ABSTRACT BACKGROUND: Epidermal growth factor receptor (EGFR) is overexpressed in a subset of human epidermal growth factor receptor 2 (HER2)-positive breast cancers, and coexpression of HER2

and EGFR has been reported to be associated with poor clinical outcome. Moreover, interaction between HER2 and EGFR has been suggested to be a possible basis for trastuzumab resistance.

METHODS: We analysed the clinical significance of EGFR overexpression and _EGFR_ gene copy number alterations in 242 HER2-positive primary breast cancers. In addition, we examined the

correlations between EGFR overexpression, trastuzumab response and clinical outcome in 447 primary, and 112 metastatic HER2-positive breast cancer patients treated by trastuzumab. RESULTS:

with poor disease-free survival in all patients, and in the subgroup not receiving adjuvant trastuzumab. In 447 HER2-positive primary breast cancer patients treated with adjuvant

trastuzumab, EGFR overexpression was also an independent poor prognostic factor. However, EGFR overexpression was not associated with trastuzumab response, progression-free survival or

overall survival in the metastatic setting. CONCLUSIONS: Epidermal growth factor receptor overexpression, but not high _EGFR_ copy number, is a poor prognostic factor in HER2-positive

primary breast cancer. Epidermal growth factor receptor overexpression is a predictive factor for trastuzumab response in HER2-positive primary breast cancer, but not in metastatic breast

cancer. SIMILAR CONTENT BEING VIEWED BY OTHERS DIFFERENTIAL RESPONSE OF HER2-POSITIVE BREAST CANCER TO ANTI-HER2 THERAPY BASED ON HER2 PROTEIN EXPRESSION LEVEL Article Open access 22

September 2023 PROGNOSTIC SIGNIFICANCE OF HER2-LOW STATUS IN HR-POSITIVE/HER2-NEGATIVE ADVANCED BREAST CANCER TREATED WITH CDK4/6 INHIBITORS Article Open access 17 April 2023 PROGNOSTIC

IMPACT OF HER2-LOW POSITIVITY IN PATIENTS WITH HR-POSITIVE, HER2-NEGATIVE, NODE-POSITIVE EARLY BREAST CANCER Article Open access 11 November 2023 MAIN Epidermal growth factor receptor (EGFR,

ERBB1) expression has been widely studied in breast cancer by immunohistochemistry (IHC), and the frequency of EGFR overexpression in breast cancer is quite variable, ranging from 7 to 43%

(Wrba et al, 1988; Suo et al, 2002; Bhargava et al, 2005; DiGiovanna et al, 2005; Nieto et al, 2007; Koletsa et al, 2010; Lv et al, 2011b; Hwangbo et al, 2013). Epidermal growth factor

receptor overexpression is associated with hormone receptor negativity, large tumour size, high histologic grade and poor clinical outcome (DiGiovanna et al, 2005; Nieto et al, 2007; Koletsa

et al, 2010; Lv et al, 2011b). In particular, coexpression of human epidermal growth factor receptor 2 (HER2) and EGFR is associated with worse survival in patients with HER2-positive

breast cancer (Suo et al, 2002; DiGiovanna et al, 2005; Nieto et al, 2007). Interaction between HER2 and other ERBB coreceptors, such as EGFR and HER3 (ERBB3), has been suggested as a

possible mechanism of resistance to trastuzumab, a humanised monoclonal antibody against the extracellular portion of the HER2 protein (Franklin et al, 2004; Diermeier et al, 2005). Upon

ligand binding, ERBB receptors form homo- or heterodimers, and these phosphorylate their cytoplasmic tyrosine kinase domains and activate intracellular signalling, leading to cell division,

motility, survival and angiogenesis (Yarden and Sliwkowski, 2001). Ritter et al (2007)generated trastuzumab-resistant BT-474 cells _in vivo_; these contained higher levels of phosphorylated

