple-negative breast cancer carries a poorer prognosis than other subtypes of invasive breast cancer.1 It is relatively sensitive to chemotherapy, but early relapse and visceral metastases are common and overall survival remains poor.4, 5 In addition, current systemic therapeutic options for triple-negative breast cancer are limited to conventional cytotoxic chemotherapy, whereas non-triple-negative breast cancer, that is, hormone receptor-positive, or HER2-positive breast cancer may have benefit from anti-hormonal or HER2-targeted therapy. The search for specific molecular targets in triple-negative breast cancer and the development of therapeutics for these targets are ongoing.

Epidermal growth factor receptor (EGFR) alterations have been implicated in the pathogenesis and progression of many malignancies including non-small cell lung cancer6, 7 and glioblastoma.8 EGFR is a well-established treatment target for colorectal cancer, non-small cell lung cancer, and squamous cell carcinoma of the head and neck. In breast cancer, EGFR overexpression has been reported in up to 78% of triple-negative breast cancers,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 more than in non-triple-negative breast cancers,12, 15 suggesting that EGFR is a potential therapeutic target for triple-negative breast cancer. EGFR tyrosine kinase inhibitors have yielded insignificant response rates in breast cancer,21, 22, 23 possibly due to the lack of patient selection in these studies; they were not restricted to breast cancers with EGFR overexpression or triple-negative breast cancers. Recently, however, EGFR-targeting monoclonal antibody, cetuximab, has improved outcomes in triple-negative breast cancers.24

EGFR gene amplification, one of the mechanisms of EGFR overexpression, is highly variable and found in up to 24% of triple-negative breast cancer.10, 11, 13, 14, 19, 25 EGFR gene mutation, another mechanism of EGFR overexpression, has been reported to be rare,11, 17, 19, 25, 26 although a recent study reported that it was present in 11% of triple-negative breast cancers.16 EGFR immunoreactivity has been presented as an independent indicator of poor prognosis in patients with triple-negative breast cancer.18, 20 However, there have been no reports on the prognostic impact of EGFR copy number alteration or mutation in triple-negative breast cancer. Intratumoral heterogeneity of EGFR protein overexpression and copy number alteration has also not been studied in triple-negative breast cancer, although it may be associated with responsiveness to EGFR-targeted therapy.

The primary goals of this study were to (1) evaluate the rates and prognostic significance of EGFR copy number alteration and mutation in triple-negative breast cancers and (2) assess intratumoral heterogeneity of EGFR protein overexpression and copy number alteration.

Materials and methods

Patients and Tissue Samples

We retrospectively examined the records of the Department of Pathology, Seoul National University Bundang Hospital from 2003 to 2011 and searched for cases of invasive triple-negative breast cancer using immunohistochemical data for standard biomarkers. Estrogen and progesterone receptors were regarded as negative if there were <1% positive tumor nuclei.27 Expression of HER2 was scored according to 2007 American Society of Clinical Oncology/College of American Pathologist guidelines28 and immunohistochemical scores of 0 or 1+ were regarded as negative. For the equivocal (2+) cases, HER2 negative status was confirmed by fluorescence in situ hybridization (FISH). After excluding cases with initial metastases, we selected 151 invasive triple-negative breast cancers from cases of surgically resected primary breast cancer. Baseline patient characteristics are summarized in Table 1. Hematoxylin- and eosin-stained slides were reviewed for each case, and the following histopathologic variables were determined: histologic subtype, T stage, nodal status, Nottingham combined histologic grade, venous invasion, lymphatic invasion, tumor border, and presence or absence of ductal carcinoma in situ component. All cases were independently reviewed by two breast pathologists (SYP and HJL). The study was approved by the institutional review board of Seoul National University Bundang Hospital (IRB No. B-1005/100-303), which waived the requirement for informed consent.

