Low incidence of EGFR and HRAS mutations in cutaneous squamous cell carcinomas of a German cohort

Abstract:  Epidermal growth factor receptor (EGFR) is highly expressed in squamous cell carcinoma (SCC). The response of patients with lung cancer to EGFR inhibitors is significantly associated with the presence of EGFR mutations. Although these drugs have already been used for the treatment of advanced cutaneous SCC, the knowledge about EGFR mutations in this cancer is limited to one previous study in the US population. We analysed the presence of EGFR and concomitant HRAS mutations in a German cohort of 31 patients with cutaneous SCC by direct sequencing of EGFR and SNaPshot analysis of concomitant RAS mutations. We found a low prevalence of EGFR mutations (1/31; 3%) and HRAS mutations (1/31; 3%). The detected P741L EGFR mutation was proven to be somatic. Our results indicate that both EGFR and HRAS mutations are rare events in the carcinogenesis of cutaneous SCC, and therefore, only a small subgroup of patients will benefit from the screening for EGFR mutations in the run‐up to targeted therapies with EGFR inhibitors.


Accepted for publication 30 May 2011
Background The incidence of cutaneous squamous cell carcinoma (SCC) of the skin in the Caucasian population is increasing. Risk factors for the development of SCC comprise chronic UV light exposure, fair skin, immunosuppression, HPV infection, chronic wounds, scarring dermatoses and various genodermatoses (1). Several molecular genetic alterations have been identified in the pathogenesis of cutaneous SCC, including chromosomal aberrations, mutations in the p53 tumor suppressor gene and oncogenic HRAS mutations (2). In addition, cutaneous SCC express the epidermal growth factor receptor (EGFR), a transmembrane type I receptor tyrosine kinase (3). Overexpression of EGFR was recently described in 25 of 32 cutaneous SCC, and EGFR amplification was observed in seven of 35 SCC (4). Impaired EGFR signalling showed decreased incidence of papillomas in a multistage skin chemical carcinogenesis mouse model (5). Previous case reports suggested that inhibition of activated EGFR by antibodies or small molecule inhibitors might be a therapeutic option in advanced cutaneous SCC (6)(7)(8). Activation of the EGFR pathway may be caused by both gene amplification and activating somatic mutations (9). The presence of EGFR mutations is of relevance for the efficiency of EGFRbased molecular targeted therapies in lung and colon cancer (10)(11)(12). EGFR mutations are also strong predictors of a better outcome with gefitinib, an EGFR tyrosine kinase inhibitor, among patients with non-small-cell lung cancer in East Asia (13).

Questions addressed
The aim of our study was to investigate the presence of oncogenic EGFR mutations and concomitant RAS mutations in a cohort of German patients with cutaneous SCC.

Sample and DNA acquisition
Thirty-one formalin-fixed paraffin-embedded cutaneous SCC (19 from men and 12 from women) were retrieved from the histologi-cal files of the Department of Dermatology, University of Regensburg. Written informed consent has been obtained from the patients, and the study was performed according to the guidelines of the local Ethics Committee of the University Clinic of Regensburg and the declaration of Helsinki. All material at first underwent routine histopathological diagnosis, and only material not needed for further diagnostic procedures was used for this study. DNA was isolated after manual microdissection following standard protocols as described previously (14).

Mutational analysis of EGFR and RAS genes
Possible EGFR mutations were studied by sequence analysis as described earlier (15). In brief, we used a nested PCR approach. The first multiplex PCR contained all forward and reverse primers for exons 18 (fwd: GCATGGTGAGGGCTGAGGTGA; rev: CCCC ACCAGACCATGAGAGGC), 19 (fwd: TGCCAGTTAACGTCTTC CTTC; rev: CCACACAGCAAAGCAGAAAC) and 21 (fwd: AGCT TCTTCCCATGATGATCTGTCC; rev: GGCAGCCTGGTCCCTGG TGTC) of EGFR. For the second multiplex PCR (exon 18 fwd: AC CCTTGTCTCTGTGTTCTTGTCCC; rev: GCCCAGCCCAGAGGC CTGTG; exon 19 fwd: AACGTCTTCCTTCTCTCTCTG; rev: CCA CACAGCAAAGCAGAAAC; and exon 21 fwd: TCCCATGATGAT CTGTCCCTCACA; rev: CAGGAAAATGCTGGCTGACCTAAAG), 1 ll from the first multiplex PCR was used as a template. Sequencing was performed in forward and reverse directions for each exon. Hot spot mutations of HRAS, KRAS and NRAS genes were screened using a highly sensitive SNaPshot Ò multiplex assay (Applied Biosystems, Carlsbad, CA, USA) as described earlier (16). In brief, two exons containing the codons 12, 13 and 61, which represent hot spot mutation loci of RAS genes in human cancer, were amplified in a multiplex PCR. The hot spot loci were then analysed by mutationspecific SNaPshot Ò probes, followed by the separation of SNaPshot Ò products on the basis of size by gel electrophoresis. Possible mutations were confirmed by a second independent PCR.

Results
An EGFR mutation was found only in 1 ⁄ 31 (3%) SCC (Table 1). This c.2222C>T transition of exon 19 resulted in a p.P741L substitution. Analysis of normal skin adjacent to this SCC showed a wild-type EGFR sequence (Fig. 1), confirming the somatic nature of this mutation and excluding a germline mutation. Interestingly, the patient with the EGFR mutation is an organ transplant recipient. The P741L mutation has not yet been described for SCC, but 2 ⁄ 81 glioblastomas previously showed this missense mutation (17).
Analysis of RAS genes showed a c.182A>T transversion in 1 ⁄ 31 (3%) SCC, resulting in a p.Q61L substitution. According to the COSMIC database (http://www.sanger.ac.uk/genetics/CGP/cosmic/ ), the Q61L mutation has been described in 67 tumors, most frequently in the skin (two SCC, one basal cell carcinoma, three malignant melanomas, 10 Spitz nevi and nine keratoacanthomas), the urinary tract and prostate cancer. No further mutations of HRAS, KRAS and NRAS genes were found at the investigated loci (Table 1).

Conclusion
The reported HRAS mutation frequency in SCC varies between 0% and 46%, with an average of <10% (18)(19)(20)(21)(22)(23). The COSMIC database reports the following mutation frequencies in cutaneous SCC: TP53 (44%), CDKN2A (27%), HRAS (9%), PTCH1 (8%), NRAS (7%), KRAS (5%), PTEN (7%) and FGFR2 (2%). Our results suggest that EGFR mutations are rare events in the pathogenesis of cutaneous SCC, confirming the results of a previous study on patients with cutaneous SCC in the US population, which detected 2.5% EGFR mutations (24). Similarly, EGFR mutations are infrequent in head and neck SCC (HNSCC) (25,26). Interestingly, oncogenic mutations appear to occur at a rather low frequency in cutaneous SCC in contrast to tumor suppressor gene mutations, whereas in benign seborrhoeic keratoses, multiple oncogenic mutations can be observed at a high frequency, in contrast to tumor suppressor gene mutations (27). Although EGFR mutations are obviously rare in cutaneous SCC, the EGFR signalling pathway represents an interesting therapeutic target, because activation can occur by alternative mechanisms (8).  Andreas Mauerer analysed the data and prepared the manuscript, Eva Herschberger performed the mutation analyses, Wolfgang Dietmaier analysed the EGFR data, Michael Landthaler discussed the data and contributed to the preparation of the manuscript and Christian Hafner designed the study, analysed the data and contributed to the manuscript preparation.