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ANTICANCER RESEARCH 29: 1327-1334 (2009)

EGFR Inhibition Using Gefitinib Is Not Active in Neuroblastoma Cell Lines

JOCHEN RÖSSLER1,2, EVA ODENTHAL1, BIRGIT GEOERGER2, AURORE GERSTENMEYER2, JEANETTE LAGODNY1, CHARLOTTE MARIE NIEMEYER1 and GILLES VASSAL2

1Division of Pediatric Hematology and Oncology, Department of Pediatrics and Adolescent Medicine, University Hospital of Freiburg, Germany; 2University of Paris XI, UPRES EA3535, Pharmacology and New Treatments of , Institut Gustave Roussy, 94805 Villejuif Cedex, France

Abstract. Background: Inhibition of the epidermal growth small cell (NSCLC), particularly in factor (EGFR), using a inhibitor such of women and non-smokers bearing EGFR as gefitinib, was suggested to be a new treatment approach for gene mutations in exons 19 and 21 (3, 4). neuroblastoma. Material and Methods: EGFR expression and Neuroblastoma, the second most frequent solid tumor of gene mutation were studied by reversetranscriptase-polxmerase childhood, still has an unacceptably poor outcome, notably chain reacting, fluorescence-activated cell sorting and gene at advanced stages (5). Therefore, new therapeutic sequencing in six neuroblastoma cell lines. In vitro cytotoxicity approaches have to be urgently explored. Within this context, of gefitinib 0.1-10 μM alone or in combination with topotecan, TKIs are under investigation for the treatment of vincristine and 9-cis retinoic acid (9cisRA) was determined by neuroblastoma (6). For example, sensitivity of has MTT proliferation assay. Results: EGFR overexpression and been studied extensively in vitro and in vivo (7-10). These gene mutations were absent in all cell lines tested. Inhibition pre-clinical evaluations suggest a potential of imatinib in of cell viability of 62-85% was found at 10 μM gefitinib, combination with , retinoic acid (RA) or concentrations that, however, can clinically not be reached. In radiotherapy (11). However, first clinical data on imatinib addition, gefitinib increased inhibitory effects of topotecan, used as a single agent in pediatric malignancies showed only vincristine and RA by 10-15% . Conclusion: Our in vitro data minor effects, including in neuroblastoma patients (12), and do not support the use of gefitinib as monotherapy in combination treatments are under discussions. Furthermore, neuroblastoma and its chemosensitizing effects appear minor. EGFR inhibition has been suggested for neuroblastoma. Ho et al. reported inhibition of EGFR autophosphorylation in New drugs targeting specific pathways of cancer cells have vitro using gefitinib by blocking the phosphoinositide-3- entered adult oncology with an enormous success (1). In this kinase (PI3K)/AKT pathway (13) and Tamura et al. showed regard, imatinib mesylate, the tyrosine kinase inhibitor (TKI) induction of (14). We investigated if gefitinib has of the ABL transcript, KIT and the platelet-derived growth cytotoxic activity as a single agent or in combination with factor receptors, was the first agent that targets specifically currently used chemotherapeutics, topotecan and vincristine, and selectively molecular pathways in malignant cells (2). as well as the differentiating agent 9cisRA. In addition, further TKIs such as gefitinib and targeting endothelial receptor (EGFR) tyrosine Materials and Methods kinase have been developed and showed efficiency in non- Cell culture. The neuroblastoma cells SH-EP, SH-SY5Y, Kelly and Lan- 5 were kindly provided by Professor M. Schwab, DKFZ Heidelberg, Germany, and SK-N-LO and SK-N-FI by Dr. A. Voigt, Department of Pediatrics, University of Jena, Germany. The lung cancer cells HCC- Correspondence to: Jochen Rössler, MD, Division of Pediatric 827 (control) were received from the DSMZ (Braunschweig, Germany). Hematology and Oncology, Department of Pediatrics and Adolescent All cells were cultured in RPMI-1640 medium (Gibco BRL, Medicine, University Hospital of Freiburg, 79106 Freiburg, Eggenstein, Germany) with 10% fetal serum (FCS) and 1% Germany. Tel: +49 7612704506, Fax: +497612704518, e-mail: penicillin/streptomycin under 5% CO2 in atmospheric air at 37˚C. [email protected] MTT proliferation assay. Gefitinib was kindly provided by Key Words: Neuroblastoma, epidermal , AstraZeneca (Macclesfield, Cheshire, UK). A total of 1×104 cells gefitinib, chemotherapy, retinoic acid. per well were plated in a 96-well-plate and cultured overnight. On

