Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity

European Journal of Cancer (2013) xxx, xxx– xxx

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journal homepage: www.ejcancer.com

Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity Adriana Amaro a,f, Valentina Mirisola a,f, Giovanna Angelini a, Alessandra Musso b, Francesca Tosetti b, Alessia I. Esposito a, Patrizia Perri a, Francesco Lanza c, Francesca Nasciuti c, Carlo Mosci c, Roberto Puzone d, Sandra Salvi e, Mauro Truini e, Alessandro Poggi b, Ulrich Pfeffer a,⇑ a

Integrated Molecular Pathology, IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy Molecular Oncology and Angiogenesis, IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy c Ocular Oncology Center, Galliera Hospital, Genoa, Italy d Clinical Epidemiology, IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy e Anatomy and Cytohistopathology, IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy b

KEYWORDS Epidermal growth factor receptor Tyrosine kinase Antibody-dependent cellular cytotoxicity Targeted therapy

Abstract Despite advances in surgery and radiotherapy of uveal melanoma (UM), many patients develop distant metastases that poorly respond to therapy. Improved therapies for the metastatic disease are therefore urgently needed. Expression of the epidermal growth factor receptor (EGFR), a target of kinase inhibitors and humanised antibodies in use for several cancers, had been reported. Forty-eight human UMs were analysed by expression profiling. Signalling was tested in three EGFR expressing UM cell lines by Western blotting using phosphorylation specific antibodies for EGFR and the downstream mediators AKT (v-akt murine thymoma viral oncogene homolog) and extracellular signal-regulated kinase (ERK). Evidence for signalling in tumours was obtained through the application of a UM-specific EGF-signature. The EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal antibody, Cetuximab, were tested for their effect on EGFR signalling. Natural killer cell mediated antibody-dependent cellular cytotoxicity (ADCC) and tumour necrosis factor a (TNF-a) release was analysed for Cetuximab.

⇑ Corresponding author: Address: Integrated Molecular Pathology, IRCCS A.O.U. San Martino – IST Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi 10, 16132 Genoa, Italy. Tel.: +39 0105737303. E-mail address: [email protected] (U. Pfeffer). f These authors have equally contributed to this work.

0959-8049/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ejca.2013.06.011

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fourteen of 48 UMs and three of 14 cell lines (over-)express EGFR, at least in part due to trisomy of the EGFR locus on chromosome 7p12. EGFR and the downstream mediator, AKT, are phosphorylated upon stimulation with EGF in EGFR expressing cell lines. EGFR over-expressing tumours but not EGFR negative tumours show an activated EGF-signature. Gefitinib inhibits EGFR and AKT phosphorylation and Cetuximab induces EGFR phosphorylation but inhibits signalling to AKT induced with EGF. Cetuximab triggers natural killer (NK) cells to lyse EGFR+ cell lines and to release TNF-a. EGFR appears suited as a novel molecular drug target for therapy of uveal melanoma. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Uveal melanoma, the most common primary intraocular malignant tumour in adults, arises from neoplastic proliferation of uveal melanocytes. Diagnosis of uveal melanoma is usually made by ophthalmoscopic examination and ultrasound. Uveal melanoma is molecularly clearly distinct from cutaneous melanoma (for a review see Ref.1). The recent identification of somatic mutations in uveal melanomas explains the molecular difference to cutaneous melanoma. BRAF mutations, frequent in cutaneous melanoma, were not detected in uveal melanomas that instead frequently carry mutations in GNAQ, GNA11 and BAP1.2–4 Several prognostic factors of disseminated relapse after initial ophthalmologic treatment have been determined, including cell morphology5 location with respect to the equator,6 retinal detachment,6 monosomy 37 and additional cytogenetic markers,8–10 syntenin protein expression11 and a specific gene expression signature.12 However, no effect of these prognostic markers on patient care can be envisaged in the absence of effective systemic therapies. Treatment options are local radiotherapy (chargedparticle beam therapy with protons or helium ions and the episcleral plaque radiation therapy) or enucleation. Uveal melanoma is defined by a poor natural outcome with a five year survival rate of 68.9%13 that is considerably worse for patients with large tumours with monosomy of chromosome 3. Chemotherapy, such as oral temozolomide and intra-arterial fotemustine used at the metastatic stage, induces very low response rates, 4.3% and 36%, respectively, and a median survival time of 6.7 and 15 months.14 No postoperative adjuvant therapies are currently available to decrease the risk of metastases. Treatment by systemic or intra-hepatic chemotherapy or partial hepatectomy only rarely prolongs life.15 The increased knowledge of molecular and genetic events associated with oncogenesis and tumour progression of ocular melanoma can lead to the identification of new therapeutic targets and agents. The preclinical investigation in relevant models is therefore mandatory to identify new therapeutic approaches. Many molecularly targeted drugs have been developed for other malignancies and if the responsiveness of uveal mela-