EGFR and EGFR/HER2 heterodimers. Application of the EGFR tyrosine kinase inhibitors, erlotinib and gefitinib, was associated with decreased phosphorylation of HER2, suggesting that

amplification of ligand-induced activation of ERBB receptors through heterodimerisation is a plausible mechanism for resistance to trastuzumab (Ritter et al, 2007). Actually, it was shown

that trastuzumab is not able to block ligand-induced EGFR/HER2 and HER2/HER3 heterodimers (Cho et al, 2003; Franklin et al, 2004). However, the significance of EGFR overexpression for

trastuzumab response is not clear in clinical settings. Gori et al (2009) evaluated the expression of EGFR in 45 HER2-positive metastatic breast cancer patients treated with trastuzumab, and

found that EGFR overexpression was not associated with response to trastuzumab, time to progression or overall survival. In contrast, in another study, EGFR expression was associated with

decreased overall survival of HER2-positive metastatic breast cancer patients treated with trastuzumab (Gallardo et al, 2012). _Epidermal growth factor receptor_ copy number alteration, one

of the mechanisms of EGFR overexpression, is also highly variable in breast cancer, with amplification frequencies up to 24% in triple-negative breast cancer (Bhargava et al, 2005; Gumuskaya

et al, 2010; Koletsa et al, 2010; Shao et al, 2011; Lv et al, 2011a; Martin et al, 2012). Recently, we reported that high _EGFR_ copy number because of _EGFR_ amplification or high polysomy

is an independent prognostic factor for poor disease-free survival in patients with triple-negative breast cancer (Park et al, 2014). However, the prognostic significance of _EGFR_ copy

number in HER2-positive breast cancer is not clear. In this study, we assessed the clinicopathologic significance of EGFR overexpression and _EGFR_ copy number alteration in a large series

of HER2-positive primary breast cancers from two institutions. In addition, we examined the correlation of EGFR expression with trastuzumab response and clinical outcome in HER2-positive

primary and metastatic breast cancer patients treated by trastuzumab. MATERIALS AND METHODS PATIENTS AND TISSUE SAMPLES We used three different sets of breast cancer samples in this study.

First, we retrospectively examined 242 cases of HER2-positive primary breast cancers, which comprised 105 cases operated on at Seoul National University Bundang Hospital (SNUBH) between 2003

and 2009, and 137 cases operated on at Asan Medical Center (AMC) between 2003 and 2004. Expression of HER2 was scored according to the 2007 ASCO/CAP guidelines: 0, no staining; 1+, weak and

incomplete membranous staining in ⩾10% of the tumour cells; 2+, weak to moderate, complete membranous staining in ⩾10% of the tumour cells and 3+, strong, complete membranous staining in

⩾30% of the tumour cells (Wolff et al, 2007). Human epidermal growth factor receptor2 positivity was defined as a score of 3+ in the IHC test, or amplification shown by fluorescence _in

situ_ hybridisation. Baseline characteristics of the patients are summarised in Supplementary Table S1. The second set comprised 447 cases of HER2-positive primary breast cancer treated with

chemotherapy and with adjuvant trastuzumab for 1 year at Asan Medical Center from 2006 to 2011, which were used for a previous study (Lee et al, 2014a). Of the 450 cases analysed in the

previous study (Lee et al, 2014a), three cases were occult breast cancers, and thus were excluded for this study. The third set was 112 cases of HER2-positive metastatic breast cancer

treated with trastuzumab at Seoul National University Bundang Hospital, Seoul National University Hospital and Asan Medical Center from 2001 to 2011 (Lee et al, 2014b). In 80 of the 112

metastatic cases, the tissue originated from the primary cancer site, and in the remaining cases it was obtained from metastatic sites. Trastuzumab was administered as the first-line

treatment for metastatic breast cancer in 99 patients (88.4%). Taxane was used most frequently used in trastuzumab-based combination chemotherapy (in 107 patients, 95.5%), and other agents

