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


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.

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).

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).

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.

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.

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.

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.