Mutational status of the epidermal growth factor receptor (EGFR) gene in thymomas and thymic carcinomas

Cancer Letters 248 (2007) 186–191 www.elsevier.com/locate/canlet

Mutational status of the epidermal growth factor receptor (EGFR) gene in thymomas and thymic carcinomas Michael Meister a, Peter Schirmacher b, Hendrik Dienemann c, Gunhild Mechtersheimer b, Philipp A. Schnabel b, Michael A. Kern b, Esther Herpel b, Elizabeth C. Xu a, Thomas Muley a, Michael Thomas d, Ralf J. Rieker b,* a

Translational Research Unit, Thoraxklinik am Universita¨tsklinikum Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany b Department of General Pathology, University Hospital, INF 220/221, 69120 Heidelberg, Germany c Department of Surgery, Thoraxklinik am Universita¨tsklinikum Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany d Department of Internal Medicine, Thoracic Oncology, Thoraxklinik am Universita¨tsklinikum Heidelberg, Amalienstr. 5, 69126 Heidelberg, Germany Received 22 May 2006; received in revised form 4 July 2006; accepted 6 July 2006

Abstract Epithelial tumours of the thymus (thymoma, thymic carcinoma) are rare tumours of the anterior mediastinum. Current treatment options of advanced stage thymomas and thymic carcinomas include a multimodal therapy with radio- and chemotherapy as well as surgery. In recent years, new therapeutic targets such as EGFR (epidermal growth factor receptor), COX-2 and KIT have emerged as new potential therapeutic targets. So far, EGFR mutational status of different subtypes of epithelial tumours of the thymus has been analyzed only inappropriately. We have investigated 20 different subtypes of thymomas (type A, AB, and B3) and thymic carcinomas for mutations in exons 18, 19, 20, and 21 of the EGFR gene and performed immunohistochemistry for EGFR. Concerning immunohistochemistry, most of the cases (17/20) had a strong positive staining. Although sequence alterations were found in four samples, none of these alterations led to amino acid changes in the tyrosine kinase domain of EGFR comparable to those in non-small cell lung cancer. Thus EGFR-expression in thymic tumours does not rely on mutations in critical functional (activation) domains of the EGFR-gene. Experimental and therapeutic approaches have to consider this difference. Ó 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Thymoma; Thymic carcinoma; EGFR; Mutation

1. Introduction *

Corresponding author. Tel.: +49 6221 56 2666; fax: +49 6221 56 5251. E-mail address: [email protected] (R.J. Rieker).

Thymic epithelial tumours are rare neoplasms located in the anterior mediastinum. Since 1999 a WHO histologic concensus has unified various

0304-3835/$ - see front matter Ó 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.07.003

M. Meister et al. / Cancer Letters 248 (2007) 186–191

aspects of previous applied histological classifications [1–3]. Type A, type AB, and type B1 thymomas exhibit a better prognosis compared to type B2 and B3 thymomas and thymic carcinomas (type C thymomas). The latter group displays a malignant behaviour [4,5]. In advanced thymomas and thymic carcinomas, a multimodal approach including radio- and chemotherapy as well as surgery is currently considered the preferential therapeutic option. However, the response rate to different chemotherapy-regimes varies from 10% to 90% [6]. Therefore novel therapeutic targets are needed to optimize the response rate in thymomas and thymic carcinomas. One promising novel target is KIT. This transmembranous tyrosine kinase receptor is expressed in about 40% of thymic carcinomas. The majority of these tumours do not contain alterations in the c-kit gene in contrast to reported mutations in other tumour entities, like gastrointestinal stroma tumour [6]. So far only one case of a metastasizing thymic carcinoma with a c-kit mutation has been published, in which a therapeutic effect of a KIT inhibitor has been observed [7]. A further promising target might be COX-2, which is overexpressed in several thymomas and thymic carcinomas [8]. The other promising target, which is expressed in thymomas and in thymic carcinomas, is the epidermal growth factor receptor (EGFR). One clinical trial is published concerning this subject. The study reports a well tolerated gefitinib treatment in 26 patients with metastatic thymomas and thymic carcinomas and a stable disease could be achieved in 14 out of 26 patients (54%, 1 PR, 14 SD). The EGFR gene was sequenced only in five patients and no activating mutations were found [9]. A paradigm for EGFR targeted therapies is nonsmall cell lung cancer (NSCLC). Several studies have shown that the majority of NSCLC patients with EGFR gene mutations responded to the tyrosine kinase inhibitors gefitinib or erlotinib [10–12] demonstrating that EGFR-mutational status may not only reveal the pathogenetic principle but may also represent a predictive marker. In thymomas and thymic carcinomas mutational analysis of EGFR in a collective of sufficient size has not yet been reported. We therefore determined the EGFR-mutational status in 20 thymic tumours of several subtypes.