noma to these drugs can be shown, clinical trials could be designed in a very straightforward manner. We and others have therefore set out to identify targets of biological therapies. Hofmann et al. tested the tyrosine kinase inhibitor, Imatinib, that is specific for c-kit, a tyrosine kinase that is frequently over-expressed but not mutated in uveal melanoma. No objective response was observed.16 Over-expression of the vascular endothelial growth factor (VEGF) has been reported17 although its expression does not correlate with metastasis.18 The basic fibroblast growth factor 2 (FGF2) and its receptor (FGFR1) have been described to activate the mitogen activated kinase, extracellular signal-regulated kinase 1 (ERK1).19 Similarly, c-met, the receptor for the hepatocyte growth factor (HGF) has been shown to be frequently over-expressed by uveal melanomas20,21 and its miRNA mediated inhibition can tame tumour cell proliferation and migration.22 The insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) have also been shown to be expressed in uveal melanoma20,23,24 and to correlate with outcome.20,24 The recent identification of somatic mutations in the gene encoding the epidermal growth factor receptor (EGFR) in uveal melanoma cases indicates a potential etiopathological role for this tyrosine receptor kinase.25 A mutated EGFR gene induces melanoma formation, including uveal melanomas, in transgenic fishes with 100% penetrance.26 EGFR expression has been reported for uveal melanoma cell lines where it increases the ability of the cells to localise to the liver.27 Hurks and coworkers described EGFR expression in human uveal melanoma and reported an inverse correlation with survival.28 These data were challenged by a study that ascribed EGFR expression exclusively to tumour associated macrophages without addressing, however, the expression on uveal melanoma cell lines.29 A more recent study reported EGFR expression on uveal melanomas using immunohistochemistry.21 In order to proceed to clinical trials, the efficacy of targeted therapies must be shown in appropriate cellular models. We present here an analysis of EGFR signalling in uveal melanoma cell lines and tumours and show the effects of the EGFR specific tyrosine kinase inhibitor, Gefitinib and the anti-EGFR monoclonal antibody, Cetuximab. Our data establish EGFR as a novel molecular drug target for therapy of uveal melanoma.

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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2. Patients, materials and methods 2.1. Patients and cell lines Tissue samples were obtained from 48 primary uveal melanomas after enucleation surgery upon approval of the institutional bioethics board and informed written consent of the patients. Samples for gene expression profiling were removed within 15 min after surgery and conserved in RNAlater (Ambion, Monza, Italy) at 20 °C until processing. Cell lines used were OCM1, OCM8,30 92.1,31 OMM1,32 MEL270, OMM2.5,33 MEL290, MEL285,34 UPMM1, UPMM3, UPMD1, UPMM2 and UPMD235 and MEL15765 (unpublished). MDA-MB-231 and A549 certified cell lines were obtained from the Italian cell line collection (ICLC, www.iclc.it). The identity of OMM, MEL and UPM cell lines and OCM cell lines was certified by microsatellite mapping performed by ICLC in accordance to published profiles.36,37 All cell lines except for MEL15765, OMM1 and OMM2.5 derive from primary uveal melanomas. OMM1 (subcutaneous), OMM2.5 (liver) and MEL15765 (liver) derive from metastases. The mutational status for GNAQ, GNA11, BAP1 and BRAF of the cell lines used is reported in Supplementary Table 1. Cell lines were cultured in RPMI 1640 (Gibco-BRL, Rockville, MD, USA) supplemented with 10% foetal bovine serum (FBS), 2 mM L-glutamine and 100 U/ml penicillin/streptomycin at 37 °C. All cell lines used in this work are available from the Italian Cell line collection (www.iclc.it).

2.2. Gene expression profiling of tumours and cell lines Tumour samples were homogenised in the tissue lyser Mixer Mill (Qiagen, Hilden, Germany) in total RNA extraction lysis buffer using RNeasy (Qiagen). RNA quality was assessed in the BioAnalyser (Agilent, St. Clara, CA). RNA Integrity Number (RIN) was evaluated and only samples with RIN P 6 were considered acceptable. cDNA synthesis was performed using T7-(dT)24 oligo primers and the Custom SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, Irvine, CA, USA). Double-stranded cDNAs were extracted with phenol–chloroform–isoamyl alcohol (25:24:1), ethanol precipitated and used to prepare cRNAs using the Bioarray High Yield RNA Transcription Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. cRNAs were purified using the RNeasy Mini Kit (Qiagen), controlled by agarose gel electrophoresis and subjected to fragmentation for 35 min at 94 °C in fragmentation buffer (40 mM Tris–acetate pH 8.1, 100 mM CH3COOH and 30 mM Mg(CH3COO)2  4H2O). Labelled cRNA was used for screening of GeneChip Human Genome U133plus2 arrays (Affymetrix, Santa Clara, CA, USA) following the instructions given by the

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provider as described previously.11 The tumour samples analysed here comprise 29 samples published before.11 Microarray data were normalised using the RMA algorithm implemented in R/Bioconductor (http://www.bioconductor.org) applying quantile normalisation. Expression data were filtered to remove non-expressed (log 2 intensity <5) or invariant (lowest 25% of standard deviation) genes. The remaining gene expression data were used for further analysis. For the identification of EGF induced transcriptional changes microarray data from EGF treated cell lines and their controls were compared to identify genes that were at least two fold induced or reduced in their expression. These genes were normalised on the mean expression values and the expression values of all samples analysed were separately summed up for down- and up-regulated genes. The correlation of the expression of this metagene and EGFR gene expression was analysed using Pearson correlation. The EGF response genes were also used to identify significantly regulated genes applying Significance Analysis of Microarrays (SAM)38 and hierarchical clustering using Pearson correlation and average linkage, performed using MeV 4.0 software (http:// www.tm4.org/mev/). Differences of median EGFR expression between clusters were tested by Mann–Whitney–Wilcoxon (MWW) tests. 2.3. aCGH The DNA was extracted using QIAamp DNA Blood Mini kit (Qiagen). DNA concentration and quality were checked in the Nanodrop ND-1000 spectrophotometer. Processing of genomic DNA was performed using the GeneChip Mapping 250 K Assay Kit (Affymetrix) following the protocol provided. 250 ng of DNA sample were digested with the restriction enzyme NspI. Adapters were ligated using T4 DNA Ligase. Whole genome amplification was performed on a BioRad MyCycler thermocycler. 90 lg of amplified and normalised polymerase chain reaction (PCR) product was fragmented and labelled. Hybridisation, washing, staining and scanning of singlenucleotide polymorphism (SNP) arrays were performed on the Affymetrix station. Quality of the samples was assessed on agarose gels before the hybridisation step. Affymetrix Genotyping Console (GTC4.1.2) was used to perform genotype call and quality control assessments. Copy number analysis and virtual karyotypes were generated using CNAG3.0.39 The 23 samples in this study had an average call rate of 96.5%, exceeding the manufacturer’s recommendation of >93%. CNAG3.0 and GTC4.1.2 software use best-fit references selected from our library of normal samples. 2.4. Western blot For signalling analyses, uveal melanoma (UM) cells were grown in serum free medium for 24 h and, where

