Background
Prostate cancer (PC) is among the most frequently diagnosed solid tumours in men, and the metastatic forms still represent the second leading cause of cancer-related death [
1,
2]. Treatment of PC by radical prostatectomy, radiotherapy and anti-androgen therapy results in long term survival in patients with localized and androgen-dependent PC. By contrast, hormone-refractory prostate cancer (HRPC) forms are associated with disease relapse and poor patient survival [
3,
4]. At present, increasing serum prostate-specific antigen (PSA) levels following treatment of primary PC is used to identify PC biochemical relapse, a condition that anticipates clinically detectable tumour progression. The identification of novel biomarkers that predict the risk of relapse or that could be used as therapeutic targets is needed.
The molecular mechanisms responsible for PC development, progression and hormone-independence are not clear yet. Several findings suggest that alterations of different pathways involving growth factor receptors play a role in this multistep process [
5,
6]. In particular, the Epidermal Growth Factor Receptor (EGFR) is frequently overexpressed in PC and this is associated with a more aggressive clinical outcome. EGFR overexpression has also been linked to the transition from androgen-responsiveness to the androgen-independent/hormone-refractory phenotype [
7,
8]. Furthermore, preclinical data have suggested that the EGFR signalling pathway can activate the androgen receptor under conditions of clinical androgen deprivation [
9]. EGFR has thus assumed considerable importance, due to overexpression in different tumour types and to its role as a drug target. A variety of anti-EGFR drugs are currently Food and Drug Administration-approved or under evaluation in clinical trials. These drugs include small inhibitory molecules such as gefitinib or erlotinib, as well as antibodies such as cetuximab and panitumumab. Gefitinib is an oral anilinoquinazolone compound that blocks the EGFR tyrosine kinase (TK) activity [
10] resulting in the inhibition of downstream signalling pathways. Clinical evidence, mostly deriving from non small cell lung cancer (NSCLC) patients, demonstrated that activating mutations in the EGFR TK domain (exons from 18 to 21) predict response to gefitinib [
11].
A recent study identified 4 novel missense mutations in exons 19, 20 and 21 of the EGFR TK domain in Korean and Caucasian PC patients. Three of them, G735S, G796S and E804G, led to an oncogenic activation promoting cell proliferation and invasion [
12].
Preclinical studies have shown activity of gefitinib against PC cell lines and xenografts [
13]. In a phase I clinical trial, 252 patients with different solid tumours, including 28 patients with HRPC, received oral gefitinib [
14]. One patient with HRPC had a measurable reduction of disease in a lymph node metastasis, palliation of disease-related pain, and a reduction in PSA [
15]. In another randomized phase II clinical trial 82 HRPC patients were treated with prednisone plus gefitinib or prednisone plus placebo [
16]. This study showed limited antitumour activity of gefitinib in HRPC patients. However, patients were not selected on the basis of EGFR status. At present, no clinical data are available on EGFR-mutated PC patients treated with gefitinib.
In an effort to provide a rationale for further studies of targeted therapies in PC, we set out to analyze EGFR protein expression, EGFR mutations and their possible correlations with clinical parameters and outcomes. For 51 of these samples we also carried out gene expression profiling with oligo-microarrays.
Discussion
In this study, we were interested in analysing EGFR expression and somatic mutations of its TK domain in prostatectomy specimens from patients with operable PC. Metastatic PC represents a challenge for the oncologist and novel therapeutic strategies are therefore warranted, especially when it evolves into the hormone-refractory status. Previous observations have pointed to deregulated EGFR function as a potentially relevant phenomenon in sustaining PC progression and the development of a hormone-refractory phenotype [
7,
8]. However, EGFR targeting agents have shown limited clinical activity in clinical trials conducted in PC patients [
16,
26‐
29]. Notably, these studies enrolled unselected patients with respect to EGFR status. EGFR targeting with TKIs, such as gefitinib and erlotinib [
10,
30], has proven successful in molecularly defined subsets of patients with non-small cell lung cancer (NSCLC), where specific mutations of the EGFR tyrosine kinase domain, but not protein expression, were predictive of the clinical efficacy [
31]. Defining the relationship between EGFR status and the clinical behaviour of PC may therefore help define a possible therapeutic role of EGFR inhibitors in this patient subset.
In our study, we found that EGFR was expressed at normal levels in healthy prostate cells in 90% of the surgical specimens. In tumour areas, EGFR overexpression was found in 36 patient specimens (36%). In line with the literature, we confirmed that EGFR overexpression was significantly associated with biochemical relapse. Furthermore, although confirmation in a larger dataset is needed, EGFR overexpression predicts TTBR independently from Gleason Score, which is an established prognostic factor in this disease.
Having only analysed an Italian Caucasian population, and due to a small sample size, this incidence of EGFR overexpression is difficult to generalize. It has been shown, for example, that Afro-American patients affected by PC expressed EGFR at higher levels than Caucasian patients [
32].