(i.e. gemcitabine, capecitabine, and vinorelbine) were combined with trastuzumab in three cases. All cases were independently reviewed by two breast pathologists (SYP and HJL), and the

following histopathologic variables were examined: histologic subtype, T stage, nodal status, Nottingham combined histologic grade and lymphvascular invasion. This study was approved by the

institutional review boards of Seoul National University Bundang Hospital, Asan Medical Center and Seoul National University Hospital; the requirement for informed consent was waived. TISSUE

MICROARRAY CONSTRUCTION For the first set, tissue microarrays (TMAs) were constructed from archival formalin-fixed, paraffin-embedded tissue blocks. Briefly, a representative tumour area

was carefully selected for each tumour from haematoxylin- and eosin-stained sections. The designated zone of each donor block was punched with a 2 mm diameter tissue cylinder, and the sample

was transferred to a recipient block. Each sample was arrayed in triplicate. For the second set, we used TMAs that were constructed in a previous study (Lee et al, 2014a). For metastatic

cases, TMAs were constructed from archival formalin-fixed, paraffin-embedded tissue blocks from resected cases. After selection of a representative tumour area, each donor block was punched

with a tissue cylinder 4 mm in diameter. For biopsy specimen, whole sections were evaluated. IMMUNOHISTOCHEMISTRY AND SCORING Formalin-fixed and paraffin-embedded tissue sections were cut,

dried, dewaxed in xylene and rehydrated through graded alcohol. Epidermal growth factor receptor expression was detected using EGFR pharmDx Kit (Dako, Capinteria, CA, USA) and scored as

follows: 0, no staining, or weak membranous staining in <10% of the tumour cells; 1+, weak membranous staining in ⩾10% of the tumour cells; 2+, moderate, membranous staining in ⩾10% of

the tumour cells; 3+, strong membranous staining in ⩾10% of the tumour cells. Both complete and incomplete membranous staining was accepted. If the triplicate TMA cores yielded different

scores, the highest score was used. In the first set, we classified tumours into two groups according to the staining pattern of EGFR: homogeneous expression (0/1+ or 2+/3+ in all TMA cores)

and heterogeneous expression (mixed pattern of 0/1+ and 2+/3+ in three TMA cores). Oestrogen and progesterone receptors were regarded as positive if there were at least 1% positive tumour

nuclei (Hammond et al, 2010). Cases with 10% or more positive staining were grouped as positive for p53. The Ki-67 proliferation index was defined as the percentage of tumour cells showing

nuclear positivity. FLUORESCENCE _IN SITU_ HYBRIDISATION ASSAYS FOR EGFR Fluorescence _in situ_ hybridisation (FISH) was performed on the TMA 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, USA). Briefly,

4-_μ_m deparaffinised TMA sections were incubated in the pretreatment solution (Abbott Molecular) at 80 °C for 30 min, and then in the protease solution (Abbott Molecular) for 25 min at 37 

°C. Probes were diluted in tDen-Hyb-2 hybridisation buffer (InSitus Biotechnologies, Albuquerque, NM, USA). Codenaturation of the probes and DNA was achieved by incubating at 75 °C for 5 min

in a HYBrite hybridisation chamber (Abbott Molecular) followed by 16-h hybridisation at 37 °C. Posthybridisation washes were performed according to the supplier protocols. Slides were

mounted in 4′,6-diamidino-2-phenylindole/antifade and viewed with a fluorescence microscope. A total of 50 tumour cells were evaluated for each core, and the genetic variables evaluated

were: _EGFR_ gene copy number, chromosome 7 copy number and average _EGFR_ gene : chromosome 7 ratio. The University of Colorado Cancer Center criteria for the _EGFR_ gene were used as

follows: disomy (⩽2 copies in ⩾90% of cells), low trisomy (⩽2 copies in ⩾40% of cells, 3 copies in 10–40% of cells, ⩾4 copies in <10% of cells), high trisomy (⩽2 copies in ⩾40% of cells,

