Background
The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that plays a crucial role in the signal transduction pathway, regulating key cellular functions such as proliferation, angiogenesis, metastasis, and evasion of apoptosis [
1,
2]. EGFR is highly overexpressed in numerous types of human cancers, including lung, stomach, and head and neck cancers, and is a strong prognostic factor [
3‐
6].
Gefitinib, a selective small-molecule EGFR tyrosine kinase inhibitor, is widely used as a second- or third-line therapy for the treatment of patients with advanced non-small cell lung cancer (NSCLC) who failed to respond to standard chemotherapy [
7]. Very recently, the European Medicine Agency has granted marketing authorization for gefitinib in patients with locally advanced or metastatic NSCLC with activating mutations of EGFR in all lines of therapy [
8]. First-line gefitinib was approved in Korea for the treatment of patients with NSCLC who harbor the EGFR mutation [
9]. However, gefitinib-induced interstitial lung disease (ILD) has been reported as a serious adverse effect [
10,
11], in addition to the common adverse effects of gefitinib including skin rash and diarrhea. To avoid the adverse effects and to effectively use the molecular targeted drug, it is necessary to accurately evaluate the tumor response early after the start of treatment. Such an evaluation method enables us to identify patients responsive to gefitinib and determine the treatment strategy: continuation or discontinuation of gefitinib therapy, or even a reduction in gefitinib dose. Indeed, re-administration at a reduced dose is a potential treatment strategy for patients who have once responded to, but later discontinued gefitinib treatment owing to severe adverse effects including ILD. The early and accurate assessment of treatment effects is particularly necessary in these patients. Recently, EGFR mutation, EGFR copy number, and EGFR protein expression are the three EGFR-related biomarkers that have been reported to be associated with the therapeutic benefit of gefitinib [
12]. However, the therapeutic effect of gefitinib is not confined to patients whose tumors harbor EGFR mutation and other predictors of efficacy of this agent. In general, about 80% of NSCLCs with EGFR mutation respond to EGFR-TKIs, whereas 10% of tumors without EGFR mutations do so [
13]. Although this observation provides highly valuable insights into the molecular mechanisms underlying sensitivity to EGFR-TKIs, none of the known clinical or molecular tumor characteristics allows the accurate prediction of tumor response at an early phase of treatment with gefitinib in an individual patient. Therefore, there is a clear need for new approaches to identify patients who will benefit from treatment with EGFR-TKIs. In this respect, imaging techniques that can be used to predict treatment outcome in an early phase of treatment are warranted.
X-ray computed tomography (CT) and magnetic resonance imaging (MRI) have commonly been used to evaluate the anti-tumor effect of cytotoxic and molecular targeted drugs by measuring tumor size. However, these anatomical imaging techniques have limited value because a relatively long time is required to obtain sufficient tumor size shrinkage with successful drug therapies. Thus, patients may have to endure adverse effects [
14] and high medical costs [
15] during the periods of desperate treatment. These limitations could be overcome using functional imaging techniques such as positron emission tomography (PET), because metabolic and physiologic changes in the tumor are likely to precede changes in size [
16]. The quantitative nature of PET also contributes to the accurate determination of functional changes. In fact, PET imaging using 2-deoxy-2-
18F-fluoro-D-glucose (
18F-FDG) is increasingly used to assess early tumor response after chemotherapy [
17]. On the other hand, the thymidine analog 3′-deoxy-3′-
18F-fluorothymidine (
18F-FLT) was also developed as a PET tracer for imaging tumor proliferation in vivo [
18].
18F-FLT uptake has been shown to reflect the activity of thymidine kinase-1 (TK1), an enzyme expressed during the DNA synthesis phase of the cell cycle. Owing to the phosphorylation of
18F-FLT by TK1, negatively charged
18F-FLT monophosphate is formed, resulting in intracellular trapping and accumulation of radioactivity [
19]. Thus, this tracer is retained in proliferating cells through the activity of thymidine kinase. Accordingly,
18F-FLT PET could more appropriately evaluate the effects of signal transduction inhibitors whose main action mechanism is the inhibition of tumor cell proliferation, as compared with
18F-FDG PET [
20]. Measurement of tumor proliferative activity by
18F-FLT PET may enable early and accurate assessment of the response to therapy with molecular targeted drugs [
21].