0250-7005/2009 $2.00+.40 1327 ANTICANCER RESEARCH 29: 1327-1334 (2009) the following day, cells were exposed to 0.1, 0.5, 1 or 10 μM Table I. Cell viability of 6 neuroblastoma cell lines 72 hours after gefitinib; 1 μM 9cisRA (Sigma, St. Louis, MO, USA); 0.5, 1 or exposure to topotecan, vincristine and 9cisRA as single agents. 10 ng/ml topotecan (SmithKline Beecham plc, Brentford, UK); or 0.5, 1 or 10 ng/ml vincristine (Pharmachemistry B.V., Haarlem, Cell line Topotecan Vincristine 9cisRA Holland). Gefitinib was dissolved in dimethyl sulfoxide (DMSO); 10 ng/ml 10 ng/ml 1 μM stock solution of topotecan or vincristine in water; 9cisRA was prepared in ethanol. Cells were treated with the indicated SH-EP 79±7% * 45±5% * 82±4% * concentrations of gefitinib, 9cisRA, topotecan and vincristine SH-SY5Y 81±5% * 51±5% * 82±1% * alone and in combinations for 72 hours. Cell viability was Lan-5 76±9% * 96±7% 84±4% * Kelly 53±5% * 34±3% * 101±6% measured by the 3-(4.5-dimethylthiazol-2-yl) diphenyltetrazolium SK-N-LO 56±2% * 40±4% * 102±9% bromide (MTT) colorimetric assay (Sigma) as described SK-N-FI 48±4% * 41±5% * 98±3% elsewhere (11). Values are defined as percentage of cell viability compared to control Reverse transcriptase-polymerase chain reaction. Total RNA was cells treated with the solvents alone. *Statistically significantly different isolated using PeqGOLD TriFast reagent (PeqLab, Erlangen, from controls. Germany). First-strand cDNA synthesis was performed with 5 μg of total RNA using Ready-To-Go T-Primed First-Strand Kit (Amersham Bioscience Europe GmbH, Freiburg, Germany). For PCR), 1 minute at 72˚C of extension and a final elongation for 3 PCR amplification 1 μl of cDNA (100 ng) was used. The primers minutes at 72˚C. After electrophoretic separation in 1% agarose gel, used for PCR were as follows: for EGFR, sense: 5’-GAC AGC the products were analyzed using an ABI sequencer (Seqlab, TAT GAG ATG GAG GAA-3’ (nucleotides 1186-1206) and Göttingen, Germany). antisense 5’-GAG TCA CCC CTA AAT GCC A-3’ (nucleotides 1385-1367); for β-actin, sense: 5’-CCA AGG CCA ACC GCG Statistical analysis. Student’s t-test for paired samples was used to AGA AGA TGA C-3’ (nucleotides 409-432) and antisense compare the means of two groups. We considered an adaptive 5’-AGG GTA CAT GGT GGT GCC GCC AGA C-3’ (nucleotides multiple testing procedures to maintain the family-wise error rate 972-995). The PCR conditions were as follows: initial of 0.05. Therefore a p-value of 0.001 was used to indicate statistical denaturation for 5 minutes at 94˚C, amplification at 35 cycles: significance. Statistical analysis was performed using SPSS for 30 seconds at 94˚C of denaturation, 30 seconds at 60˚C of Windows 14.0.1 (SPSS Inc., Chicago, IL, USA). annealing, 1 minute at 72˚C of extension and a final elongation for 10 minutes at 72˚C. β-Actin as a constitutively expressed gene product was used as control. The PCR