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Research Letter |

EGFRvIII Expression in Squamous Cell Carcinoma of the Skin FREE

Piotr J. Dziunycz, MD1; Zelmira Lazarova, MD2; Nathan Duncan, BS2; Stuart Wong, MD3; Marcy Neuburg, MD2; Günther F. L. Hofbauer, MD1; Edit B. Olasz, MD, PhD2
[+] Author Affiliations
1Department of Dermatology, University Hospital Zurich, Switzerland
2Department of Dermatology, Medical College of Wisconsin, Milwaukee
3Department of Oncology, Medical College of Wisconsin, Milwaukee
JAMA Dermatol. 2013;149(10):1240-1242. doi:10.1001/jamadermatol.2013.5230.
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Published online

Cutaneous squamous cell carcinoma (SCC) is a common cancer with an estimated 200 000 new cases annually.1 It is usually readily curable. However, a small subset of SCC tumors (approximately 10%, or 20 000 cases annually), termed “high-risk SCC,” carries an elevated risk of metastasis and death and accounts for nearly all the mortality associated with SCC.2 Epidermal growth factor receptor (EGFR) is overexpressed in a large percentage of SCC. Several therapeutic trials using EGFR blockade (with agents such as lapatinib, erlotinib, or panitumumab) to treat high-risk or metastatic SCCs are ongoing.3 Cetuximab, a chimeric IgG1 monoclonal antibody against EGFR, has been considered and used in the treatment of SCC; however, formal trials have not been performed, and good data are lacking.4 The most common form of mutant EGFR, called EGFRvIII, has been described in several cancers, including head and neck cancer. It has been shown that EGFRvIII contributes to enhanced growth of SCC and resistance to EGFR inhibitor drugs.5The aim of this study was the assessment of wild-type EGFR (wtEGFR) and EGFRvIII expression in skin SCC and its correlation with tumor biology.

Ethical approval was obtained from the institutional review board for processing specimens from clinically indicated excisions. All participants provided their written consent to participate in the research study. A total of 73 SCC samples for the analysis of EGFR messenger RNA (mRNA) expression levels were derived from Mohs surgery. Total RNA was extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Previously described primers5 flanking the deletion of exons 2 to 7, which generate bands for both wtEGFR and EGFRvIII, were used for standard reverse transcriptase–polymerase chain reaction. Polymerase chain reaction products were separated and visualized on a 2% agarose gel containing ethidium bromide.

Immunohistochemical staining was performed on tissue microarray composed of 11 normal skin samples and 240 SCC samples with monoclonal anti-wtEGFR IgG rabbit antibody (EP38Y; Abcam) and rabbit monoclonal anti-EGFRvIII (L8A4) antibody. Immunoreactivity was rated as 1 for weakly positive (<25% of the tumor mass stained), 2 for moderately positive (25%-75% of the tumor mass stained), and 3 for strongly positive (>75% of the tumor mass stained). Statistical analysis was performed using GraphPad Prism 5.0 and Microsoft Excel 2000. P < .05 was considered statistically significant. Differences in the immunoreactivity between the groups were calculated using Mann-Whitney test or analysis of variance.

High Expression of wtEGFR Protein in Skin SCC

The immunohistochemical staining of wtEGFR (Figure 1A-C) showed its high expression in cutaneous SCC as well as in the epidermis of normal skin. There was, however, no detectable difference in the expression between the 2 groups (Figure 1D; P = .30) Although the expression varied within the group of SCC, no significant differences in the EGFR expression have been detected when compared the well, moderately, and poorly differentiated tumors to each other (Figure 1E; P = .09).

Place holder to copy figure label and caption
Figure 1.
Representative Immunohistochemical Staining of SCC Tumor Samples

Paraffin-embedded tumor samples were stained with mouse anti-epidermal growth factor receptor (EGFR) (31G7) antibodies, which recognize the wild-type EGFR (wtEGFR). Mouse monoclonal IgG1 was used as an isotype control. Immunoreactivity was rated as (A) 1 for weakly positive (<25% of the tumor mass stained), (B) 2 for moderately positive (25%-75% of the tumor mass stained), and (C) 3 for strongly positive (>75% of the tumor mass stained) staining. D, EGFR immunostaining scores of normal skin, in situ, and invasive squamous cell carcinoma (SCC). E, No significant differences in the expression of wtEGFR have been detected when comparing normal skin with SCC or when comparing well, moderately (mod), and poorly differentiated (diff) SCC. Error bars in D and E indicate standard deviations.