3 copies in ⩾40% of cells, ⩾4 copies in <10% of cells), low polysomy (⩾4 copies in 10–40% of cells) and high polysomy (⩾4 copies in ⩾40% of cells). For gene amplification, the criterion

was a ratio of _EGFR_ to chromosome 7 of ⩾2 or ⩾15 copies of _EGFR_ per cell in ⩾10% of the analysed cells (Cappuzzo et al, 2005). For further analysis, the patients were divided into two

groups according to _EGFR_ copy number as follows: low _EGFR_ copy number (disomy, low trisomy, high trisomy and low polysomy) and high _EGFR_ copy number (high polysomy and gene

amplification). ASSESSMENT OF TRASTUZUMAB RESPONSE FOR HER2-POSITIVE METASTATIC BREAST CANCER Responses to trastuzumab-based therapy were evaluated every 8 to 12 weeks using Response

Evaluation Criteria in Solid Tumours criteria version 1.1 (Eisenhauer et al, 2009). Clinical benefit was defined as complete response, partial response or stable disease over at least 6

months. Time to progression was defined as the time from the initiation of trastuzumab treatment to disease progression, and overall survival as the time from initiation of trastuzumab

treatment to patient’s death from any cause. STATISTICAL ANALYSIS Statistical significance was assessed using Statistical Package, SPSS version 18.0 for Windows (SPSS, Chicago, IL, USA). The

correlation between EGFR overexpression and _EGFR_ gene : chromosome 7 ratio was determined by Spearman’s correlation analysis. The associations of EGFR overexpression or high _EGFR_ copy

number with clinicopathological characteristics of the tumours were analysed using Student’s _t_-test, Fisher’s exact test or the _χ_2 test, depending on the test conditions. Survival curves

were estimated using the Kaplan–Meier 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 included in a multivariate analysis using a Cox proportional hazards regression model, and the hazard ratio (HR) and its 95% confidence interval

(CI) were assessed for each factor. _P_-values of <0.05 were considered statistically significant. All _P_-values reported are two-sided. RESULTS CORRELATION BETWEEN EGFR OVEREXPRESSION

AND EGFR COPY NUMBER ALTERATION IN HER2-POSITIVE PRIMARY BREAST CANCER The EGFR IHC and FISH results obtained in the 242 HER2-positive primary breast cancers are shown in Supplementary Table

S2. Epidermal growth factor receptor protein expression was not evaluable in 5 of the 242 cases, because of the loss of TMA cores. Of the other 237 cases, 149 (62.9%) did not express EGFR

protein (IHC score 0); 30 (12.7%) were scored as 1+, another 30 (12.7%) as 2+ and 28 cases (11.8%) were scored as 3+. Of the 58 cases with EGFR expression of 2+ or more, 27 showed

heterogeneous EGFR expression in three TMA cores. _Epidermal growth factor receptor_ copy number data were available in 232 cases; they were unavailable in the remainder because of the

detachment of TMA cores or inadequate hybridisation. _Epidermal growth factor receptor_ gene amplification and high polysomy were detected in 11 (4.7%) and 13 cases (5.6%), respectively. The

distribution of EGFR IHC scores and _EGFR_ copy number did not differ between the two institutions (_χ_2 square test, _P_=0.723, _P_=0.664, respectively). We compared the results of EGFR

IHC and _EGFR_ FISH in each TMA core. Epidermal growth factor receptor protein overexpression of 3+ and 2+ or more was associated with high _EGFR_ copy number (all _P_<0.001; Table 1). In

a correlation analysis, a weak but significant positive correlation was found between EGFR IHC score and _EGFR_: chromosome 7 ratio (_ρ_=0.282, _P_<0.001). Of the 11 cases in which