products were analyzed Results after electrophoretic separation in 1% agarose gel and visualized by ethidium bromide staining. Expression and mutation analysis of EGFR. EGFR expression at the RNA level was present in the Fluoresence-activated cell sorting analysis. Sub-confluent neuroblastoma cell lines SH-EP, SH-SY5Y, Kelly and SK- neuroblastoma cells were harvested from culture flasks and washed N-LO comparable to the control lung cancer cells HCC-827, twice in phosphate-buffered saline. Approximately 5×105 cells were as shown by RT-PCR (Figure 1A), while EGFR was not stained with a polyclonal rabbit anti-EGFR (#2232; Cell Signaling Technologies, Inc., Danvers, USA) followed by a polyclonal goat detectable in Lan-5 or SK-N-FI cells. However, cell surface anti-rabbit fluorescein (FITC)-conjugated antibody (Jackson expression of the EGFR protein was demonstrated by FACS ImmunoResearch Laboratories, Newmarket, UK) for surface analysis only in SH-SY5Y (Figure 1B). Mutation analysis receptor detection. Data from a minimum of 1×104 cells were of exon 19 and 21 of the EGFR gene showed no mutation in acquired and analyzed with CellQuest software (BD Biosciences the 6 neuroblastoma cell lines tested (Figure 1C). Immunocytometry Systems, San Jose, USA). Inhibition of cell viability by gefitinib. Cell viability Sequencing. Genomic DNA was prepared using PeqGOLD TriFast significantly decreased after treatment for 72 hours only at reagent (PeqLab). A nested two-step PCR was performed using 1 10 μM gefitinib, the highest concentration tested (Figure μl of gDNA and the following primers (15): exon 19 external PCR, 2A). No antiproliferative effect was seen in Kelly or SK-N- sense 5’-AAA TAA TCA GTG TGA TTC GTG GAG-3’, antisense: 5’-GAG GCC AGT GCT GTC TCT AAG G-3’; exon 19 internal LO. The treatment of HCC-827 cells showed a distinct PCR, sense: 5’-GTG CAT CGC TGG TAA CAT CC-3’, antisense: stronger growth inhibition (Figure 2B). In conclusion, the 5’-TGT GGA GAT GAG CAG GGT CT-3’; exon 21 external PCR: IC50 of gefitinib in HCC-287 cells is <0.1 μM while no IC50 sense: 5’-GCA GCG GGT TAC ATC TTC TTT C-3’, antisense: 5’- was reached in the 4 most gefitinib-sensitive neuroblastoma CAG CTC TGG CTC ACA CTA CCA G-3’; exon 21 internal PCR, cell lines using concentrations up to 10 μM. sense: 5’-GCT CAG AGC CTG GCA TGA A-3’, anti-sense: 5’- CAT CCT CCC CTG CAT GTG T-3’. The PCR conditions were as Inhibition of cell viability by gefitinib and topotecan. Cell follows: initial denaturation for 15 minutes at 94˚C, amplification at 35 cycles: 20 seconds at 94˚C of denaturation, 30 seconds of viability measured by MTT test after 72 hours significantly annealing (at 58˚C for exon 19 external PCR, at 57˚C for exon 21 decreased in all neuroblastoma cells tested at therapeutically internal PCR and at 60˚C for exon 19 internal and exon 21 external relevant topotecan concentrations up to 10 ng/ml (Table I).