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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indicated, stimulated with 10 nM EGF in the presence or absence of the tyrosine kinase inhibitor Gefitinib (1 lM), or 2 lg/ml of the humanised monoclonal antiEGFR antibody Cetixumab, or 2 lg/ml of the antiCD20 antibody Rituximab for the times indicated. The Gefitinib and Cetuximab concentrations were chosen in accordance to several recently published functional studies.40,41 The cells were lysed in RIPA buffer containing protease inhibitors. Protein concentration was determined with the DC Protein Assay kit (Bio-Rad). Equal amounts of samples were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS– PAGE), transferred to nitrocellulose and probed at 4 °C overnight with the following anti-human antibodies (Cell Signaling Technology, Beverly, MA): rabbit polyclonal anti-phospho-AKT-1 (Ser473), AKT, phosphoERK1/2 (Thr202/Tyr204), ERK1/2, EGFR, rabbit monoclonal anti-phospho-EGFR (Tyr1068). After washing, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (GE-Healthcare, Milano, Italy) and specific complexes were revealed by enhanced chemiluminescence (ECL, GE-Healthcare). An anti-GAPDH antibody conjugated to horseradish peroxidase (Novus Biologicals, Littleton, CO) was used as a loading control for all samples. 2.5. Real time PCR cDNA was synthesised with oligo dT primers from 1 lg of total RNA with Superscript II (RT Invitrogen). For semi-quantitative real time PCR (RT-PCR) 2 ll of cDNA were amplified with 2.5 IU of Taq Polymerase (Roche), using the following primers: 50 -ACTGCTGCCACAACCAGTG (forward) and 50 GGCTTCGTCTCGGAATTTG (reverse). Real time PCR was performed on LightCycler480 II (Roche Applied Science) using 10 ll of LightCycler 480 SYBR Green I Master (Roche Applied Science), 2 ll of cDNA (5 diluted), 0.3 lmol sense and antisense primers in a final reaction volume of 20 ll. After amplification, melting curves with 65 steps of 15 s and 0.5 °C increase were performed. Expression data were normalised on the mean of GAPDH, RPII and G6PDH gene expression data. Relative expression values were obtained using Qgene software as described previously.42 2.6. Leucocyte cell separation, flow cytometry, antibodydependent cellular cytotoxicity (ADCC) and tumour necrosis factor a (TNF-a) production assays PBMC (peripheral blood monocytes) were obtained after Ficoll-Hypaque density centrifugation of blood samples derived from healthy volunteers as previously described.8 Natural killer (NK) cells were isolated from PBMC using the negative selection Rosettesep NK iso-

lation kit (Stemcell Technologies, Vancouver, Canada) according to the manufacturer’s instruction. Ex vivo isolated NK cell populations were 70–95% CD16+CD56+ and 100% CD3 (n = 8). Flow cytometry was performed as described.35 Briefly, NK cells were incubated with Cetuximab or Rituximab (as isotype matched control antibody) at 2.0 lg/ml for 30 min at 4 °C, then washed and incubated again for 30 min at 4 °C with an anti-human immunoglobulin Alexafluor647-labelled antiserum (Invitrogen, 2 lg/ml). After extensive washes, samples were run on a CyAN ADP cytofluorimeter (Beckman-Coulter) and analysed with the Summit 4.3 computer programme (Beckman-Coulter). Results are expressed as Log far-red fluorescence intensity (arbitrary Units) versus number of cells. Ex vivo isolated NK cells were used as effector cells in ADCC assay. This assay was performed by adding Cetuximab or Rituximab antibodies (20–2.0–0.2 lg/ml) to a 4 h standard cytolytic assay. Antibody concentrations were selected according to the plasma concentrations observed in Cetuximab treated patients.43,44 Tumour target cells were labelled with sodium 51Chromate, seeded in 96-V-bottomed microplates with effector cells (E)/target (T) at different E:T ratios in 200 ll of volume and specific lysis was calculated as described.45 Production of TNF-a was determined by enzyme-linked immunosorbent assay (ELISA) from SN of NK-melanoma cells cultured at 1:1 ratio alone or with Cetuximab or Rituximab at 37 °C for 24 h. Two lg/ml of Cetuximab was the optimal antibody amount in inducing TNF-a production as determined in preliminary experiments. The amount of TNF-a in each SN was calculated from a standard curve determined with recombinant TNF-a.