Table 1 Baseline characteristics of 151 patients with triple-negative breast cancer
Full size table

Tissue Microarray Construction

We used tissue microarrays to evaluate EGFR protein expression and EGFR copy number alteration. All slides were reviewed and the most representative tumor section was selected for each case. Tissue microarrays were conducted in two different ways. At first, we constructed large core (4-mm diameter) tissue microarrays (Superbiochips Laboratories, Seoul, Korea) using 42 cases of triple-negative breast cancer to test EGFR protein overexpression and gene copy number. We then constructed tissue microarrays from three different representative tissue cores (2-mm diameter) of 109 triple-negative breast cancers to evaluate the heterogeneity of EGFR protein expression and copy number alteration.

Immunohistochemical Analyses and Scoring

Expression of standard biomarkers including estrogen receptor, progesterone receptor, HER2, p53, and Ki-67 was evaluated in full sections at the time of diagnosis or in tissue microarray sections for missing data during the study. EGFR and cytokeratin 5/6 were evaluated using tissue microarrays. Tissue sections (4 μm) were cut, dried, deparaffinized, and rehydrated following standard procedures. EGFR expression was detected by using EGFR pharmDxTM (Dako). Immunohistochemical staining for the other biomarkers was performed in a BenchMark XT autostainer (Ventana Medical Systems, Tucson, AZ) using an i-View detection kit (Ventana Medical Systems) for estrogen receptor (1:100; clone SP1; Labvision), progesterone receptor (1:70; PgR 636; Dako), HER2 (1:700; polyclonal; Dako), p53 (1:600; D07; Dako), Ki-67 (1:250; MIB-1; Dako), and cytokeratin 5/6 (1:50; clone D5/16 B4; Dako). EGFR expression was scored as follows: 0, no staining or weak membranous staining in <10% of the tumor cells; 1+, weak membranous staining in ≥10% of the tumor cells; 2+, moderate, membranous staining in ≥10% of the tumor cells; 3+, strong membranous staining in ≥10% of the tumor cells. Both complete and incomplete membranous staining was accepted, and 2+ or more staining was considered to represent EGFR overexpression. If the tissue microarray cores yielded a different score, the highest score for the case was used. For cytokeratin 5/6, cases with any positive membranous staining were defined as positive. For p53, cases with 10% or more positive staining were grouped as positive. For the Ki-67 proliferation index, cases with 50% or more positive tumor cells were regarded as having high indices.

FISH Assays for EGFR

To evaluate EGFR copy number alteration, we performed FISH on tissue microarray samples with commercially available locus-specific and chromosome enumeration probes (CEPs) (LSI EGFR SpectrumOrange probe (7p12) and CEP 7 SpectrumGreen probe (7p11.1-q11.1)) (Abbott Molecular, Des Plaines, IL).

FISH was performed as reported for HER2 amplification.29 Briefly, 4-μm deparaffinized tissue microarray sections were incubated in pretreatment solution (Abbott Molecular) at 80 °C for 30 min, then in protease solution (Abbott Molecular) for 25 min at 37 °C. Probes were diluted in tDen-Hyb-2 hybridization buffer (InSitus Biotechnologies, Albuquerque, NM). Co-denaturation of the probes and DNA was achieved by incubating at 75 °C for 5 min in a HYBriteTM (Abbott Molecular) followed by 16-h hybridization at 37 °C. Post-hybridization washes were performed according to supplier protocols. Slides were mounted in 4′,6-diamidino-2-phenylindole/anti-fade and viewed with a fluorescence microscope.

At least 50 non-overlapping tumor cells were evaluated for each tissue microarray core. EGFR copy number was classified into six categories, as described previously30, 31: disomy (≤2 copies in >90% of cells); low trisomy (≤2 copies in ≥40% of cells, three copies in 10–40% of cells, and ≥4 copies in <10% of cells); high trisomy (≤2 copies in ≥40% of cells, three copies in ≥40% of cells, and ≥4 copies in <10% of cells); low polysomy (≥4 copies in 10–40% of cells); high polysomy (≥4 copies in ≥40% of cells); and gene amplification (presence of tight EGFR gene clusters and a ratio of the EGFR gene to chromosome 7 of ≥2, or ≥15 copies of EGFR per cell in ≥10% of cells). For further analysis, the patients were divided into groups according to EGFR copy number as follows: low EGFR gene copy number (disomy, low trisomy, high trisomy, and low polysomy) and high EGFR gene copy number (high polysomy and gene amplification).