_EGFR_ was amplified, five scored as 3+, three as 2+ and three as 0 (Supplementary Table S3 and Figure 1). CLINICOPATHOLOGIC CHARACTERISTICS OF CASES WITH EGFR OVEREXPRESSION OR HIGH EGFR

COPY NUMBER We examined the clinicopathologic significance of EGFR overexpression by two different criteria (3+; 2+ or more), because there is no standardised cutoff point for EGFR

overexpression in breast cancer. Clinicopathologic characteristics of tumours showing EGFR overexpression or high _EGFR_ copy number are summarised in Table 2. Epidermal growth factor

receptor overexpression was significantly correlated with hormone receptor negativity, and higher Ki-67 proliferation index, irrespective of the criteria for positivity (EGFR overexpression

of 3+, _P_<0.001, _P_=0.014, respectively; EGFR overexpression of 2+ or more, _P_<0.001, _P_=0.003, respectively). High _EGFR_ copy number tended to be associated with hormone receptor

negativity, but this did not reach statistical significance (_P_=0.078). PROGNOSTIC SIGNIFICANCE OF EGFR OVEREXPRESSION AND EGFR COPY NUMBER INCREASE At the time of the analysis, the median

follow-up for patients was 98.3 months (range, 1–152 months). There were 41 (16.9%) recurrences and 1 (0.4%) cancer-related death as the first event. In survival analysis, EGFR

overexpression defined as 3+ (_P_<0.001; Figure 2A) and 2+ or more (_P_=0.025; Figure 2B) was associated with worse disease-free survival of the patients. However, high _EGFR_ copy number

was not associated with disease-free survival (_P_=0.521; Figure 2C). The overall survival of the patients was not affected by EGFR expression or _EGFR_ copy number alteration (Figures

2D–F). Pattern of EGFR overexpression (heterogeneous or homogeneous) was not associated with survival of the patients, either. Besides EGFR overexpression (3+, 2+ or more), high T stage and

lymph node metastasis were associated with poor disease-free survival (Table 3). In multivariate analysis including EGFR overexpression (3+), high T stage and lymph node metastasis, both

lymph node metastasis (pN0 _vs_ pN1∼pN3; HR, 5.611; 95% CI, 2.262–13.357; _P_<0.001) and EGFR overexpression (0∼2+ _vs_ 3+; HR, 3.013; 95% CI, 1.481–6.131; _P_=0.002) were found to be

independent prognostic factors of poor disease-free survival. However, in multivariate analysis including EGFR overexpression (2+ or more), only lymph node metastasis (HR, 5.465; 95% CI,

2.235–13.363; _P_<0.001) was an independent prognostic indicator of poor disease-free survival. In the subgroup analyses, according to adjuvant trastuzumab therapy, EGFR overexpression of

3+ was also an independent prognostic factor of poor disease-free survival (0∼2+ _vs_ 3+; HR, 3.065; 95% CI, 1.480–6.348; _P_=0.003) in the subgroup not receiving adjuvant trastuzumab

(_n_=209), but EGFR overexpression of 2+ or more and high _EGFR_ copy number were not associated with disease-free survival. However, as only a small number of patients (_n_=33) were treated

with adjuvant trastuzumab, we could not determine the prognostic and predictive significance of EGFR overexpression in this subgroup. Therefore, we additionally analysed EGFR overexpression

in an independent set of HER2-positive primary breast cancer patients treated with adjuvant trastuzumab. CORRELATION BETWEEN EGFR OVEREXPRESSION AND CLINICAL OUTCOME IN HER2-POSITIVE

PRIMARY BREAST CANCER PATIENTS TREATED WITH ADJUVANT TRASTUZUMAB In the second set, which comprised 447 HER2-positive primary breast cancer patients treated with adjuvant trastuzumab for 1

year, EGFR expression data were not available in five cases, because of the loss of TMA cores. Of the other 442 cases, 42 (9.5%) were scored as 1+, 46 (10.4%) as 2+ and 30 cases (6.8%) as

3+. Epidermal growth factor receptor overexpression also showed correlations with high histologic grade, hormone receptor negativity and high Ki-67 labelling index (Supplementary Table S4).