3. Results 3.1. EGFR expression and signalling in uveal melanoma When analysing gene expression microarray data of primary uveal melanomas, we observed a highly heterogeneous expression of EGFR ranging, after normalisation, from a fluorescence intensity of 52 to 5254 (mean = 883, median = 361) reported in Fig. 1a. Expression below 200 (18 cases) corresponds to spurious expression; eight cases express elevated levels (>2000) of the gene. Reverse transcription polymerase chain reaction confirmed differential EGFR expression in a series of uveal melanomas (Fig. 1b). In order to study EGFR expression in vitro, we analysed a panel of certified uveal melanoma cell lines using real time PCR. The cell lines showed highly variable levels of EGFR expression: MEL285 and MEL290 cell lines expressed elevated levels of the gene, UPMM1, UPMM2, UPMM3 and UPMD1 and UPMD2, MEL15765 and OCM8 cells expressed low levels and in the other cell lines (OMM1, OMM2.5, 92.1, OCM1 and MEL270), no

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fig. 1. Epidermal growth factor receptor (EGFR) expression and signalling. The EGFR is expressed at highly variable levels in primary human uveal melanomas as revealed by microarray gene expression analysis (A) and real time polymerase chain reaction (RT-PCR) (B) and human uveal melanoma cell lines analysed using RT-PCR (C). Expression is indicated as intensity values for microarray data and as relative expression as measured using three housekeeping genes for calculation of DDct values for RT-PCR. EGFR gene expression corresponds to expression of the receptor protein as revealed by Western blotting. The breast cancer cell line MDA-MB 231 and the lung adenocarcinoma cell line A549 have been included as positive controls (D). Densitometry of Western blot EGFR protein expression normalised on GAPDH loading control (E).

EGFR transcripts were detectable (Fig. 1c). Western blot analysis using antibodies specific for EGFR and for GAPDH as a loading control revealed high expression of the receptor protein in MEL285 and MEL290 cells and lower expression in UPMM3 cells (Fig. 1d). The breast cancer cell line MDA-MB-231 and the lung adenocarcinoma cell line A549 were added as positive controls and showed expression levels comparable to that observed in MEL285 and MEL290 cells (Fig. 1d and e). EGFR expression of the tumours did not correlate with other tumour and patient parameters available (age, sex, tumour location, dimension, height, spindle or epitheloid cell morphology). Similarly, there was no significant difference in EGFR expression between tumours with and without monosomy of chromosome 3. We analysed the chromosomal status by aCGH for 23 of the 48 tumours analysed. Ten of these tumours were monosomic, among which one, four and five tumours with high, intermediate and low EGFR expression, respectively. Additional three tumours showed partial losses in chromosome three and two of these showed high EGFR expression (data not shown). To determine whether EGFR expressed on human uveal melanoma can deliver an intracellular signal, we performed microarray gene expression profiling of MEL285 in the presence or absence of 10 ng/ml EGF in the culture medium; the MDA-MB-231 cell line was used as a prototype of EGFR-mediated sig-

nalling. Genes that are at least 2-fold differentially expressed upon treatment in both cell lines were identified by comparing the expression profiles. 632 probesets were identified and the expression heatmap is shown in Fig. 2a. We then extracted the expression values for these genes from the expression profiles of 48 uveal melanoma samples and, after removing 224 genes that were expressed below the arbitrary threshold of 25 (for the expression values of the remaining 408 genes see Suppl. Table 2), we calculated a metagene by adding the expression values after normalisation on the mean for each sample, separately for upand down-regulated genes. The expression values of this metagene were plotted over the expression values of EGFR for the 48 samples and compared by Pearson’s correlation showing a correlation coefficient r = 0.5024 (P = 0.0003; Fig. 2b). The correlation is particularly strong for those samples where the metagene assumes positive values whereas there is no correlation between EGFR expression and the EGFmetagene for values below zero of the latter. This could indicate that some EGF induced genes are also induced by other factors in cells that express only low levels of EGFR. This is highly likely since many signalling pathways, among which the pathway containing GNAQ and GNA11, use overlapping sets of downstream mediators and targets. We then identified EGF responsive genes that are differentially expressed between uveal melanomas with high

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fig. 2. Epidermal growth factor receptor (EGFR) signalling in human uveal melanomas. The gene expression profiles of EGFR treated cell lines MEL285 and MDA-MB-231 as compared to the untreated controls show differential expression of many genes (A). The metagene calculated using the EGF regulated genes shown in panel A correlates with the expression of EGFR in human uveal melanomas indicating that many genes that are responsive to EGF in the cell lines are also differentially expressed in EGFR expressing tumours (B). Resampling statistics applied to the expression values of EGF regulated genes in uveal melanomas identify 54 genes that are significantly differentially expressed in tumours with high versus low EGFR expression values (FDR < 1%). Hierarchical clustering of these genes yields four clusters (C). These clusters show significantly differential EGFR expression levels (D).

