Immunohistochemical Characterization of Three Monoclonal Antibodies Raised against the Epidermal Growth Factor and Its Receptor in Non-Small-Cell Lung Cancer: Their Potential Use in the Selection of Patients for Immunotherapy

Abstract

Adequate methods to identify which lung cancer patients are most likely to benefit from the targeted drugs against both epidermal growth factor receptor/epidermal growth factor (EGFR/EGF) are needed. For this reason, we evaluated both the tissue reactivity of ior egf/r3 monoclonal antibody (Mab) in human lung carcinomas and its biological activity in NCI-H125 cells. Additionally, we assessed the tissue expression of EGF using two Mabs, CB-EGF1 and CB-EGF2. The overexpression of EGFR was detected in 33.33% and 62.71% of small-cell lung carcinoma (SCLC) and non-small-cell lung carcinoma (NSCLC), respectively. The ability of ior egf/r3 Mab to bind the extracellular domain of EGFR inhibiting cell proliferation and inducing apoptosis in NCI-H125 cells was also demonstrated. The EGF expression was observed in about 17% and 70% of SCLC and NSCLC, respectively. However, differences in the reactivity of CB-EGF1 and CB-EGF2 were evidenced. A dual expression of EGFR and EGF was observed in 16.67% and 57.63% of SCLC and NSCLC patients, respectively. But, a correlation between them was only obtained in NSCLC. Our results permit to recommend the development of diagnostic kits using ior egf/r3 and/or CB-EGF1 Mabs in order to achieve a better selection of patients to EGFR/EGF-targeting treatment.

Figure 1

Kaplan-Meier estimate of overall survival among NSCLC patients showing different stages of the disease (P = 0.0405; Log rank test).

Figure 2

Kaplan-Meier estimate of overall survival among NSCLC patients showing different level of EGFR expression (P = 0.9560; Log rank test).

Figure 3

Hematoxylin and eosin staining of squamous cell carcinoma of the lung (a). Note: the intense reaction of ior egf/r3 Mab is mainly located in cell membrane and also in the cytoplasm of malignant epithelial cells (b) (inset on the upper-right corner, 1000x magnification). An intense immunostaining with both CB-EGF1 and CB-EGF2 Mabs was also evidenced (c and d, resp.). Black bar =100 μm.

Figure 4

Antiproliferative activity of ior egf/r3 Mab in cultures of NCI-H125 cells. (a) Known numbers of NCI-H125 cells were treated with the indicated concentrations of anti-EGFR Mab and cultured for 72 h. The maximum of cell growth inhibition was obtained at 100 μg/mL. Data points, means of triplicate samples; bars, SE. (b) Cells cultures were incubated for 72 with ior egf/r3 Mab at 100 μg/mL following 3 different treatment designs (Materials and Methods). See: The statistical significant differences between treatment 1 and 3 (S phase, 0.0149; ANOVA). (c) and (d) Representative flow cytometry histograms of the NCI-H125 cells treated with the irrelevant Mab and with ior egf/r3 Mab, respectively. (d) Note an increase in the percentage of cells undergoing apoptosis (area in gray color) as well as the decrease in the percentage of cells in S phase (area in blue color).