Introduction
Epidermal growth factor receptor (EGFR) and HER2 are both members of the ErbB tyrosine kinase receptor family which also comprises HER3 and HER4. When stimulated by the presence of a ligand, ErbB receptors homodimerize or heterodimerize with other members of the family, and through transphosphorylation, initiate intracellular signaling cascades [
1]. Although HER2 has no known ligand, it is believed to be a preferred dimerization partner for EGFR and HER3 [
2]. As members of the ErbB family are often deregulated in a number of malignancies [
3], they have been an attractive option for targeted therapy. EGFR is thought to be highly expressed in as many as 90 % of the head and neck squamous cell carcinoma (SCCHN) tumors [
4], while the expression of HER2 is more variable [
5,
6].
One of the earliest agents to target the ErbB network system was cetuximab, a chimeric monoclonal antibody to EGFR. After demonstrating efficacy in preclinical models [
7], cetuximab earned FDA approval for locally advanced as well as recurrent or metastatic SCCHN [
8‐
11]. Another potential therapeutic target is the intracellular tyrosine kinase domain of ErbB receptors. First generation small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib and erlotinib, specifically target the tyrosine kinase of EGFR and are approved for clinical use in lung cancer. Gefitinib demonstrated excellent activity in preclinical SCCHN models [
12,
13]. In phase 2 trials, gefitinib had a response rate of 9–10 % in patients with recurrent or metastatic SCCHN [
14]. Erlotinib had similarly promising preclinical results [
15] and slightly worse response rates in clinical studies [
16].
Afatinib is an irreversible TKI that targets all kinase competent ErbB family members (EGFR, HER2, and HER4; HER3 lacks essential catalytic residues and displays weak to no activity) [
17]. Early in vitro studies of afatinib in human tumor cell lines demonstrated significant activity as well as an increase in the proportion of cells in the sub-G0/G1 phase of the cell cycle [
18,
19]. Effective antitumor activity of afatinib has also been shown in multiple xenograft models using human cell line-derived xenografts and transgenic mouse models [
19,
20]. Phase I trials in patients with nonsmall cell lung cancer (NSCLC) and other solid tumors found afatinib to be well tolerated, with the most common adverse effects being diarrhea, rash, fatigue, and nausea [
21,
22]. Although not designed to determine clinical response, there was a decrease in tumor size in 50 % of patients [
22]. At this point, besides early evaluations assessing afatinib in combination with radiotherapy in one single model system (FaDu) [
23], no work characterizing the effect of afatinib in a panel of SCCHN models has been reported.
Materials and methods
Viability assays
Cell Titer Blue assays (Promega, Madison, WI) were used to determine cell viability. Briefly, cells were seeded at 1000 cells/well on 96-well plates, incubated for 24 h in complete media, maintained in serum-free conditions for 24 h, and treated with afatinib or cetuximab for 72 h at 37 °C. Then, Cell Titer Blue reagent was added to each well and incubated for 2 h at 37 °C before 3 % SDS was added. Cell viability was quantified by scanning absorbance at 570 nm in a microplate reader (Bio-Tek, Winooski, VT).
Tumor xenografts treated with afatinib only or multiple ErbB-targeting agents
Mice were allowed to adjust to conditions at least for 5 days before they were used for experiments. They were housed in Macrolon type III cages in groups of ten under standardized specific pathogen-free (SPF) conditions at 21.5 ± 1.5 °C temperature and 55 ± 10 % humidity. Standardized diet (PROVIMI KLIBA) and autoclaved tap water were provided ad libitum. HN5 or FaDu cells were injected into the right flank of 6-week-old female BomTac:NMRI-Foxn1nu mice (Taconic, Denmark). Tumor take was monitored over time, and animals with established tumors (50–100 mm3) were randomized to the treatment groups (n = 10/group). Mice were treated with either vehicle or afatinib, daily or every other day or in a weekly alternating schedule (afatinib only experiment) or with vehicle or other agents daily (multiple agent experiment). Tumor volumes and body weights were recorded three times a week, and median tumor volumes as well as change in body weight were plotted over time.
Tumor xenografts treated with afatinib and cetuximab
HN5 and SCC25 cell line xenograft experiments were conducted as previously described [
24]. Briefly, female athymic nude mice (Harlan, Indianapolis, IN) were maintained in a pathogen-free animal facility in accordance with the University of Chicago Animal Care and Use Committee. Mice received standard laboratory rodent food and water as desired. All handling procedures were conducted in a laminar flow biosafety hood. At 6–7 weeks of age, mice were injected subcutaneously in the right flank with HN5 or SCC35 cells. Drug treatment was initiated when mean tumor volumes reached 200 to 250 mm
3. Mice were treated with cetuximab at 30-mg/kg body weight two times per week, via i.p. injection and/or afatinib at 12.5-mg/kg body weight once per day via oral gavage, or equal volume of diluents (control). For tumor growth analysis, tumor size was measured with a vernier caliper. Tumor volumes were calculated with the formula V = 0.52 × L × W
2, where L and W represent the length and the width of the tumor (mm). The animals were monitored two times per week for body weight and tumor volume.