Taken together, we aim to apply
18F-FLT PET for monitoring the antiproliferative effect of gefitinib. Several studies have shown that
18F-FLT PET is useful for the early evaluation of tumor response to anti-EGFR targeted therapy such as erlotinib and cetuximab [
22‐
24]. However, there have been no studies on the usefulness of
18F-FLT PET for monitoring the antiproliferative effect of gefitinib, except for two reports [
25,
26]. Sohn et al. demonstrated that
18F-FLT PET can predict early responses to gefitinib treatment in patients with advanced pulmonary adenocarcinoma [
25]. The effect of gefitinib on
3H-FLT uptake in vitro was studied previously by Su et al. [
26]. Although several studies have indicated the ability of
18F-FLT or
3H-FLT to detect the effect of gefitinib [
25,
26], whether changes in
18F-FLT uptake can reflect the effect of gefitinib by comparing the level of
18F-FLT uptake with those of other proliferation or predictive markers, such as Ki-67 or phosphorylated EGFR, in an early phase of treatment has not been fully validated under a pathological condition.
Thus, in the present study, to determine whether early changes in 3H-FLT uptake can reflect the antiproliferative effect of gefitinib, we determined the changes in 3HFLT uptake level after the start of treatment at different doses of gefitinib in comparison with those in 18F-FDG uptake, Ki-67 expression, and phospho-EGFR levels in a human tumor xenograft (EGFR-dependent human tumor xenograft model, A431).
Discussion
After the treatment with two different doses of gefitinib, the
3H-FLT uptake levels in the tumor were significantly decreased at an early time point (Table
1). These early changes in tumor proliferation activity were confirmed by our pathological studies that including immunohistochemical staining of the Ki-67 (Figure
2) and phospho-EGFR assay (Figure
3). There was no statistically significant difference in tumor size between pre- and post-treatments in each group. Thus, the measurement of tumor proliferative activity using
3H-FLT may enable early accurate assessmentof the response to therapy with a molecular targeted drug, gefitinib, in human tumor xenografts.
Kawano et al. reported that the phospho-EGFR expression level significantly correlates with the response to gefitinib treatment [
34]. They showed that a high level of basal EGFR activation (phospho-EGFR) is an important indicator of sensitivity to gefitinib. When ligands bind to the receptor, the molecule is phosphorylated (phospho-EGFR) by constitutive tyrosine kinases, activating downstream pathways [
35]. Gefitinib blocks EGFR tyrosine kinases and prevents epidermal growth factor-induced proliferation of cultured cells. It inhibits growth and causes regression in human tumor xenografts overexpressing EGFR [
29]. In our study, these effects of gefitinib were confirmed by the phospho-EGFR assay and analysis of
3H-FLT uptake in the tumor. Namely, phosphor-EGFR expression level was markedly decreased after the gefitinib treatment, which was accompanied by the reduction in
3H-FLT uptake level. Shen, et al. [
36] also reported that the expression level of phospho-EGFR in lung cancer cells treated with gefitinib for 2 days was lower than that in non-treated cells. Su et al. [
26] reported that the growth inhibitory effect of gefitinib was parallel to the inhibition of EFGR phosphorylation in a gefitinib-sensitive cell line (NSCLC H3255). These data strongly support our results in confirming the proof of the mechanism of the EGFR inhibitor gefitinib. Thus, our findings suggest that
3H-FLT can reflect EGFR activation and can be a predictor of the tumor response to gefitinib in human tumor xenograft.
Several clinical trials have demonstrated that
18F-FLT can be used for imaging a various tumor types and that there is strong correlation between
18F-FLT uptake level and proliferation index (Ki-67) in individual tumors [
37‐
40]. Although TK1 is not a specific proliferation marker, TK1 is regulated within the cell cycle [
41], and the
18F-FLT uptake level within tumors usually reflects the fraction of tumor cells in the S-phase, in which the TK1 expression level is the highest. The TK1 activity is high in proliferating cells and low in dormant cells. In our study, the antiproliferative effect of gefitinib was confirmed by the Ki-67 and
3H-FLT uptake in the tumor. Namely, the expression level of Ki-67 was markedly decreased after the gefitinib treatment, which was accompanied by a reduction in
3H-FLT uptake level.