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Figure 1. EGFR expression and EGFR gene mutation in 6 neuroblastoma cell lines. A, EGFR mRNA as detected in SH-EP, SH-SY5Y, Kelly, SK-N-LO and the HCC-827 control cells by RT- PCR. B, EGFR protein as detected by FACS analysis in SH-SY5Y and HCC-827 cells. C, Sequencing of the EGFR gene shows a deletion in exon 19 (E746_A750del) in the control cell line HCC, but none in the neuroblastoma cell lines.

SK-N-FI showed decreased cell viability in a dose- Inhibition of cell viability by gefitinib and vincristine. Treatment dependent manner, with a maximum inhibition of 48±4% at with 10 ng/ml vincristine alone inhibited cell viability in all 10 ng/ml topotecan. Simultaneous administration of 1 μM neuroblastoma cells tested, except in Lan-5 cells (Table I). gefitinib and topotecan in SK-N-FI resulted in increased Gefitinib at concentrations of 0.5 and 1 μM sensitized Lan-5 for inhibitory effects with 79±8% viability for the combination the treatment with 10 ng/ml vincristine, resulting in 79±5% and compared to 91±6% for 0.5 ng/ml topotecan alone (Figure 67±5% viability compared to 96±7% with vincristine alone 3A). Enhanced inhibition was also observed for 0.5 and 1 (Figure 3B). Simultanous administration of 0.5 or 1 μM μM gefitinib with 1 ng/ml topotecan with 89±7% viability gefitinib with 1 and 10 ng/ml vincristine resulted in an increased for topotecan alone and 78±6% and 76±5% , respectively, inhibitory effect over single vincristine treatment in all other for the combinations. neuroblastoma cells tested (data not shown).

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Figure 2. Effect of gefitinib as single agent in 6 neuroblastoma cell lines. A, Neuroblastoma cells were treated with 0.1, 0.5, 1 or 10 μM gefitinib for 72 hours. Cell viability was determined by MTT test. Optical density (OD) corresponds to the number of viable cells. Experiments were performed independently three times; the mean value and the standard deviation are shown. Asterisks indicate a statistically significant decrease in the cell viability when compared to cells treated with DMSO alone (p<0.01). B, Effect of gefitinib on HCC-827 cell line.

Inhibition of cell viability by gefitinib and 9cisRA. Treatment SH-SY5Y cells. However, no additional effects on the with 1 μM 9cisRA showed no effects on the neuroblastoma inhibitory activity of gefininib with RA were observed in cell lines Kelly, SK-N-LO and SK-N-FI, while significantly SH-EP and the 9cisRA-resistant cell lines (data not shown). reduced viability was found in SH-EP, SH-SY5Y and Lan-5 (Table I) cells. Co-administration of 1 μM 9cisRA and 1 or Discussion 0.5 μM gefitinib increased the inhibitory effects on cell viability of the RA-sensitive Lan-5 cells from 84±4% EGFR is an interesting survival signalling pathway in viability for RA alone to 75±6% and 74±8% for the neuroblastoma (16-18) and was used successfully as a target combinations (Figure 4). Similar effects were observed in to enhance the oncolytic potency of conditionally replicative

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Figure 4. Effect of gefitinib in combination with 9cisRA in Lan-5. The RA-sensitive Lan-5 cell line was treated with 1 μM 9cisRA and 0, 0.1, 0.5 and 1 μM gefitinib for 72 hours. Cell viability was determined by MTT test. Results are presented as viable cells as a percentage of the control cells treated with the equivalent solvents. Experiments were performed three times; the mean value and the standard deviation are shown. Asterisks indicate a statistically significant decrease in the cell viability after combination treatment when compared to viability of cells treated only with 1 μM 9cisRA (p<0.01).