At the time of the analysis, the median follow-up for patients was 49 months (range, 18–104 months). There were 34 (7.6%) recurrences. In survival analysis, the patients with

EGFR-overexpressing tumour had shorter disease-free survival time than those without it (3+, _P_=0.013; 2+ or more, _P_=0.002; Figures 3A and B). The overall survival of the patients was

also significantly affected by EGFR overexpression, showing poor overall survival (3+, _P_<0.001; 2+ or more, _P_<0.001; Figures 3C and D). In this second set, high T stage, lymph node

metastasis and lymphovascular invasion were also associated with poor disease-free or overall survival of the patients (Supplementary Table S5). In multivariate analysis, both EGFR

overexpression (3+; 2+ or more) and lymphovascular invasion were found to be independent prognostic factors for poor disease-free and overall survival of the patients (Table 4). CORRELATION

BETWEEN EGFR OVEREXPRESSION AND RESPONSE TO TRASTUZUMAB-BASED THERAPY IN HER2-POSITIVE METASTATIC BREAST CANCER IN THE THIRD SET We also examined EGFR overexpression in HER2-positive

metastatic breast cancers treated with trastuzumab-based chemotherapy to evaluate the predictive significance of EGFR overexpression, as well as its prognostic significance. Epidermal growth

factor receptor IHC failed in 5 of the 112 cases. Epidermal growth factor receptor expression was 2+ in 7.5% (8 out of 107) and 3+ in 17.8% (19 out of 107). Epidermal growth factor receptor

overexpression was also associated with hormone receptor negativity (3+, _P_=0.002; 2+ or more, _P_<0.001; Supplementary Table S6). The clinical benefit of trastuzumab treatment did not

differ between the cases in which there was EGFR overexpression and those in which there was not (3+, 84.2% _vs_ 84.3%, _P_=1.000; 2+ or more, 84.6% _vs_ 84.2%, _P_=1.000). Epidermal growth

factor receptor overexpression was not associated with progression-free survival irrespective of the criterion used (3+; HR, 1.177; 95% CI, 0.682–2.033; _P_=0.559 (Figure 4A); 2+ or more,

HR, 1.140; 95% CI, 0.694–1.870; _P_=0.605 (Figure 4B)). Although EGFR overexpression tended to be associated with decreased overall survival, the effect did not reach statistical

significance (3+; HR, 1.733; 95% CI, 0.925–3.244; _P_=0.086 (Figure 4C); 2+ or more; HR, 1.561; 95% CI, 0.870–2.800; _P_=0.135 (Figure 4D)). DISCUSSION Epidermal growth factor receptor

alterations occur at an advanced stage of malignancy characterised by metastatic competence, and EGFR is thought to promote cancer cell migration and invasion (Masuda et al, 2012). In the

present study, we have shown that EGFR overexpression is a poor prognostic factor in two independent sets of HER2-positive primary breast cancer patients. In the first set, patients with

EGFR-overexpressing tumours showed poor disease-free survival, and the poor prognostic impact of EGFR overexpression (3+) was also applied to the subgroup not receiving trastuzumab.

