Abstract 4958: Presence of amphiregulin autocrine-loop predicts sensitivity of <i>EGFR</i> wild type cancers to gefitinib and cetuximab
Authors: Kimio Yonesaka, Kreshnik Zejnullahu, Alison J Homes, Bruce E Johnson, Pasi A Janne
Journal: Cancer Research (2008)

Abstract Background: Epidermal growth factor receptor (EGFR) is a therapeutic target in non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC). Patients with NSCLC with activating mutations in EGFR typically have significant tumor regressions with gefitinib. However approximately 30% of patients treated with gefitinib develop stable disease even without EGFR mutations. EGFR mutations are rare in HNSCC. As EGFR can be activated by one of its ligands (amphiregulin, EGF, TGFα), we examined the relationship of EGFR ligands and the efficacy of gefitinib and cetuximab using EGFR wild type NSCLC, HNSCC cell lines and in tissue samples from NSCLC pts treated with gefitinib or erlotinib. Methods: Amphiregulin (AR), EGF and TGFα in cell culture media were analyzed with ELISA assays in 18 cell lines, 14 NSCLC (4 EGFR mutant; 10 EGFR wild type) and 4 HNSCC cell lines. The in vitro sensitivity was analyzed using MTS assay. EGFR, HER3, Akt, ERK1/2, Cyclin D1 and phosphorylation of these were analyzed with Western blotting. Cell cycle was analyzed by flow cytometry. Tissue samples from 29 pt with EGFR wild type NSCLC were studied using IHC. These are treated with gefitinib or erlotinib. AR expression was scored (0-400) by multiplying extent (0-100%) and intensity (0-4) of staining. Results: TGFα (range, 0 to 4.8pg/ml) and EGF (0 to 2.2pg/ml) was detected at negligible level in all cell lines except Calu3 (TGF; 116.6pg/ml). The presence of AR varied significantly among the cell lines (range, 4.6 to 1625.8pg/ml). All 4 EGFR mutant cell lines had negligible levels of AR (&amp;lt;50.3pg/ml). Seven cell lines of the 14 cell lines with wild-type EGFR had &amp;gt; 250 pg/ml (718.4 to 1625.8pg/ml) of AR and all were sensitive to gefitinib with an IC50 of &amp;lt; 1 μM (range, 0.10 to 0.33). In addition, they were also sensitive to cetuximab (mean growth inhibition following10ug/ml cetuximab 56.8%, range, 33.1 to 87.1 %). In contrast cell lines producing &amp;lt; 250 pg/ml of AR were resistant to both gefitinib (IC &amp;gt; 1 μM) and cetuximab (mean growth inhibition following 10ug/ml cetuximab 1.0%, range -4.4 to 15.3%). The AR producing cell lines underwent G1/S arrest following either gefitinib or cetuximab. AR producing cell lines exhibited dose dependent decrease in p-EGFR, p-ERK 1/2 and cyclin D1 following gefitinib or cetuximab. AR non-producing cell lines had no effect on these proteins following treatment. IHC of tissue samples showed 9/10 samples from pts, developed stable disease.following gefitinib, had high AR expression (cut off score 100), while only 3/19 samples had high AR expression from pts with progressive disease (p&amp;lt;0.001). Conclusion: Our findings suggest that in EGFR wild type NSCLC and HNSCC cell lines the presence of an AR autocrine loop is a major determinant of in vitro sensitivity to gefitinib and cetuximab. AR expression may be a suitable biomarker to select EGFR wild type NSCLC patients likely to benefit from gefitinib or erlotinib.