Analysis of EGFR Mutation

DNA was extracted from five formalin-fixed, paraffin-embedded tissue sections (10 μm) containing a representative portion of tumor tissue using the QIAamp DNA FFPE Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. DNA (50 ng) was amplified in a 20-μl reaction containing 10 μl of 2 × HotStarTaq Master Mix (Qiagen), including PCR Buffer with 3 mM MgCl2, 400 μM each dNTP, and 0.3 μM of each primer (Exon 18F: 5′-CCA TGT CTG GCA CTG CTT T-3′, 18R: 5′-CAG CTT GCA AGG ACT CTG G-3′; Exon 19F: 5′-TGT GGC ACC ATC TCA CAA TTG-3′, 19R: 5′-GGA CCC CCA CAC AGC AA-3′; Exon 20F: 5′-GGT CCA TGT GCC CCT CCT-3′, 20R: 5′-TGG CTC CTT ATC TCC CCT CC-3′; Exon 21F: 5′-CCA TGA TGA TCT GTC CCT CA-3′, 21R: 5′-AAT GCT GGC TGA CCT AAA GC-3′). Amplifications of EGFR exons 18–21 were performed using a 15-min initial denaturation at 95 °C; followed by 35 cycles of 30 s at 94 °C, 30 s at 59 °C, and 30 s at 72 °C, and a 10-min final extension at 72 °C. PCR products were purified with a HiYieldTMGel/PCR DNA Extraction Kit (Real Biotech Corporation, Taiwan).

DNA templates were processed for sequencing with ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster, CA) with both forward and reverse sequence-specific primers. Purified PCR products (20 ng) were used in a 10-μl sequencing reaction containing 1 μl BigDye Terminator v3.1 and 0.1 μM PCR primer. Sequencing reactions were performed using 25 cycles of 10 s at 96 °C, 5 s at 50 °C, and 4 min at 60 °C. Sequence data were generated with the ABI PRISM 3730 DNA Analyzer (Applied Biosystems). Sequences were analyzed with Sequencing analysis 5.4. software (Applied Biosystems).

Statistical Analysis

Statistical significance was assessed using Statistical Package, SPSS version 15.0 for Windows (SPSS Inc., Chicago, IL). Concordance of EGFR protein overexpression or EGFR copy number alteration in different tissue microarray cores of a tumor were analyzed by the kappa test. The associations of EGFR protein expression or copy number alteration with clinicopathologic tumor characteristics were analyzed by Fisher’s exact test or the Chi-square test, depending on test conditions. Survival curves were estimated using the Kaplan–Meier product-limit method, and the significance of differences between survival curves was determined using the log-rank test. Covariates that were statistically significant in the univariate analysis were then included in the multivariate analysis using the Cox proportional hazards regression model, and the hazard ratio and its 95% confidence interval were assessed for each factor. P-values <0.05 were considered statistically significant. All reported P-values are two-sided.

Results

EGFR Protein Expression, Copy Number Alteration, and mutation

Of the 151 triple-negative breast cancers, 41 cases (27%) were scored as 3+, 56 (37%) were scored as 2+, 24 (16%) were scored as 1+, and the remaining 30 (20%) were scored as 0 by EGFR immunohistochemistry (IHC) (Figure 1). EGFR FISH revealed gene amplification in 3 (2%) cases, high polysomy in 47 (31%) cases, low polysomy in 69 (46%) cases, high trisomy in 4 (3%) cases, low trisomy in 25 (17%) cases, and disomy in 3 (2%) cases (Figure 1). EGFR mutation was observed in 4 (3%) out of 151 triple-negative breast cancers (Table 2; Figure 2). Three cases had L858R mutation in EGFR exon 21, of which one harbored another mutation, G719A in EGFR exon 18. The remaining case had a V786M mutation in EGFR exon 20, along with EGFR high polysomy and strong (3+) EGFR overexpression.

