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

Disclosures

Mod Pathol. 2014;27(9):1212-1222. 

In This Article

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.

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 al[11] and Toyama et al[17] 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).[9–20] 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.

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