Furthermore, in the second set, EGFR overexpression was associated with poor disease-free and overall survival of the patients treated with adjuvant trastuzumab. Therefore, these results

indicate that EGFR overexpression has a prognostic and predictive value in HER2-positive primary breast cancers. However, there are, at present, no validated scoring systems for EGFR protein

overexpression assessed by IHC. We therefore analysed the clinical significance of EGFR overexpression using two different cutoff values (3+, 2+ or more). We found that prognostic impact of

EGFR overexpression using two different criteria was similar, although not same. Previous studies that showed prognostic significance of EGFR overexpression in HER2-positive breast cancer

also used different cutoff values for EGFR expression (Suo et al, 2002; DiGiovanna et al, 2005; Nieto et al, 2007), and even any membranous staining was found to be significant (DiGiovanna

et al, 2005). However, they used different clones and different staining methods for EGFR IHC. In this study, we used EGFR pharmDx Kit, which is approved for the identification of colorectal

cancer patients eligible for treatment with cetuximab or panitumumab, but any membranous staining or expression of 1+ or more had no prognostic impact on the survival of the patients. Thus,

most reliable cutoff values for EGFR overexpression and its standard staining method should be determined to predict the prognosis of HER2-positive breast cancer patients, but, in the

study, EGFR overexpression of 3+ was found to be most predictive. We also revealed that the clinicopathologic characteristics of EGFR-overexpressing HER2-positive breast cancers were same

irrespective of the cutoff value used. Especially, the association of EGFR overexpression with hormone receptor negativity was clearly demonstrated in the three different sets of

HER2-positive breast cancers. Recently, the Cancer Genome Atlas (TCGA) Network analysed primary breast cancers with various platforms, and showed that there were at least two types of

clinically HER2-positive breast cancers (Cancer Genome Atlas N, 2012). One corresponded to the subtype which is enriched for _HER2_ mRNA and in which there is high expression of receptor

tyrosine kinases including _EGFR_, _HER2_ and _FGFR4_. The other is the luminal mRNA subtype with high expression of _GATA3_, _BCL2_ and _ESR1_, the so-called luminal cluster of genes. In

our study, the correlation between EGFR overexpression and hormone receptor negativity is consistent with the results of TCGA analysis, and suggests that HER2-positive breast cancers have

different features depending on their hormone receptor status. The relationship between EGFR overexpression and high _EGFR_ copy number has not been clearly defined. In this study, analysis

of TMA cores revealed a positive correlation between _EGFR_ gene status and EGFR protein overexpression. In particular, cases with high levels of _EGFR_ amplification (case nos. 196 and 224;

Supplementary Table S3) yielded 3+ EGFR IHC results in all three cores evaluated. However, our data also revealed that EGFR overexpression had low specificity and low positive predictive

value for high _EGFR_ copy number as not all tumours with high _EGFR_ copy number overexpressed EGFR and not all EGFR-overexpressing tumours had high _EGFR_ copy numbers. Furthermore, while

EGFR overexpression was associated with poor prognosis in HER2-positive primary breast cancer patients, high _EGFR_ copy number did not have prognostic significance. These results suggest

that besides increased _EGFR_ gene copy number, EGFR overexpression may be induced by other mechanisms, such as mutation, aberrant transcription or translational modification. Epidermal

growth factor receptor overexpression did not affect the response to trastuzumab and progression-free survival in patients with HER2-positive metastatic breast cancers, although EGFR

overexpression tended to be associated with poor overall survival. In this study, predictive value of EGFR overexpression for trastuzumab response in metastatic setting seems to be different

from that in an adjuvant setting. However, the third metastatic breast cancer set has some limitations; not all patients were treated by the same protocol, and most of the tissues used for

analysis were primary tumours even though there were possibilities of change in EGFR status during tumour progression. In HER2-positive primary breast cancer, EGFR overexpression was

associated with worse survival in the patients not treated with trastuzumab, as well as those treated with adjuvant trastuzuamb. Therefore, the association of EGFR overexpression with poor

clinical outcome in patients receiving adjuvant trastuzuamb cannot be solely explained by resistance to trastuzumab caused by EGFR overexpression. The utility of EGFR overexpression as a

predictive biomarker for traststuzumab response should be validated carefully in further large-scale studies. In the light of the known interplay between EGFR and HER2, EGFR inhibitor