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2. Material and methods 2.1. Tissue and clinical data Tissue samples obtained by thymectomy from 20 different patients with epithelial tumours of the thymus were selected from the tissue bank of the Institute of Pathology, University of Heidelberg, Germany (Table 1). Diagnoses were established according to histological criteria of the WHO classification including immunohistochemistry. All samples were fixed in 4% formaldehyde and paraffin embedded via routine procedure. They were staged according to Masaoka et al. [13]. The 20 tumours split into the following subtypes: 7 type A thymomas, the A component of 8 type AB thymomas, 3 type B3 thymomas and 3 thymic carcinomas (type C thymomas) of different stages according to Masaoka (I–IV). The age of the patients ranged from 48 to 88 years. Eight patients were male and 12 patients were female (Table1). 2.2. Mutational analysis of the EGFR gene For each case, the hematoxyline and eosine stained slides were reviewed to localize tumour regions with less than 10% lymphocyte-infiltration and P90% tumour cell content. From each case one paraffin block was selected and a 16 gauge needle for bone marrow aspiration was used to obtain paraffin wax embedded tissue cores (2 mm diameter, 3 mm height) for DNA extraction. Genomic DNA was isolated from paraffin-embedded tissue samples after deparaffinization with xylene using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendation. The DNA was eluted twice with 50 ll 10 mM Tris–HCl pH 8,0. Exons 18, 19, 20, and 21 of the human EGFR gene were amplified by nested PCR using oligonucleotides designed according to previously published sequences [10,11]: exon 18 (first PCR, forward 5 0 -CAAATGAG CTGGCAAGTGCCGTGTC-3 0 and reverse 5 0 -GAGTT TCCCAAACACTCAGTGAAAC-3 0 ; nested PCR forward 5 0 -CAAGTGCCGTGTCCTGGCACCCAAGC-3 0 and reverse 5 0 -CCAAACACTCAGTGAAACAAAGA G-3 0 ); exon 19 (first PCR, forward 5 0 -AAATAATC AGTGTGATTCGTGGAG-3 0 and reverse 5 0 -GAGGCC AGTGCTGTCTCTAAGG-3 0 ; nested PCR forward 5 0 -GT GCATCGCTGGTAACATCC-3 0 and reverse 5 0 -TGTGG AGATGAGCAG GGTCT-3 0 ); exon 20 (first PCR, forward 5 0 -CCATGAGTACGTATTTTGAAACT-3 0 and reverse 5 0 -CATATCCCCATGGCAAACTCTTGC-3 0 ; nested PCR forward 5 0 -GAAACTCAAGATCGCATTC ATGC-3 0 and reverse 5 0 -GCAAACTCTTGCTATCCCA GGAG-3 0 ); exon 21 (first PCR, forward 5 0 -GCAGCGGG TTACATCTTCTTTC-3 0 and reverse 5 0 -CAG CTCTGG CTCACACTACCAG-3 0 ; nested PCR forward 5 0 -GCTC AGAGCCTGGCATGAA-3 0 and reverse 5 0 -CATCCTC

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Table 1 Clinical and genetic characteristics of thymoma and thymic carcinoma patientsa Age

sex

stage

Hist.