Figure 1
figure 1

Two representative examples of high EGFR copy number. A case of triple-negative breast cancer with EGFR amplification (a) and strong (3+) EGFR overexpression (b). Another case of triple-negative breast cancer with EGFR high polysomy (c) and strong (3+) EGFR overexpression (d).

Full size image
Table 2 Summary of triple-negative breast cancer patients harboring EGFR mutations
Full size table
Figure 2
figure 2

Missense mutations of EGFR exon 20 and exon 21. (a) Substitution of G to A at mRNA coding nucleotide sequence 2356, resulting in valine to methionine substitution at amino acid codon position 786 (V786M). (b) Substitution of T to G at mRNA coding nucleotide sequence 2573, resulting in leucine to arginine substitution at amino acid 858 (L858R).

Full size image

The results of EGFR IHC and EGFR FISH were compared in each tissue microarray core from 151 cases. EGFR protein overexpression correlated with EGFR gene amplification and high polysomy (P<0.001; Table 3). The sensitivity and specificity of EGFR overexpression (immunoreactivity of more than 2+) for high EGFR gene copy number were 83% and 48%, respectively. The sensitivity and specificity of EGFR IHC 3+ for EGFR gene amplification were 100% and 78%, respectively.

Table 3 Correlation of EGFR protein expression and copy number alterations in each tissue microarray core
Full size table

Intratumoral Comparison of EGFR Protein Expression and EGFR Copy Number

Of the 109 triple-negative breast cancers with three tissue microarray cores, 86 cases with three different cores and 19 cases with two different cores were available for comparison of EGFR protein expression within a tumor; the remainder was excluded owing to the loss of tissue microarray cores. For EGFR FISH, 94 cases with three tissue microarray cores and 9 cases with two tissue microarray cores were available; the remainder was excluded due to FISH failure or core detachment. Intratumoral agreement of EGFR protein overexpression or copy number alteration was analyzed in these cases. Overall, the concordance rate of intratumoral EGFR expression (0, 1+ vs 2+, 3+) was 90% (94/105) with a mean kappa value of 0.836 (P<0.001) (Table 4). The concordance rate of intratumoral EGFR copy number alteration (low gene copy number vs high gene copy number) was 86% (89/103) with a mean kappa value of 0.793 (P<0.001) (Table 5). Specifically, the three cases with EGFR gene amplification showed homogenous EGFR gene amplification and strong (3+) EGFR protein expression in all tissue microarray cores. There was no clinicopathologic difference between cases with intratumoral heterogeneity and those with homogeneity for EGFR protein expression or EGFR copy number alteration.

Table 4 Analysis of intratumoral concordance of EGFR protein expression in triple-negative breast cancers
Full size table
Table 5 Analysis of intratumoral concordance of EGFR copy number alteration in triple-negative breast cancers
Full size table

Clinicopathologic Features According to EGFR Protein Expression, Copy Number, and Mutation

We explored the relationship of EGFR protein expression, copy number gain, and mutation with the clinicopathologic variables of triple-negative breast cancer (Table 6). EGFR overexpression (2+ or 3+) was significantly associated with lower stage (P=0.018). However, EGFR copy number and mutation did not correlate with any clinicopathologic variable. We also investigated the prognostic utility of EGFR alteration. At the time of analysis, the median follow-up was 4.9 years (range, 0.1–9.0 years). There were 7 (5%) loco-regional recurrences, 12 (8%) distant metastases, and 1 (1%) cancer-related death as the first event. In Kaplan–Meier survival analyses, the patients with high EGFR copy number had shorter disease-free survival than those without it (P=0.027; Figure 3; Table 7). However, EGFR overexpression and EGFR mutation were not associated with disease-free survival (Table 7). In multivariate analysis including stage and EGFR copy number alteration, high stage (stage I and II vs stage III; hazard ratio, 2.815; 95% confidence interval, 1.022–7.751; P=0.045) and high EGFR gene copy number (low gene copy number vs high gene copy number; hazard ratio, 2.569; 95% confidence interval, 1.063–6.208; P=0.036) remained as independent prognostic indicators of poor disease-free survival.

