High EGFR Gene Copy Number Predicts Poor Outcome in Triple-negative Breast Cancer

Heae Surng Park; Min Hye Jang; Eun Joo Kim; Hyun Jeong Kim; Hee Jin Lee; Yu Jung Kim; Jee Hyun Kim; Eunyoung Kang; Sung-Won Kim; In Ah Kim; So Yeon Park


Mod Pathol. 2014;27(9):1212-1222. 

In This Article

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 guidelines[28] 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.

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 previously:[30,31] 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.