and low EGFR expression levels applying resampling statistics (Significance Analysis of Microarrays). Fifty-four genes of the cell line derived EGF-signature were differentially expressed in a statistically significant manner (FDR < 1%) in uveal melanomas with different EGFR expression levels. Hierarchical clustering of these genes using the expression values of the 48 samples analysed reveals the presence of two main clusters one of which had three evident subclusters (Fig. 2c). The EGFR expression of the samples in these clusters is shown in Fig. 2d. Cluster 1 mainly contains uveal melanomas that express higher than median levels of EGFR, samples of cluster 2 express intermediate levels of the receptor and the clusters 3 and 4 contain samples with low EGFR expression. These median differences are highly significant (MWW P = 0.0001). These data show that many genes that are responsive to EGF in cell lines are also differentially expressed in tumours with different EGFR expression levels. It is therefore highly likely that the EGFR pathway is actively signalling in uveal melanomas. 3.2. Copy number alterations of the EGFR locus on chromosome 7p11.2 We performed copy number alteration analyses for 23 of the samples analysed by gene expression profiling using Affymetrix 250 K SNP microarrays designed for

the analysis of single nucleotide polymorphisms. This analysis reveals that eight of these samples showed copy number alterations of chromosome 7 that encompassed the EGFR locus at 7p11.2. In all these cases the EGFR locus was present in three copies. The length of the amplified region was highly variable spanning from a complete trisomy of chromosome 7 (four cases) to 1 Mb containing the EGFR gene (Fig. 3). The analysis of EGFR expression showed a trend towards higher expression in cases with chr.7p11.2 amplification that, however, did not reach statistical significance. 3.3. Inhibition of EGFR signalling in uveal melanoma cell lines In order to understand whether the EGFR specific kinase inhibitor, Gefitinib and the humanised monoclonal anti-EGFR antibody, Cetuximab, can be used to inhibit EGFR signalling we analysed their effect on uveal melanoma cell lines. We used the two uveal melanoma cell lines, MEL285 and MEL290 with the highest EGFR gene expression values and UPMM3 cells with low but detectable expression (see Fig. 1) as models and the breast cancer cell line MDA-MB-231 as prototype EGFR positive cell line. The Western blot protein expression and phosphorylation analysis of EGFR, AKT and ERK in comparison to the loading control

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fig. 3. Copy number alteration (CNA) analyses of 23 uveal melanomas. Copy number alterations are frequent for chromosome 7 in uveal melanomas. In eight cases three copies of the locus 7p11.2 containing epidermal growth factor receptor (EGFR) (red bar) are observed. These CNAs range from 1 Mb to the whole chromosome in length. Copy number gains (all three copies) are shown above the chromosome banding pattern and a single very short copy number loss (one copy left) is shown below the chromosome. The genes contained in the minimal interval of approximately 1 Mb with copy number gain in all eight cases that have a copy number gain encompassing EGFR is shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

GAPDH is reported in Fig. 4 (MEL285) and Supplementary Fig. 1 (MEL290, UPMM3 and MDA-MB231). The treatments had, if any, only slight effects on the level of EGFR, AKT and ERK1/2 protein expression. Background phosphorylation of the receptor was reduced by the addition of the EGFR specific tyrosine kinase inhibitor, Gefitinib, for 2 min or 2 h in the absence of EGF (Fig. 4, lanes 3 and 5). The addition of the antiEGFR antibody Cetuximab for 2 min or 2 h slightly induced EGFR phosphorylation in the absence of EGF (Fig. 4, lanes 2 and 4). The treatment with EGF for 2 min (lane 6) induced EFGR phosphorylation. EGF induced phosphorylation of EGFR was unchanged in the presence of Cetuximab (lane 7) but reduced by the treatment with Gefitinib for 2 h. (lane 8). Phosphorylation of AKT reflects the phosphorylation status of EGFR and EGF induced phosphorylation of AKT is reduced by Gefitinib and Cetuximab, indicating that EGF induces active EGFR signalling that can be opposed by the tryrosine kinase inhibitor and by the anti-EGFR antibody. Phosphorylation of the MAP-kinases ERK1/2 does not show clear effects after treatment with EGF and/or inhibition of EGFR signalling by Gefitinib or Cetuximab. Very similar EGFR signalling patterns are observed for MEL290 and MDA-MB-231 cells (Suppl. Fig. 1) that express levels of EGFR similar to MEL285 cells. We performed the same analysis with UPMM3 cells that show lower expression levels of EGFR that is, however, phosphorylated in the presence of the ligand. The drugs show similar, though less evident effects as in the two other cell

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lines (Suppl. Fig. 1). In MDA-MB-231, MEL285, MEL290 and UPMM3 cell lines Gefitinib substantially decreases EGF induced AKT phosphorylation. The decrease of ERK1/2-phosphorylation, whose basal levels are generally high in all four models analysed, by Gefitinib is more pronounced in MEL290 and UPMM3 relative to MDA-MB-231 cells. EGF-induced EGFR phosphorylation is more effectively inhibited by Cetuximab in MEL290 and UPMM3 than in MEL285 and MDAMB-231 cells. The different effects observed for the single cell lines most probably depend on the well-known crosstalk of EGFR with the signalling of other growth factor receptor tyrosine kinases and of G-protein coupled receptors.46 UPMM3 cells carry a GNAQ mutation, MDAMB-231 cells a BRAF mutation and MEL285 and 290 cells probably have other yet unknown mutations in related signalling pathways that also lead to the activation of ERk1/2. We have sequenced the exons 18–21 of EGFR, known to harbour activating mutations in nonsmall cell lung cancer (NSCLC), in MEL285, MEL290 and UPMM3 cell lines but did not find any mutation. MDA-MB-231 cells have wildtype EGFR.