Table 6 Clinicopathological correlation according to EGFR alteration in 151 triple-negative breast cancer patients
Full size table
Figure 3
figure 3

Disease-free survival and EGFR copy number in triple-negative breast cancer. Cases with high EGRF copy number show significantly poorer disease-free survival in comparison to subjects with low EGFR copy number.

Full size image
Table 7 Univariate analyses for disease-free survival
Full size table

Discussion

EGFR is frequently overexpressed in triple-negative breast cancer and clinical trials of EGFR-targeting agents are underway in patients with triple-negative breast cancer. However, the rate of EGFR copy number alteration and mutation, and the underlying mechanisms of EGFR overexpression are unclear, and their prognostic significance is poorly defined in triple-negative breast cancer. We evaluated the rates of EGFR gene alteration, their clinical implications in prognosis, and the intratumoral agreement of EGFR protein overexpression and gene copy number in triple-negative breast cancer.

EGFR gene amplification and high polysomy were reported in up to 24% and 27% of triple-negative breast cancer, respectively (Table 8).10, 11, 13, 14, 19, 25 However, the frequency of EGFR gene amplification is quite variable, even though most studies used the same, University of Colorado Cancer Center criteria.30, 31 We also assessed EGFR amplification by the University of Colorado Cancer Center criteria and found that EGFR amplification was quite rare, being present in only 2% of cases, which is consistent with some previous studies.10, 19, 25 However, EGFR high polysomy was found in 31% of cases, which is relatively higher than in previous studies. This result may be related to the heterogeneity of high polysomy in some cases, which will be discussed later. The frequency of EGFR gene copy number gain in the previous studies appears to be greater in a subset of triple-negative breast cancers, ie, metaplastic carcinoma and triple-negative breast cancer with basal-like feature. In this study, the rate of high EGFR gene copy tended to be higher in metaplastic carcinoma than the other tumors (58% vs 30%, P=0.063, data not shown); however, there were no significant differences in EGFR copy number gain between basal-like and non-basal-like, triple-negative breast cancers.

Table 8 Review of previous studies on EGFR alterations in triple-negative breast cancer
Full size table

The correlation between EGFR protein expression by IHC and EGFR gene copy number gain is controversial. Several groups that used in situ hybridization technique reported significant correlations between EGFR protein overexpression and high gene copy in triple-negative breast cancer.10, 13, 14, 19 However, Martin et al11 and Toyama et al17 showed no correlation between EGFR immunoexpression and increased EGFR gene copy number. In this study, EGFR overexpression was generally correlated with high EGFR copy number. However, about half of the cases with low EGFR gene copy number showed EGFR overexpression. Of the 202 tumor cores showing EGFR overexpression, only 76 (38%) revealed high EGFR copy number. Even if EGFR overexpression was defined as IHC 3+, only 44 (55%) of 80 cores with EGFR IHC 3+ showed EGFR high polysomy or gene amplification. That is, the specificity and positive predictive value of EGFR overexpression for high EGFR gene copy number were relatively low. These findings are contrary to the close correlation between HER2 overexpression and HER2 amplification; HER2 overexpression is mostly attributable to HER2 gene amplification.28, 32 Moreover, eight cases (11 tumor cores) with no EGFR immunoreactivity had high EGFR polysomy. Therefore, EGFR IHC alone has limited value in defining the group of triple-negative breast cancer patients with increased EGFR gene copy number.