Figure 3. Effect of gefitinib in combination with topotecan in SK-N-FI and vincristine in Lan-5. Cells were cultured for 72 hours in the neuroblastoma underlies post-transcriptional regulation and presence of different concentrations (0, 0.5 and 1 ng/ml) of topotecan (A) and vincristine (B) in combination with 0.5 and 1 μM gefitinib. Cell that EGFR overexpression is rare. Moreover, we extended viability was determined by MTT test. Results are presented as viable the findings on the absence of gene mutations in the tyrosine cells as a percentage of the control cells treated with equivalent kinase domain of this receptor in neuroblastoma. solvents. Experiments were performed three times; the mean value and For in vitro cytotoxicity effects of gefitinib, we tested the standard deviation are shown. Asterisks indicate statistically all neuroblastoma cell lines under normal growth significant differences between viability of cells treated with topotecan or vincristine alone and in combination with gefitinib (p<0.01). conditions (10% FCS), including the EGFR non- expressing cells. We found significant inhibitory effects only at a high gefitinib concentration (10 μM) which was independent of the presence of EGFR expression. In adenoviruses (19). To date, two in vitro studies reported on a contrast, Ho et al. found an IC50 of 0.05 μM gefitinib in potential therapeutic approach using gefitinib for EGFR the EGFR-expressing NLF and SH-SY5Y cell lines when targeting. Based on EGFR and EGF receptor-ligand they were cultured with 10% FCS (13). Tamura et al. interaction Ho et al. suggested a role of this pathway for cell demonstrated the IC50 of gefitinib in the EGFR positive proliferation although most of the neuroblastoma cells KP-N-TK and KP-N-SIFA cell lines to be 1.2 μM when analyzed did not express EGFR levels higher than that of they were cultured with 2% FCS, with IC50s of 10 and 12 normal fetal brain (13). In the second report, EGFR protein μM, respectively, when cells were cultured at 10% FCS. expression was shown for 8 out of 10 cell lines at various Moreover, apoptotic effects of gefitinib were only found at levels, however much below those of the A431 epidermoid concentrations greater than 30 μM for the two cell lines cell line (14). Our own study confirms that RNA (14). It is important to note that the clinically achievable expression of EGFR does not always lead to detectable plasma concentration in patients receiving gefitinib at the EGFR surface protein expression. Four out of six cell lines maximum tolerated dose was 1 μM (20), thus the in vitro expressed EGFR mRNA, but only SH-SY5Y showed EGFR effects observed in our studies may not be relevant for protein expression, suggesting that EGFR expression in neuroblastoma patients.