EGFR

Ex 18

Int 18

Ex 19

Int 19

Ex 20

Ex 20 Q787Q CAG ! CAA

Ex 21

1

66

m

III

A

3

wt

wt

hom

wt

75 71 67 68 58 68 49 74 48

f m m m f m f f f

IV III b II II III II II II

A A A A A AB AB AB AB

3 3 3 3 3 3 3 3 3

wt wt wt wt wt wt wt wt wt

wt wt wt wt wt wt wt wt wt

het hom het het hom hom hom hom het

wt wt wt wt wt wt wt wt wt

11 12 13 14 15 16 17 18

56 68 48 88 62 58 66 69

m f f f f m m f

II III II III II III b I

AB AB AB AB B3 B3 B3 C

3 3 1 3 3 3 3 0

wt wt wt wt wt wt wt wt

Wt wt wt wt wt wt wt wt

wt 164996 G!A wt wt wt wt wt wt wt wt 164996 G!A wt wt wt wt wt wt wt

wt

2 3 4 5 6 7 8 9 10

wt 164250 C!T wt wt wt wt wt wt wt wt 164169 G!A wt wt wt wt wt wt wt

wt

hom

wt wt wt wt wt wt

hom het hom het hom hom

19

57

f

IV

C

3

wt

wt

wt

wt

wt

wt

20

58

f

II

C

2

wt

wt

wt

wt

wt

wt

wt wt wt wt wt wt wt wt R836R CGG ! CGT R836R CGG ! CGT

#

Wt wt wt wt wt wt wt wt wt

a

Ex, exon; Int, intron; b, biopsy; f, female; m, male; Hist., histologic subtype according to WHO (C, thymic carcinoma); EGFR: 0, negative staining; 1, weakly positive staining; 2, moderately positive staining; and 3, strongly positive staining. wt, wild type; het, heterozygous; hom, homozygous; nucleotide numbering according to Acc. # AF288738.

CCCTGCATGTGT-3 0 ). PCR reactions were carried out in a volume of 20 ll containing 2 ll of genomic DNA from the second eluate using HotStar Taq DNA Polymerase (Qiagen). Incubations were done at 95 °C for 15 min, then 40 cycles of 95 °C for 20 s, 60 °C for 30 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min. The integrity of the amplified fragments was checked by 2% TAE gel electrophoresis. PCR products were purified using the High Pure PCR Product Purification Kit (Roche, Mannheim, Germany). Finally, PCR products were directly sequenced in both directions (Medigenomix, Munich, Germany). Retrieved sequences were subjected to a BLAST analysis [14] and electropherograms were checked manually. 2.3. Immunohistochemistry Tissue microarrays (TMA) were constructed from paraffin–wax-embedded blocks of twenty specimens of the thymus. A tissue arrayer device (Beecher Instruments, WI, USA) was used. All investigated cases were reviewed and representative tumor areas were marked in the corresponding paraffin wax blocks. Concerning the type AB thymomas at least four areas were marked (two times the A component and two times the B component). The diameter of the cylinders was 1.2 mm.

For the EGFR staining, the EGFR pharmDx kit (Dako) was used. The staining was performed according to the manufacturer’s instructions. The membranous intensity of EGFR was scored for each specimen on a scale of 0 to 3: 0, negative staining; 1, weakly positive staining; 2, moderately positive staining; and 3, strongly positive staining [8]. The staining intensity was evaluated for the maximum intensity among positive cells (‘‘maximum intensity of staining’’) [8]. 2.4. Statistical analyses With respect to stage and histological subtype, the differences in the frequency distribution of the intensity were analyzed using the Kruskal–Wallis Test. A difference was considered statistically significant if the p-value of the corresponding statistical test did not exceed 5% (p 6 0.05). 3. Results 3.1. Sequencing results of exons 18 to 21 of the EGFR gene No mutations in the tyrosine kinase domain of EGFR that are currently considered pathogenic and predictive of a therapeutic response to small molecule tyrosine kinase

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4. Discussion

Fig. 1. Sequencing results. Single nucleotide polymorphism at codon 787 in exon 20 for patients #8, 10, and 19 (a) and at codon 836 in exon 21 for patient #19 (b)

inhibitors (gefitinib, erlotinib) were detected in the DNA samples analyzed in this study (Table 1). However, we identified in two patients at codon 836 (Arg; CGG to CGT) in exon 21 of the EGFR gene a variant that was also present in the corresponding non-tumourous normal tissue. Furthermore we detected a single nucleotide polymorphism at codon 787 (Gln; CAG to CAA), with 6 patients out of 20 being heterozygous and 11 out of 20 being homozygous for the CAA variant (Fig. 1). 3.2. Sequencing results of introns 18 to 21 of the EGFR gene In addition, several variations in intronic sequences (Table 1) were found. Variations found were localised in one patient in intron 18 at position 164250 (C to T) and intron 19 at position 164996 (G to A), one patient in intron 18 at position 164169 (G to A) and intron 19 at position 164996 (G to A). These changes were also present in the respective non-tumour tissues. No alterations were found in introns 20 and 21. 3.3. Immunohistochemistry results of the investigated cases In Table 1 the results of EGFR immunohistochemistry were given for the 20 investigated cases. All thymomas except one exhibited a strong membranous staining. One thymic carcinoma was negative; the other two had a moderate and strong membranous staining. There was no difference between the EGFR expression and stage (p > 0.0547) as well as histology (p > 0.0587).

