Profiling of EGFR mRNA and protein expression in 471 breast cancers compared with 10 normal tissues: A candidate biomarker to predict EGFR inhibitor effectiveness

Dear Editor, EGFR plays a pivotal role in the control of complex cellular events such as cell growth, apoptosis, differentiation, angiogenesis, migration and metastasis. EGFR mutations, EGFR amplification/overexpression and increased EGFR ligand production can lead to EGFR pathway activation during malignant transformation of many cell types, resulting in resistance to cytotoxic drugs and EGFR inhibitors. EGFR inhibitors are used to treat colorectal and lung cancer but are only beneficial in patients with colorectal tumors harboring wild-type KRAS or lung tumors harboring EGFR mutations. Clinical trials of EGFR-targeting treatments are underway in patients with breast cancer and particularly ‘‘triple-negative’’ breast cancer (TNBC) 3,4 without identified activating EGFR gene alteration and with a still controversial EGFR expression status. We quantified EGFR mRNA by quantitative RT-PCR and EGFR protein by immunohistochemistry (IHC) in a large series of patients who had undergone partial or complete mastectomy for unilateral invasive primary breast cancer. EGFR mRNA was quantified in 471 breast tumors and in 10 normal breast tissue specimens obtained during reduction mammoplasty. EGFR expression was normalized to the housekeeping gene TBP (which encodes the TATA box-binding protein) and the median relative expression level in 10 normal breast tissues was attributed a value of 1. As the EGFR mRNA expression values for all 10 normal breast samples were between 0.6 and 1.6, values of 2 or more were considered to represent overexpression and values of 0.5 or less underexpression in breast cancer tissues. Based on hormonal receptors (HR : ER and PR) and ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) status, we subdivided the population (n 1⁄4 471) into the following four groups: HRþ (ERþ or/and PRþ)/ERBB2þ (n 1⁄4 56), HRþ (ERþ or/and PRþ)/ERBB2 (n 1⁄4 296), HR (ER and PR )/ERBB2þ (n 1⁄4 48) and HR (ER and PR )/ERBB2 (n 1⁄4 71). We also determined the mutation status of PIK3CA, which is the only oncogene mutated in the tyrosine kinase receptor (TRK) -RAS-PI3K-AKT-mTOR signaling pathway in breast cancer and that might thus serve as a biomarker to predict sensitivity to TRK inhibitors. IHC was performed on a subgroup of 88 samples, using mouse monoclonal anti-EGFR clone 2-18C9 (EGFR pharmDx kit; Dako France, Trappes, France). EGFR protein expression status was obtained by comparing neoplastic cells with normal breast cells of terminal ductulo-lobular units. Overall, EGFR mRNA was markedly underexpressed in the breast tumors relative to the normal breast tissues (HRþ/ ERBB2þ: 80%; HRþ/ERBB2 : 89%; HR /ERBB2þ: 73%; HR /ERBB2 : 42%; Tables 1 and 2). EGFR mRNA was overexpressed in only 11% of TNBC (HR /ERBB2 ) carcinomas and in almost none of the tumors in the other three subgroups (Tables 1 and 2). EGFR protein was underexpressed in 52 (59.1%) of the 88 tumors tested (HRþ/ ERBB2þ: 53%; HRþ/ERBB2 : 76.5%; HR /ERBB2þ: 40%; HR /ERBB2 : 27.7%; Tables 1 and 2). EGFR was found normal in 23 tumors (26.1%) and overexpressed in only 13 cases (14.8%), all belonging to the TNBC group. IHC revealed EGFR expression on cell membranes (mainly submembranes)