3.4. Cetuximab triggers NK cells to antibody-dependent cellular cytotoxicity and TNF-a release upon interaction with EGFR+ uveal melanoma cell lines The anti-cancer activity of Cetuximab has also been ascribed to antibody-dependent cellular cytotoxicity (ADCC) where the antibody essentially acts by making the tumour cell visible to the host’s immune system. Natural killer (NK) cells can induce lysis of antibody decorated tumour cells. We asked whether this process is active on uveal melanoma cells. Cetuximab recognises the EGFR expressed on uveal melanoma cells as assessed by indirect immunofluorescence and analysis on flow cytometer (Fig. 5). When the EGFR positive cell lines MEL285 or MEL290 were incubated with Cetuximab, in the presence of ex vivo isolated NK cells, ADCC was triggered. The ADCC effect was detected at different effector/target ratios with a wide range of Cetuximab antibody concentrations (20–2.0 and 0.2 lg/ml). The EGFR negative cell line OCM8 did not trigger ADCC of NK cells as expected. It is of note that ADCC of UPMM3 cell line, which express very low levels of EGFR, was not elicited in the presence of Cetuximab (Fig. 5). Upon activation, NK cells can produce anti-tumour soluble factors as tumour necrosis factor a (TNF-a). We therefore measured the release of TNF-a during the interaction between EGFR positive uveal melanoma cells and NK cells in the presence of Cetuximab; TNF-a was found in cell culture supernatants when NK cells were exposed to Cetuximab-treated EGFR positive uveal melanoma cells (Fig. 6). Incubation of NK cells with Cetuximab alone did not induce the release of TNF-a (Fig. 6). Again, UPMM3 cells, that

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fig. 4. Effects of Gefitinib and Cetuximab on epidermal growth factor receptor (EGFR) signalling. The effects of the tyrosine kinase inhibitor, Gefitinib and of the humanised monoclonal anti-EGFR antibody, Cetuximab, on EGFR signalling have been analysed by Western blotting for the uveal melanoma cell line MEL285. Antibodies against EGFR, AKT and extracellular signal-regulated kinase 1/2 (ERK1/2) and their phosphorylated forms have been used. The cell lines have been treated with Gefitinib or Cetuximab for 2 min, 2 h and/or with EGF for 2 min as indicated. Staining with anti-GAPDH antibodies has been used to monitor protein loading. (A) Western blot analyses, (B) densitometric analysis of the blot. Similar analyses for uveal melanoma (UM) cell lines MEL290 and UPMM3 and the breast cancer cell line MDA-MB231 are shown in Supplementary Fig. 1.

express low levels of EGFR, did not trigger a statistically significant release of TNF-a in the presence of Cetuximab. This would indicate that the extent of activation of NK cells depends on the presence of a threshold amount of EGFR molecule decorated with Cetuximab. The anti-CD20 antibody, Rituximab, used as an isotype matched negative control, did neither trigger ADCC nor TNF-a release (not shown). 4. Discussion No effective adjuvant therapies have been reported for uveal melanoma and when patients develop metasta-

ses their survival is short and response to existing therapies is very low. New therapies, both for the adjuvant setting and for metastatic disease, are therefore urgently needed. The putative driving mutations in the G-proteins GNAQ and GNA11 as well as in the tumour suppressor BAP1 have only recently been identified.2–4 At present no specific drugs that target these genes are available. Wu and colleagues recently proposed targeting of protein kinase C (PKC) that transduces the signal from GNAQ to the mitogen activated kinase (MAPK) ERK1/2 using the PKC inhibitor enzastaurin.47 At least until specific drugs for uveal melanoma will be available, the use of other molecularly targeted drugs could reduce

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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Fig. 5. Cetuximab triggers antibody-dependent cellular cytotoxicity (ADCC) of ex vivo isolated natural killer (NK) cells against uveal melanoma cells. Cytolytic activity of ex vivo isolated NK cells from two donors representative of eight tested, was assessed in a 4 h cytolytic assay at the indicated effector/target (E:T) ratios in the presence of different concentration of Cetuximab (20–2.0–0.2 mg/ml). Cytolysis in the absence of antibody is shown for comparison (nil). Results are shown as per cent specific 51Cr release and are the mean of triplicate samples. The FACS histograms of the reactivity of Cetuximab with the four uveal melanoma cell lines (MEL285, MEL290, UPMM3 and OCM8) analysed are shown in the inlets. The reactivity of Cetuximab is shown as the grey histogram overlaid with the same cells labelled with Rituximab as an isotype matched control antibody. In each subpanel the percentage of positive cells and the mean fluorescence intensity (MFI) of the grey histograms are indicated. Results are expressed as number of cells (y-axis) versus MFI in arbitrary units (x-axis).

Fig. 6. Cetuximab triggers the release of TNF-a from ex vivo natural killer (NK) cells co-cultured with human uveal melanoma cell lines. Ex vivo NK cells were co-cultured for 24 h with uveal melanoma cells (ratio 2:1) either in the presence of Cetuximab or not. Culture supernatants were harvested after 24 h and analysed for the content of TNF-a with a specific enzyme-linked immunosorbent assay (ELISA) kit. Results are the mean ± SD of data obtained with 4 different donors of ex vivo NK cells and are expressed as pg/ml. Statistical significance applying a two tailed ttest is shown.

the risk of metastasis and/or improve the survival of patients with metastatic disease, provided that the targets are present in uveal melanomas. Almost certainly, this will require the personalisation of the treatments given the heterogeneous expression of most targets. Following this approach, Hofmann and colleagues have analysed the effects of Imatinib mesylate on uveal

melanomas that over-express c-KIT and its ligand, stem cell factor, SCF. The treatment showed no effects in a small pilot study on 12 patients, probably because of the absence of activating mutations in c-KIT.16 Danielli et al. treated 13 unselected UM patients with the antiCTLA4 antibody, Ipilimumab and observed two cases of stable disease plus an additional case stabilised after