Most studies encompassing Caucasian, European, and Japanese patients report a lack of EGFR mutation in triple-negative breast cancer (Table 8).11, 17, 19, 25, 26 However, Teng et al reported the presence of EGFR mutation, specifically exon 19 deletions and exon 21 missense (L858R) mutations, in 11% (8/70) of triple-negative breast cancer samples from predominantly Chinese patients.16 In this study, EGFR mutation was found in 3% (4/151) of triple-negative breast cancer samples from Korean patients. As suggested by Lamy and Jacot,33 wide variations in the rate of EGFR mutation in different populations may reflect geographic or ethnic differences in the presence of EGFR mutation. However, most studies encompass small series; well-organized, large-scale studies are needed to determine the origin of EGFR mutation variation in triple-negative breast cancer. Of the four cases with EGFR mutation, three carried a missense mutation of exon 21 (L858R), one of which with a coexisting missense mutation of exon 18 (G719A), and remaining case carried a missense mutation of exon 20 (V786M). Missense mutations such as G719A/S and L858R and exon 19 deletions are well-known predictors of sensitivity to tyrosine kinase inhibitors in non-small cell lung cancer,34 so the presence of L858R and G719A mutations in this study population suggests gefitinib or erotinib therapy may be beneficial in these selected triple-negative breast cancer patients. In our study, there was disagreement between EGFR immunostaining and the presence of EGFR mutation, similar to the report of Teng and colleagues,16 suggesting that positivity in EGFR IHC cannot predict EGFR mutation in triple-negative breast cancer.

In this study, high EGFR copy number was significantly associated with poor disease-free survival and acted as an independent poor prognostic factor. Although EGFR overexpression has been presented as a poor prognostic indicator in triple-negative breast cancer,18, 20 high EGFR copy number has no reported prognostic impact in patients with triple-negative breast cancer. The mechanism by which high EGFR copy number contributes to the progression of triple-negative breast cancer is unclear. However, EGFR copy number gain might be one of the accumulating genetic alterations during tumor progression and EGFR activation induced by EGFR copy number gain may contribute to tumor aggressiveness in triple-negative breast cancer. It was suggested that EGFR activation drives migration and invasion of tumor cells through epithelial–mesenchymal transition and alters chemosensitivity by rewiring the apoptotic signaling network.35 However, the utility of high EGFR copy number as a predictive biomarker for EGFR-targeted therapy and as a prognostic factor for triple-negative breast cancer should be validated in large studies.

EGFR protein expression in triple-negative breast cancer is quite variable, ranging from 13 to 78% (Table 8).9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 In this study, we used EGFR pharmDxTM (Dako), which is an approved anti-EGFR antibody for identification of colorectal cancer patients eligible for treatment with cetuximab or panitumumab. Moderate to strong membranous staining in >10% of tumor cells was regarded as EGFR overexpression and was found in 97 (64%) of 151 triple-negative breast cancer cases, comparable to previous studies. However, in contrast to previous studies suggesting prognostic implications of EGFR overexpression in triple-negative breast cancer,18, 20 we did not observe a survival difference associated with EGFR protein expression. This conflicting result may be attributable to differences in the antibodies used and different definitions of EGFR overexpression.

We evaluated intratumoral concordance of EGFR protein expression and gene copy number alteration in triple-negative breast cancer using different tissue microarray cores within a tumor. EGFR protein overexpression and EGFR copy number alteration were highly concordant in different tumor areas within each triple-negative breast cancer. All tumors with EGFR gene amplification demonstrated homogeneous EGFR protein overexpression within a tumor, implying triple-negative breast cancer patients with EGFR gene amplification would be excellent candidates for EGFR-targeted therapy. On the other hand, 12 of 30 cases with high polysomy showed discordant copy number results in at least one core of three tumor cores. It appears that EGFR high polysomy is relatively heterogeneous within a tumor, possibly due to the chromosomal instability of triple-negative breast cancer.

In summary, EGFR mutation was a rare event in triple-negative breast cancers, but high EGFR copy number including EGFR amplification and high polysomy was relatively frequent and correlated with EGFR overexpression. Intratumoral heterogeneity of EGFR protein overexpression and EGFR copy number alteration was not significant. More importantly, high EGFR copy number was associated with poor clinical outcome of the patients with triple-negative breast cancer. Our results suggest that evaluation of EGFR copy number can be useful for predicting outcomes in patients with triple-negative breast cancer and selecting patients for anti-EGFR-targeted therapy.