gefitinib has been tried in combination with trastuzuamb to treat patients with HER2-positive metastatic breast cancer (Arteaga et al, 2008; Somlo et al, 2012). However, this combination of

gefitinib and trastuzumab was not found to be more effective than traststuzumab alone. Moreover, EGFR expression was not associated with response to treatment. However, in those studies, the

number of enrolled patients was too small to permit meaningful statistical analysis. Hence, the utility of EGFR overexpression as a predictive biomarker for EGFR-targeted therapy and as a

prognostic factor for HER2-positive metastatic breast cancer should be validated in large studies. There are treatments that may improve the clinical outcome of patients with HER2-positive

breast cancers overexpressing EGFR. A dual EGFR/HER2 tyrosine kinase inhibitor, lapatinib, has been shown to be useful after previous treatments such as trastuzumab in cases with progressive

metastatic HER2-positive disease (Geyer et al, 2006). Fabi et al (2013) evaluated _EGFR_ gene copy number and responses to lapatinib, and identified increased _EGFR_ copy number as a

positive predictive factor for lapatinib response. In addition to lapatinib, other small-molecule inhibitors targeting ERBB family members, such as afatinib and neratinib, are being

investigated in ongoing trials in the context of trastuzumab-resistant metastatic HER2-positive breast cancer (Gradishar, 2013). In conclusion, our study showed that EGFR protein

overexpression is an independent poor prognostic factor in HER2-positive primary breast cancer, and it is also a predictive factor for trastuzumab response in HER2-positive primary breast

cancer, but not in metastatic breast cancer. CHANGE HISTORY * _ 06 JANUARY 2015 This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as

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signalling network. _Nat Rev Mol Cell Biol_ 2: 127–137. Article  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS This research was supported by the Basic Science Research Program

through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant No. NRF-2012R1A1B4000557) and by Seoul National University Bundang

Hospital Research Fund (Grant No. 11-2012-007). AUTHOR INFORMATION Author notes * H J Lee and A N Seo: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department

of Pathology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi, 463-707, Korea H J Lee, A N Seo, E J Kim, M H Jang & S Y Park * Department of

Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul, 138-746, Korea H J Lee & G Gong * Department of Medical Oncology, Seoul

National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam-si, Gyeonggi, 463-707, Korea Y J Kim & J H Kim * Department of Medical Oncology, Seoul National University

College of Medicine, 28 Yeongon-dong, Jongno-gu, Seoul, 110-799, Korea Y J Kim, J H Kim & S-A Im * Department of Surgery, Seoul National University Bundang Hospital, 300 Gumi-dong,

Bundang-gu, Seongnam-si, Gyeonggi, 463-707, Korea S-W Kim * Department of Surgery, Seoul National University College of Medicine, 28 Yeongon-dong, Jongno-gu, Seoul, 110-799, Korea S-W Kim *

Department of Pathology, Seoul National University College of Medicine, 28 Yeongon-dong, Jongno-gu, Seoul, 110-799, Korea H S Ryu, I A Park & S Y Park * Department of Medical Oncology,

Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul, 138-746, Korea K H Jung * Department of Surgery, Asan Medical Center, University of

Ulsan College of Medicine, 88 Olympic-ro 43-gil, Songpa-gu, Seoul, 138-746, Korea H J Kim Authors * H J Lee View author publications You can also search for this author inPubMed Google

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Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Lee, H., Seo, A., Kim,

E. _et al._ Prognostic and predictive values of EGFR overexpression and _EGFR_ copy number alteration in HER2-positive breast cancer. _Br J Cancer_ 112, 103–111 (2015).

https://doi.org/10.1038/bjc.2014.556 Download citation * Revised: 23 August 2014 * Accepted: 02 October 2014 * Published: 28 October 2014 * Issue Date: 06 January 2015 * DOI:

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currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * breast carcinoma * gene amplification * EGFR * HER2 *

trastuzumab * immunohistochemistry