The role of EGFR in thymomas and thymic carcinomas has remained relatively unexplored despite the high frequency of EGFR overexpression. Immunohistochemical studies of EGFR expression showed often no correlation with histological subtype and stage in thymomas and thymic carcinomas, however, an overexpression is more often found in thymomas than in thymic carcinomas [15–18]. A similar result was found in our study, where only one thymic carcinoma had a score of 3. Thymic carcinoma and type B3 thymoma specimens showed a higher average number of EGFR gene copies per cell than other histological types in FISH analysis [18]. Further studies are needed to elucidate the lack of concordance of EGFR in regard to FISH analysis and immunohistochemistry. These results of overexpression may render anti-EGFR antibody therapy a feasible novel treatment option in epithelial tumors of the thymus [6]. NSCLC patients with somatic mutations in the EGFR gene show a high response rate and clinical benefit from an EGFR inhibitor therapy, however to date the EGFR mutational status in thymomas and thymic carcinomas has remained largely obscure. In our study, we investigated several subtypes of thymomas except for type B1 and type B2 thymomas, as these tumours have a large number of lymphocytes. The presence of lymphocytes would probably mask a mutation in the subsequent analysis [1,2]. None of the 20 investigated cases showed mutations in the EGFR tyrosine kinase domain. The observed polymorphism at exon 20 has been described by other investigators previously [19]. In spite of the results obtained in our study, EGFR tyrosine kinase inhibitors (EGFR TKIs) can currently be discussed as a therapeutic option in epithelial tumours of the thymus. Retrospective studies demonstrated that approximately 18% of the patients with no mutations in EGFR show a partial response to gefitinib in NSCLC [20]. Furthermore, patients with squamous cell carcinomas of the head and neck have responded favourably to EGFR TKIs in combination with other agents [21–23]. Considering the fact that the thymus is derived [24] from endodermal stem cells of the pharyngeal pouches (the neck region), a benefit resulting from treating patients with thymomas and thymic carcinomas with EGFR inhibitors is still conceivable. Results of a clinical trial of heavily pre-treated patients with advanced thymomas and thymic carci-

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nomas show that a stable disease with gefitinib could be achieved in more than half of these patients. In this study including 26 patients, no mutation was observed in 5 patients where EGFR gene mutational status was investigated [9]. Predicting the response rate of thymomas and thymic carcinomas, perhaps FISH analysis may be a good tool since malignant subtypes of these tumours show an increased EGFR gene copy number [18]. In NSCLC patients, increased EGFR gene copy number are associated with a better clinical outcome in case of gefitinib treatment [25–27]. Perhaps alternative pathways other than activating mutations may play a role in the EGFR overexpression in thymomas and thymic carcinomas. Emerging evidence suggests a direct interaction between EGFR signalling and COX-2 activity [28–31]. In addition, a significant reduction of tumour growth in vivo and in vitro has been shown using a combined treatment of COX-2 and EGFR inhibitors in a hypopharyngeal tumor cell line [32,33]. In a recent study an up-regulation of COX-2 could be demonstrated in several subtypes of thymomas and thymic carcinomas [8]. Further, a weak correlation with EGFR expression could be found indicating a possible interaction between these two proteins [8]. Somatic mutations in EGFR tyrosine kinase domain may be predictive for the tumour responsiveness to gefitinib or erlotinib. Our experiment results indicate that these genetic modifications are at best uncommon in thymomas or thymic carcinomas and clarified that immunohistochemistry is not predictive of mutational status of EGFR. Several clinical studies have demonstrated that non-small cell lung cancer patients do profit from EGFR tyrosine kinase inhibitors though lacking the mutations. Therefore, EGFR tyrosine kinase inhibitors should be taken into account as a therapeutic option in thymomas and thymic carcinomas in a multimodal treatment management. References [1] J . Rosai, L.H. Sobin (Eds.), Histological Typing of Tumors of the Thymus. World Health Organization. International Histological Classification of Tumours, Springer Verlag, Heidelberg, 1999. [2] W.D. Travis, E. Brambilla, H.K. Mu¨ller-Hermelink, C.C. Harris (Eds.), World Health Organization Classification of Tumours. Pathology and genetics of tumors of the Lung, Pleura, Thymus and Heart, IARC Press, Lyon, France, 2004.

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