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Since the EGFR pathway is a major cell survival pathway 2 Capdeville R, Buchdunger E, Zimmermann J and Matter A: in response to chemotherapy, we intended to evaluate the Glivec (STI571, imatinib), a rationally developed, targeted potential of gefitinib in combination with cytotoxic agents anticancer drug. Nat Rev Drug Discov 1: 493-502, 2002. currently used in neuroblastoma. We found an increase of 3 Pao W, Miller V, Zakowski M, Doherty J, Politi K, Sarkaria I, Singh B, Heelan R, Rusch V, Fulton L, Mardis E, Kupfer D, approximately 10% of the inhibitory effects when gefitinib was Wilson R, Kris M and Varmus H: EGF receptor gene mutations administered simultaneously with topotecan and vincristine are common in lung from ‘never smokers’ and are compared to inhibition with these agents alone. In the associated with sensitivity of tumors to gefitinib and erlotinib. vincristine-resistant Lan-5 cell line, vincristine sensitivity was Proc Natl Acad Sci USA 101: 13306-13311, 2004. achieved by addition of 0.5 or 1 μM gefitinib. Sensitizing 4 Ho C, Murray N, Laskin J, Melosky B, Anderson H and Bebb effects of gefitinib in neuroblastoma has previously been shown G: Asian ethnicity and adenocarcinoma histology continues to for cisplatin with enhanced growth inhibition of 5-15% when predict response to gefitinib in patients treated for advanced non- small cell carcinoma of the lung in North America. Lung Cancer gefitinib was combined (14). The chemosensitizing effect of 49: 225-231, 2005. gefitinib could be due to activation of the EGFR signaling by 5 Berthold F, Hero B, Kremens B, Handgretinger R, Henze G, chemotherapeutics (21), or due to down-regulation of the ATP- Schilling FH, Schrappe M, Simon T and Spix C: Long-term binding cassette transporter BCRP and the multiple drug results and risk profiles of patients in five consecutive trials resistance MDR1 membrane proteins that regulate cellular drug (1979-1997) with stage 4 neuroblastoma over 1 year of age. efflux by gefitinib (22). Furthermore, we showed that gefitinib Cancer Lett 197: 11-17, 2003. increased the cytotoxic effect of 9cisRA by 10-15% in RA- 6 Rossler J, Geoerger B, Taylor M and Vassal G: Small molecule tyrosine kinase inhibitors: potential role in pediatric malignant sensitive but not in RA-resistant neuroblastoma cells. EGFR solid tumors. Curr Cancer Drug Targets 8: 76-85, 2008. down-regulation has been described during neuronal 7 Vitali R, Cesi V, Nicotra MR, McDowell HP, Donfrancesco A, differentiation and could explain this effect of RA as a Mannarino O, Natali PG, Raschella G and Dominici C: c-Kit is differentiating agent (23). We looked for EGFR expression preferentially expressed in MYCN-amplified neuroblastoma and after 9cisRA treatment of the six neuroblastoma cell lines, its effect on cell proliferation is inhibited in vitro by STI-571. which was not altered (data not shown). In summary, gefitinib Int J Cancer 106: 147-152, 2003. enhanced moderately cytotoxic effects of chemotherapeutics 8 Beppu K, Jaboine J, Merchant MS, Mackall CL and Thiele CJ: and RA, and even reduced chemoresistance in one case. Effect of imatinib mesylate on neuroblastoma tumorigenesis and vascular endothelial growth factor expression. J Natl Cancer Inst Limited activity of gefitinib as a single agent in vivo has been 96: 46-55, 2004. shown in a panel of 10 pediatric tumor xenografts, including 9 Te Kronnie G, Timeus F, Rinaldi A, Crescenzio N, Spinelli M, three neuroblastoma models (24). However, the use of gefitinib Rosolen A, Ricotti E and Basso G: Imatinib mesylate (STI571) in combination with the topoisomerase I inhibitor irinotecan interference with growth of neuroectodermal tumour cell lines resulted in more than additive activity in an EGFR-negative does not critically involve c-Kit inhibition. Int J Mol Med 14: neuroblastoma xenograft model. A clinical chemosensitizing 373-382, 2004. effect for gefitinib has been suggested in a heavily pre-treated 10 Meco D, Riccardi A, Servidei T, Brueggen J, Gessi M, Riccardi R and Dominici C: Antitumor activity of imatinib mesylate in relapsed stage 4 neuroblastoma patients, when combined with neuroblastoma xenografts. Cancer Lett 228: 211-219, 2005. topotecan and cyclophosphamide (25). 11 Rossler J, Zambrzycka I, Lagodny J, Kontny U and Niemeyer Targeting EGFR using gefitinib as a single agent appears CM: Effect of STI-571 (imatinib mesylate) in combination with to have a minor interest for the treatment of neuroblastoma. retinoic acid and gamma-irradiation on viability of The moderate sensitizing effect of EGFR targeting with neuroblastoma cells. Biochem Biophys Res Commun 342: topotecan, vincristine and 9cisRA in vitro needs to be further 1405-1412, 2006. explored for its clinical relevance. In addition, pathways other 12 Bond M, Bernstein ML, Pappo A, Schultz KR, Krailo M, Blaney SM and Adamson PC: A phase II study of imatinib than EGFR signaling should be explored in preclinical studies mesylate in children with refractory or relapsed solid tumors: A to bring forward new treatment protocols for neuroblastoma. Children’s Oncology Group study. Pediatr Blood Cancer 50: 254-258, 2008. Acknowledgements 13 Ho R, Minturn JE, Hishiki T, Zhao H, Wang Q, Cnaan A, Maris J, Evans AE and Brodeur GM: Proliferation of human We would like to thank Yann Lecluse, Villejuif and Marco Teller, neuroblastomas mediated by the Freiburg, for excellent technical assistance, as well as Peter Noellke receptor. Cancer Res 65: 9868-9875, 2005. for help with statistical analysis. 14 Tamura S, Hosoi H, Kuwahara Y, Kikuchi K, Otabe O, Izumi M, Tsuchiya K, Iehara T, Gotoh T and Sugimoto T: Induction of References apoptosis by an inhibitor of EGFR in neuroblastoma cells. Biochem Biophys Res Commun 358: 226-232, 2007. 1 Steeghs N, Nortier JW and Gelderblom H: Small molecule tyrosine 15 Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto kinase inhibitors in the treatment of solid tumors: an update of RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska recent developments. Ann Surg Oncol 14: 942-953, 2007. FG, Louis DN, Christiani DC, Settleman J and Haber DA:

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Activating mutations in the epidermal growth factor receptor 22 Leggas M, Panetta JC, Zhuang Y, Schuetz JD, Johnston B, Bai F, underlying responsiveness of non-small cell lung cancer to Sorrentino B, Zhou S, Houghton PJ and Stewart CF: Gefitinib gefitinib. N Engl J Med 350: 2129-2139, 2004. modulates the function of multiple ATP-binding cassette 16 Janet T, Ludecke G, Otten U and Unsicker K: Heterogeneity of transporters in vivo. Cancer Res 66: 4802-4807, 2006. human neuroblastoma cell lines in their proliferative responses to 23 Wu JX and Adamson ED: Inhibition of differentiation in P19 basic FGF, NGF, and EGF: correlation with expression of growth embryonal carcinoma cells by the expression of vectors encoding factors and growth factor receptors. J Neurosci Res 40: 707-715, truncated or antisense EGF receptor. Dev Biol 159: 208-222, 1995. 1993. 17 Layfield LJ, Thompson JK, Dodge RK and Kerns BJ: Prognostic 24 Stewart CF, Leggas M, Schuetz JD, Panetta JC, Cheshire PJ, indicators for neuroblastoma: stage, grade, DNA ploidy, MIB-1- Peterson J, Daw N, Jenkins JJ, III, Gilbertson R, Germain GS, proliferation index, p53, HER-2/neu and EGFR – a survival Harwood FC and Houghton PJ: Gefitinib enhances the antitumor study. J Surg Oncol 59: 21-27, 1995. activity and oral of irinotecan in mice. Cancer Res 18 Meyers MB, Shen WP, Spengler BA, Ciccarone V, O’Brien JP, 64: 7491-7499, 2004. Donner DB, Furth ME and Biedler JL: Increased epidermal 25 Donfrancesco A, Jenkner A, De Ioris MA, Ilari I, Castellano A, growth factor receptor in multidrug-resistant human De Laurentis C, Garganese MC, Milano GM and Dominici C: neuroblastoma cells. J Cell Biochem 38: 87-97, 1988. Prolonged response to oral gefitinib, cyclophosphamide, and 19 Geoerger B, van Beusechem VW, Opolon P, Morizet J, Laudani topotecan in heavily pretreated relapsed stage 4 neuroblastoma: L, Lecluse Y, Barrois M, Idema S, Grill J, Gerritsen WR and a case report. J Pediatr Hematol Oncol 29: 799-803, 2007. Vassal G: Expression of p53, or targeting towards EGFR, enhances the oncolytic potency of conditionally replicative adenovirus against neuroblastoma. J Gene Med 7: 584-594, 2005. 20 Nakagawa K, Tamura T, Negoro S, Kudoh S, Yamamoto N, Yamamoto N, Takeda K, Swaisland H, Nakatani I, Hirose M, Dong RP and Fukuoka M: Phase I pharmacokinetic trial of the selective oral epidermal growth factor inhibitor gefitinib (‘Iressa™’, ZD1839) in Japanese patients with solid malignant tumors. Ann Oncol 14: 922-930, 2003. 21 Knight LA, Di Nicolantonio F, Whitehouse P, Mercer S, Sharma S, Glaysher S, Johnson P and Cree IA: The in vitro effect of Received August 26, 2008 gefitinib (‘Iressa™’) alone and in combination with cytotoxic Revised December 17, 2008 chemotherapy on human solid tumours. BMC Cancer 4: 83, 2004. Accepted January 29, 2009

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