Quantification of TGFA, EGFR, and AREG mRNA expression and EGFR copy number by real-time PCR
The real-time PCR method for quantification of
EGFR gene expression and copy number has been reported previously [
25,
26].
FISH assays
FISH assay methods and analysis were previously described [
24]. Briefly, for the evaluation of the
EGFR gene copy number (GCN) alterations, dual-color FISH assays were conducted using an
LSIEGFR SpectrumOrange:CEP7SpectrumGreen Probe mixture (Vysis/Abbott Molecular, Des Plaines, IL).
HER2 amplification was studied using the Vysis PathVysion
HER2 DNA Probe Kit according to manufacturer recommendations (Abbott Molecular, Des Plaines, IL).
CEP7 or
CEP17 probes were used to distinguish true gene amplification from
EGFR or
HER2 gene copy number gain (gene polysomy) and alterations in number of chromosome 7 or 17 homologs. The absolute number of each signal, the mean copy number of signal per cell, the ratios of
EGFR to CEP7 or
HER2 to
CEP17, and the percentage of cells with given copy number of each signal per cell were calculated. Cells with a gene to chromosome signal ratio <2 were considered nonamplified, whereas those with a ratio greater than 2.0 (or ≥15 copies of
EGFR per cells in ≥10 % of cells) were considered as having true amplification. Cells with ratios near cutoff points were equivocal or low amplified.
Western blotting
Western blots on cell lysates were performed as previously described [
24]. Visualization and quantification were performed with Odyssey Infrared Imaging System (Li-Cor Biosciences). Experiments were repeated at least three times. PTEN antibody was purchased from Cell Signaling Technology, Inc. (Danvers, MA). Actin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary goat anti-rabbit IgG IRDye antibody was purchased from LI-COR Biosciences (Lincoln, NE). Secondary mouse IgM IRDye antibody was purchased from Rockland Immunochemicals Inc. (Gilbertsville, PA).
Discussion
In vitro, SCCHN cell lines show a range of sensitivities to afatinib. The four most sensitive cell lines, SCC58, SQ20B, SCC25, and HN5, show amplification of
EGFR by FISH analysis and increased mRNA copy number by qPCR. This suggests that afatinib is most effective in cell lines where EGFR is amplified and possibly acts as a driver of cell growth. EGFR gene copy numbers have not been correlated with clinical activity of EGFR inhibitors in SCCHN; however, this has not been tested in a prospective study. Our data presented here and in the past [
26] indicate that a prospective trial is warranted. When afatinib IC
50 values are compared to those from gefitinib (Table
1), one sees that the order of increasing resistance is almost identical, the exceptions being SCC25 and SCC58, both of which are sensitive to afatinib but resistant to gefitinib. This suggests that afatinib may be a better therapeutic choice for cancers expressing high levels of EGFR and may relate to the broader ErbB inhibitory scope of afatinib as compared to gefitinib. Because gefitinib inhibits EGFR only, while afatinib targets EGFR, HER2, and HER4, one possibility could reside in differences due to HER2 signaling. However, none of the four most sensitive lines show an increased gene copy number for HER2 by FISH ruling out amplification as a possible mechanism. HER3 has been implicated as a mechanism of resistance to EGFR blockade in SCCHN in one paper [
27]. As afatinib has been shown to block transphosphorylation of HER3 in vitro [
19], it may be able to address this potential resistance mechanism. Indeed, afatinib activity was seen in cetuximab refractory patients in a phase II trial [
28] suggesting a lack of cross-resistance in some instances.
PTEN expression was also not at play in afatinib resistance, as expression levels were similar in all cell lines. The main factors which could explain the differences in efficacy are the irreversible binding mode and the anticipated tighter blockade of the ErbB signaling network by afatinib. However, a measure of caution should be used in interpreting in vitro data, as SCC35, which exhibited the highest IC
50 to afatinib (Table
1), was still sensitive to the drug in vivo (Fig.
5).
Gefitinib does block phosphorylation of AKT and ERK in SCC25 and SCC58 [
26, data not shown], possibly pointing to signaling through STAT3 downstream. Elucidation of this mechanism could point to a more specific subset of cases in which afatinib is more effective than gefitinib. De Greve et al. [
29] showed an objective response to afatinib in three patients with mutations in the tyrosine kinase domain of HER2 even after other ErbB targeting treatments had failed. Afatinib may be active in the presence of multiple genetic aberrations, which render other TKIs ineffective.
Indeed, afatinib was more effective than lapatinib, erlotinib, and neratinib in the HN5 tumor xenograft experiment. These three TKIs were only tested on one cell line, limiting broad conclusions about mechanism. However the combination of irreversible binding mode and broad ErbB signaling blockage is provided by afatinib alone among the TKIs tested here.
With these experiments, we were unable to show added benefit forcombination therapy with cetuximab in the most resistant cell lines to afatinib. Afatinib and cetuximab were very effective at arresting growth and decreasing tumor volume in vivo, both as single agents and in combination. In fact, these treatments worked so well that any benefit from the combination of drugs could not be seen.