The
epidermal growth factor receptor (EGFR) overexpressed in approximately 80% of non-small cell
lung cancers (NSCLC) is a target for novel
therapeutics. Concurrent chemoradiation is the current standard of care for treatment of patients with locally advanced NSCLC. However, < 20% of patients remain disease-free at 5 years despite this aggressive treatment.
Cetuximab is a humanized
monoclonal antibody that recognizes the human EGFR, and in previous studies, inhibited the growth of EGFR-expressing human
cancer cell lines. In this report, we investigated the cellular and molecular effects of
cetuximab alone and in combination with radiation and/or
chemotherapy in human NSCLC cell lines with varying levels of EGFR overexpression in vitro and in vivo.
EXPERIMENTAL DESIGN: We evaluated the EGFR status of a panel of human NSCLC
cancer cell lines by immunohistochemistry and flow cytometry. We then evaluated
cetuximab effects on growth, cell cycle distribution, and downstream intracellular signaling molecules in this panel of NSCLC
cancer cell lines. NSCLC cell lines were treated with
cetuximab alone or in combination with radiation,
chemotherapy, or chemoradiation to determine the cooperative effects of
cetuximab both in vitro and in vivo in athymic nude mice bearing NSCLC xenografts.
RESULTS:
Cetuximab alone inhibited the in vitro growth of some but not all EGFR-expressing NSCLC cell lines in a dose-dependent manner. Flow cytometric analysis of cell cycle distribution after 24 hours of
cetuximab treatment revealed a shift into the G(0)/G(1) phase of the cell cycle in
cetuximab-sensitive EGFR-expressing cell lines and at concentrations that were growth-inhibitory. There were no cell cycle changes in the EGFR-negative cell lines. After 4 hours of exposure,
cetuximab reduced
epidermal growth factor (
EGF)-induced phosphorylation of EGFR (pEGFR) and HER-2 (pHER2) in
cetuximab-sensitive cell lines but not in
cetuximab-resistant cell lines.
Cetuximab reduced
EGF-induced phosphorylation of
extracellular signal-regulated kinase-1/2 (pERK) in all EGFR-expressing cell lines. In the absence of
EGF,
cetuximab alone increased the level of pEGFR and pHER2 above that seen in untreated control cells in both sensitive and resistant cell lines that were EGFR- and HER2-positive, but not in EGFR- or HER2-negative lines. Despite the
cetuximab-induced increase in phosphorylation of EGFR and HER2, peak
EGF-induced levels of pEGFR and pHER2 were reduced by
cetuximab in the
cetuximab-sensitive lines but not in the resistant lines. Cooperative (combination index values < 1.0) growth inhibitory effects were observed in vitro combination assays with
cetuximab and radiation only in
cetuximab-sensitive NSCLC cell lines. A lack of cooperation was seen in
cetuximab-insensitive NSCLC cell lines. Similar findings were observed with in vitro combination studies of
cetuximab plus
cisplatin or
paclitaxel. In nude mice bearing EGFR-expressing,
cetuximab-sensitive, NSCLC cell line xenografts,
cetuximab plus radiation induced a marked improvement in
tumor growth inhibition over either agent alone. The growth inhibitory effects of
cetuximab-radiation were similar to the growth inhibitory effects of concurrent chemoradiation. Triple combination
therapy of
cetuximab and chemoradiation yielded a nonsignificant advantage in
tumor growth control over doublet combinations (
cetuximab and radiation or chemoradiation) in vivo.
CONCLUSIONS: Similar results in
tumor growth inhibition observed in mice treated with
cetuximab-radiation and
cisplatin-radiation provide a rationale for the clinical investigation of
cetuximab with concurrent radiation in selected patients with locally advanced NSCLC. Local
tumor control and treatment toxicity should be evaluated between
cetuximab-radiation and chemoradiation regimens. Proper patient selection will be critical to the success of such trials and further studies are needed to identify optimal patient selection criteria.