Because
18F-FLT PET findings reflect the proliferation of tumor cells, this method is more suitable for detecting the early therapeutic effect than conventional modalities such as CT and MRI, which are based on sequential measurements of tumor size. Recently, several investigators used
18F-FLT PET to evaluate treatment responses in animal models or humans following molecular targeted therapy [
22,
42]. However, the potentials of
18F-FLT PET for monitoring the antiproliferative effect of gefitinib have not been clarified. Our present findings suggest that
3H-FLT can predict the therapeutic effect of gefitinib at a very early time point (2-days after the start of gefitinib treatment) during which changes in tumor size cannot be detected yet. Su et al. [
26] also showed that a marked decrease (~ 90%) in
3H-FLT uptake in NSCLC H3255 cells was observed 2 days after exposure to two different doses of gefitinib. The in vitro data supported our results in confirming the proof of the mechanism of the EGFR inhibitor gefitinib. Because molecular targeted drugs are used for patients with advanced stage cancer, it is very important to determine their therapeutic effects as early as possible. If the therapeutic effects can be predicted at a very early time point, it will be possible to select the clinically optimal treatment and reduce medical costs in advance. In the present study,
3H-FLT uptake level significantly decreased in a dose-dependent manner after the treatment with gefitinib. If the therapeutic effects can be predicted quantitatively and dose-dependently,
18F-FLT PET can also be applied to evaluate the therapeutic effect of gefitinib re-administration with dose reduction in patients who have once responded to but later discontinued this treatment owing to severe adverse events including ILD. As the precise management of a gefitinib responder having severe adverse events remains to be established,
18F-FLT PET may provide a potential means for the management of gefitinib responders having severe adverse events.
It is very important to compare the level of
3H-FLT uptake with that of
18F-FDG uptake, as
18F-FDG is the most widely used tracer for tumor imaging by PET. In our study, however, the
18F-FDG uptake level in the tumor was not significantly reduced by the treatment with gefitinib. In addition, the
18F-FDG uptake level in the tumor was lower than those in most of the other organs including the heart, brown fat, and kidneys. Mamede et al. reported that the level of
18F-FDG uptake by the tumors in immunodeficient (athymic nu/nu) mice was significantly lower than that in immunocompetent mice [
43]. Therefore, our mouse model, BALB/c athymic nude mice bearing human epidermoid cancer (A431), dose not seem to be suitable for evaluating the potentials of
18F-FDG. Further studies, including comparisons between
18F-FLT and
18F-FDG uptake levels in mouse or rat allograft tumor models and in patients, are necessary to compare the potentials of
18F-FLT PET and
18F-FDG PET and to demonstrate the advantages of
18F-FLT PET for the early and accurate detection of the antiproliferative effect of gefitinib.
It seems better to measure the mice tumors in 3 dimensions (3.14/6 × (length × width × depth)) rather than using the smallest diameter for both the width and the depth (3.14/6 × longest diameter × (smallest diameter)2). Thus, to measure accurate tumor volumes, in vivo studies are necessary. Other limitations of our study were that 3H-FLT was used instead of 18F-FLT and only one tumor model (A431) was used to compare the uptake of 3H-fluorothymidine with the uptake of 18F-FDG, Ki67 and phospho-EGFR after the treatment with two different doses of gefitinib. 18F-FLT and other tumor models should be used to confirm our present results.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SZ designed the experiments, drafted the manuscript and performed the data analysis, immunohistochemical staining of Ki-67, and phospho-EGFR protein immunoassay. YK conceived of the study, performed data analysis and revised the manuscript. YZ and ST helped in the study design, and also acquired and analyzed data. KH, TT, TS, and DH participated in discussion and data interpretation. NT conceived of the study and revised the manuscript. All authors read and approved the final manuscript.