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an initial progression.48 Inhibitors of the hepatocyte growth factor receptor, MET,49 and histone deacetylase inhibitors50 have also been shown to be active on UM cells in vitro but have not yet been tested in a clinical trial. Here, we show that the epidermal growth factor receptor, EGFR, can be a target for therapy of uveal melanoma, since it is expressed at high levels in approximately one fourth of the primary tumours analysed. EGFR engaged with EGF shows a classical signalling via phosphorylation of AKT and MAP-kinases in vitro in uveal melanoma cell lines; further, the analysis of EGF induced genes in uveal melanoma cell lines compared to that found in primary tumour tissue specimens may indicate that EGFR-mediated signalling can be effective in vivo as well. The signal is not derived from macrophages since several of the tumours with high EGFR expression were free of macrophages. EGFR expression correlated with an adverse outcome in studies reporting EGFR expression in human uveal melanomas28 and in xenografts of uveal melanoma cell lines.27 This was, however, not confirmed in an analysis performed by Mallikarjuna and coworkers21 where only one of five metastatic tumours showed EGFR expression. In our series of cases we did not find a significant correlation between EGFR expression and the poor prognosis marker, chromosome 3 monosomy, or disease free survival. Given the fact that only few samples showed strong EGFR expression larger cohorts must be analysed in order to rule out any correlation of EGFR expression with tumour progression. Non-small cell lung cancers (NSCLCs) carry mutations in the ATP-binding domain of EGFR which increase the affinity of the receptor to both, ATP and specifically designed tyrosine kinase inhibitors (TKI) such as Gefitinib.51 This mutation is likely to play a major role in tumour development in NSCLC and inhibition of this signalling strongly affects tumour growth that depends on the activation of this pathway (oncogenic addiction). Gefitinib inhibits EGFR signalling also in cells without EGFR mutations yet only at concentrations 10- to 100-fold higher than those needed for cell lines carrying a mutated EGFR allele.52 Some uveal melanomas might also depend on EGFR signalling since EGFR mutations have been reported for two cases of uveal melanoma.25 In these cases Gefitinib or other EGFR specific TKIs could be active. Most uveal melanomas show constitutive activation of the MAP-kinases ERK1/2 (see Fig. 4), probably as a consequence of the initiating mutations in the G-proteins GNAQ and GNA11 that also signal via MAP-kinases. The activation of the MAP-kinase pathway by other upstream signalling molecules makes the effect of EGFR inhibition less likely. However, mutation of GNAQ or GNA11 is not sufficient to induce a metastatic phenotype and the cross-talk between EGFR and G-protein coupled recep-

tors46 might contribute to the acquisition of more aggressive growth characteristics and dissemination. The anti-EGFR humanised antibody Cetuximab has been designed to inhibit EGFR signalling53 and, in contrast to the TKIs, its activity is similar in cells carrying wild type and mutated EGFR.40 As described for other cells54 and xenografts,55 we show here that Cetuximab activates the EGFR inducing its phosphorylation but inhibits EGF-induced downstream signalling and phosphorylation of AKT in uveal melanoma cells. Cetuximab probably induces EGFR dimerisation leading to receptor phosphorylation yet in contrast to what happens after ligand induced dimerisation, the dimer is not internalised.54 Internalisation has been described as a crucial step in EGFR signalling.56 In addition to its effect on signalling, Cetuximab has also been shown to induce tumour cell lysis by natural killer cells of the host in head and neck and colon cancer.57,58 This activity, known as antibody-dependent cellular cytotoxicity (ADCC), depends on the presence and concentration of the target on the tumour cell membranes as well as on the presence of anti-tumour effector lymphocytes expressing the Fcc receptor IIIA (CD16), such as NK cells. Consequent to tumour cell injury, these effector lymphocytes release the anti-tumour cytokine, tumour necrosis factor a (TNF-a) that concurs in the elimination of tumour cells. Not all therapeutic antibodies appear to elicit ADCC that has consistently been described for Cetuximab but, for example, not for the anti-vascular endothelial growth factor receptor antibody, Bevacizumab.59 Our results show a specific, concentration dependent ADCC for uveal melanoma cells when exposed to NK cells from different donors. This activity is evident using freshly isolated NK cells and Cetuximab at concentrations similar to or lower than those obtained in the plasma of Cetuximab-infused patients. These data strongly suggest that NK cells of patients with metastatic uveal melanoma can be activated to kill EGFR positive melanoma cells in the presence of antiEGFR antibodies. This killing may be regulated both by soluble factors and negative signals mediated through the interaction between HLA-I antigens expressed on melanoma cells and corresponding inhibitory receptors expressed on NK cells.60,61 Thus, it will be relevant to determine whether these regulatory mechanisms may influence Cetuximab triggered ADCC and consequently the clinical response of patients suffering from uveal melanoma. Clinical evidence for ADCC is commonly collected by the analysis of Immunoglobulin G Fc Receptor IIIA, FccR and polymorphisms, which influence the affinity of FccR for IgG and thereby influence the efficacy of ADCC dependent therapies. These analyses have led to contrasting evidence for ADCC mediated effects of antiEGFR antibodies in colon cancer.62,63 Dual targeting

Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011

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using anti-EGFR antibodies and EGFR specific TKIs has shown synergistic effects in EGFR wild type colon carcinomas58 that might be important in the light of evidence that ADCC alone might not be sufficient for the control of tumour growth.64 The uveal melanoma cell lines used in this study can only partially reflect the typical molecular status of uveal melanomas. Mel285 and 290 cells do not carry GNAQ/11 mutations and hence represent 15% of uveal melanomas that does not carry such mutations. Of the three monosomic cell lines that we are aware of, only one (UPMM3) showed an appreciable EGFR expression. UPMM3 cells carry a mutation in GNAQ (Q209P), but the effect of Gefinitib on EGFR was limited (see Suppl. Fig. 1). Several monosomic tumours showed high or intermediate EGFR expression (data not shown). For a limited number of cases we also have the information on GNAQ (but not GNA11) mutation. We found one case with high, five with intermediate and one case with low EGFR expression (data not shown). Given the low number of cases for which aCGH and mutational data were available we cannot rule out whether the level of EGFR expression is associated with monosomy of chromosome 3 or GNAQ mutation. These data, taken together, build the rationale for a clinical trial with anti-EGFR antibodies or EGFR specific TKIs. In analogy to what has been observed for lung cancer, TKIs alone are unlikely to show effects in the absence of EGFR mutations. However, less specific TKIs targeting several ERB-B (v-erb-b2 erythroblastic leukemia viral oncogene homolog) family members might be active. Uveal melanoma is a rare cancer and the recruitment for patients for clinical trials might be difficult. Given the increasing number of drugs whose targets are expressed by a portion of uveal melanomas, trials in which chemotherapy alone is compared with tailored therapy, should be envisaged. Tailored therapies would consist in molecularly targeted drugs in addition to standard chemotherapy administered following a patient selection based on the presence of given targets. Role of funding source The sponsors of this study, Compagnia San Paolo di Torino and the Regione Liguria, had no influence on study design, execution of the study, data analysis and interpretation. Conflict of interest statement None declared. Acknowledgements This work was supported by grants from the Compagnia San Paolo di Torino and “PO CRO Fondo Soci-

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ale Europeo Regione Liguria 2007–2013 Asse IV – Capitale Umano” to U.P. A.A. is recipient of a postdoctoral fellowship from the Regione Liguria, V.M. was recipient of a doctoral fellowship from the Regione Liguria and AIE is recipient of a doctoral fellowship from the University of Genoa (PhD programme in Biotechnology). We thank Mariella Dono, Genova, for EGFR sequencing. We also acknowledge the contribution of tumour samples by Dr. Sarah Coupland and Dr. Bertil Damato, Liverpool, that have, however, not been used in this study. We thank Dr. Martine Jager, Leiden, Dr. Cristina Maccalli, Milan and Dr. Michael Zeschnigk, Essen, for providing cell lines. We thank Renata Scarzello for secretarial assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.ejca.2013.06.011. References 1. Zeschnigk M, Lohmann DR. Prognostic testing in uveal melanoma. In: Pfeffer U, editor. Cancer genomics: molecular classification, prognosis and response prediction. Dordrecht: Springer Science and Business Media; 2013. p. 79–96. 2. Van Raamsdonk CD, Griewank KG, Crosby MB, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med 2010;363(23):2191–9. 3. Van Raamsdonk CD, Bezrookove V, Green G, et al. Frequent somatic mutations of GNAQ in uveal melanoma and blue naevi. Nature 2009;457(7229):599–602. 4. Harbour JW, Onken MD, Roberson ED, et al. Frequent mutation of BAP1 in metastasizing uveal melanomas. Science 2010;330(6009):1410–3. 5. McLean IW, Zimmerman LE, Evans RM. Reappraisal of Callender’s spindle a type of malignant melanoma of choroid and ciliary body. Am J Ophthalmol 1978;86(4):557–64. 6. Desjardins L, Levy-Gabriel C, Lumbroso-Lerouic L, et al. Prognostic factors for malignant uveal melanoma. Retrospective study on 2,241 patients and recent contribution of monosomy-3 research. J Fr Ophtalmol 2006;29(7):741–9. 7. Prescher G, Bornfeld N, Hirche H, Horsthemke B, Jockel KH, Becher R. Prognostic implications of monosomy 3 in uveal melanoma. Lancet 1996;347(9010):1222–5. 8. Prescher G, Bornfeld N, Becher R. Two subclones in a case of uveal melanoma. Relevance of monosomy 3 and multiplication of chromosome 8q. Cancer Genet Cytogenet 1994;77(2):144–6. 9. Horsthemke B, Prescher G, Bornfeld N, Becher R. Loss of chromosome 3 alleles and multiplication of chromosome 8 alleles in uveal melanoma. Genes Chromosomes Cancer 1992;4(3):217–21. 10. Metzelaar-Blok JA, Jager MJ, Moghaddam PH, van der Slik AR, Giphart MJ. Frequent loss of heterozygosity on chromosome 6p in uveal melanoma. Hum Immunol 1999;60(10):962–9. 11. Gangemi R, Mirisola V, Barisione G, et al. Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression. PLoS One 2012;7(1):e29989. 12. Onken MD, Worley LA, Tuscan MD, Harbour JW. An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma. J Mol Diagn 2010;12(4):461–8.

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Please cite this article in press as: Amaro A. et al., Evidence of epidermal growth factor receptor expression in uveal melanoma: Inhibition of epidermal growth factor-mediated signalling by Gefitinib and Cetuximab triggered antibody-dependent cellular cytotoxicity, Eur J Cancer (2013), http://dx.doi.org/10.1016/j.ejca.2013.06.011