Mutational analysis revealed that 13 out of 100 patients had tumours carrying mutations in the EGFR TK domain. Two of these mutations (exon 19 T751I and exon 21 G863D) have been previously identified as predictors of response to small TKIs in NSCLC patients [
22,
23]. Another one, the V851I mutation, has been previously described in NSCLC patients refractory to TKIs treatment [
24]. The E804G mutation that we found on exon 20, was previously described by Cai et al. in PC specimens [
12]. These authors identified four novel somatic mutations in the EGFR tyrosine kinase domain of PC patients: G735S, G796S, E804G and R841K. They investigated their oncogenic potential by establishing stable clonal NIH3T3 cells expressing these mutations and WT EGFR to determine their ability to increase cell proliferation and invasion. Amongst them, the E804G mutation resulted as the most active and significant somatic missense mutation in the TK domain, being associated with the greatest growth potential, and by the highest transformation and invasion ability [
12]. Q820R, G796V, P782L, F788L, A839V, L828M, F856Y, F856L are novel mutations and further exploration of their effect on the activation of the downstream EGFR pathway is warranted.
Gene expression profile analysis failed to identify genes that were differently expressed in tumours with high vs low levels of EGFR, or in EGFR mutated vs non mutated tumours. The small sample size and the heterogeneity of mutations may have limited the ability of this analysis to capture significant gene expression patterns. Interestingly, when we restricted the analysis to EGFR-overexpressing tumours (EGFR
high), we could identify a 79 gene signature distinguishing mutated from non mutated samples. Three biological processes (lipid and primary metabolic processes and the inflammatory response) are altered and they may help discriminate between the two classes. Further studies could reveal the role of EGFR mutations in the regulation of these specific genes. Another 29 genes were found to be differently modulated in mutated tumours according to their EGFR expression level (high vs low). Four of the genes that were expressed at lower levels in EGFR
high samples have been previously linked to PC. The expression of U19/EAF2 in xenograft prostate tumours markedly induced apoptosis and inhibited tumour growth
in vivo [
33]. Moreover, the disruption of androgen-dependent growth regulation via U19/EAF2 down-regulation has been found to be commonly associated with PC progression. These observations suggest that U19/EAF2 may act as a tumour suppressor gene. Another finding revealed that the prostate specific ATP-binding cassette transporters ABCC4 was found to be expressed at higher levels in PC than in benign prostate tissue and decreased expression was found after androgen ablation [
34]. Zhao et al. found a marked reduction in KLK3 expression levels in androgen-independent, compared with androgen-sensitive PC cell lines [
35]. Finally, it was demonstrated that ANXA3 protein expression decreases from benign prostatic hypertrophy to localised pre-neoplastic lesions [
36]. Among the genes that we found to be expressed at higher levels in EGFR
high tumours, one has been previously linked to PC. FOXC1, a member of forkhead transcription factors (FOX), was found to be expressed at significantly high levels in androgen-independent PC xenografts [
37]. Our data, which are mostly concordant with previous observations, suggest that EGFR overexpression may result in a more aggressive tumour behaviour [
38], through deregulated function of these genes.
We acknowledge that these are preliminary data based on a small number of cases [
12,
39], with consequent limitations in the generalizability of our results. For example, the prevalence of EGFR mutations that we have found is in the range of that reported by other authors analyzing PC or cholangiocarcinoma [
12,
40]. Compared to other EGFR-driven diseases like NSCLC, where the prevalence of EGFR mutations has been reported to be as high as 30%, in PC EGFR mutations are less frequent. A larger sample size is therefore required to collect sufficient mutated cases to perform a more powerful analysis of the impact of mutations on prognosis or other biological features of tumours. Similarly, a larger number of mutated cases may result in the ability of gene expression analysis to capture significant gene expression patterns. In our study, because of the limited availability of fresh frozen material, the gene expression profile analysis was feasible in tumour samples from just 8 patients.
In summary, we found that 36% of the patients in this series had EGFR-overexpressing tumours and that this feature was significantly associated with biochemical relapse. Of the 13 tumours harbouring an EGFR mutation, all belonged to the high Gleason Score group. Of the identified mutations, some have been previously shown to predict the antitumor activity of small molecule tyrosine-kinase inhibitors. Further investigations on the novel mutations that we have identified may reveal new therapeutic targets. Finally, we identified a gene list in EGFR-mutated patients associated with EGFR expression.
Competing interests
All authors disclose any association that poses a conflict of interest in connection with the manuscript.
Microarray data are available on GEO (GSE 14206).
Authors' contributions
CPN designed the study, carried out the experiments, and drafted the manuscript; GM performed PCR and mutational analysis; MMG performed microarray experiments; FM performed statistical analyses and reviewed the final version of the manuscript; RS performed immunohistochemistry experiments; YP and GC supervised the study and supported with data interpretation; BT and LM performed and analyzed the immunohistochemical data; GC performed statistical analysis of microarray data; MA participated in design and coordination of the study.
All authors have read and approved the final manuscript.