Graphic Jump Location
No Detection of EGFRvIII in Either Primary or Metastatic SCC

None of the examined SCC samples expressed mRNA for EGFRvIII. We detected wtEGFR mRNA in all samples (data not shown). Similarly, in the immunohistochemical analysis of both invasive and in situ SCC, none of the examined samples showed detectable expression of the mutated EGFRvIII protein. The representative SCC staining is shown in Figure 2A-C. Since EGFRvIII has been previously detected in metastatic tumors, the staining was next extended to the metastases of cutaneous SCC; however, none of the metastatic SCC demonstrated EGFRvIII immunoreactivity (Figure 2D-F).

Place holder to copy figure label and caption
Figure 2.
Representative Immunohistochemical Staining of Cutaneous SCC and SCC Metastasis

Representative Immunohistochemical staining of cutaneous squamous cell carcinoma (SCC) (A-C), and SCC metastasis for type III EGFR deletion mutant (EGFRvIII) and wild-type EGFR (D-F). (EGFR indicates epidermal growth factor receptor.) Metastatic SCCs were collected (n = 6 from 4 patients), paraffin embedded, and stained with hematoxylin-eosin (D), mouse anti-EGFR (31G7) (E), and mouse anti-EGFRvIII (L8A4) antibodies (F). Mouse monoclonal IgG1 was used as isotype control. None of the specimens studied expressed EGFRvIII. NR6M cells transduced with EGFRvIII served as a positive control.

Graphic Jump Location

Mutations in the EGFR gene are commonly found in many types of human tumors, and deletions in the extracellular domain are the most frequent. The most common of these is the type III EGFR deletion mutant (EGFRvIII).5 It has been shown that EGFRvIII contributes to enhanced growth and resistance in targeting wtEGFR. Since EGFR seems to be an attractive target for the therapy of high-risk SCC, it is of high importance to analyze the presence of the potentially resistant mutant variant (EGFRvIII) in the skin SCC.

In this study of the screening of a large number of samples, EGFRvIII has not been detected. Our data are consistent with those of previous studies performed on smaller numbers of samples. Previous studies showed only very low incidence of EGFR mutations overall.6 Interestingly, however, even the selected group of particularly high risk SCC (eg, metastatic SCC did not show expression of the mutated variant of EGFR).

The study confirms the high expression of the wtEGFR in the cutaneous SCCs. Similarly to previous findings, we did not find any correlation between EGFR expression level and the histologic differentiation of the tumors. EGFR is a validated therapeutic target in other cancers, and approved drugs targeting EGFR exist. These findings may provide a therapeutic opportunity for a small subset of patients, with advanced SCC refractory to conventional treatment.

Corresponding Author: Piotr J. Dziunycz, MD, Department of Dermatology, Medical College of Wisconsin, 9200 W Wisconsin Ave, Milwaukee, WI 53226 (ebolasz@mcw.edu).

Published Online: August 28, 2013. doi:10.1001/jamadermatol.2013.5230.

Author Contributions: Dr Olasz had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Dziunycz, Wong, Neuburg, Hofbauer.

Acquisition of data: Lazarova, Duncan, Neuburg, Hofbauer, Olatz.

Analysis and interpretation of data: Dziunycz, Lazarova, Wong, Hofbauer, Olatz.

Drafting of the manuscript: Dziunycz, Hofbauer.

Critical revision of the manuscript for important intellectual content: Lazarova, Duncan, Wong, Neuburg, Hofbauer, Olatz.

Obtained funding: Neuburg, Hofbauer.

Administrative, technical, or material support: Dziunycz, Lazarova, Duncan, Neuburg, Hofbauer, Olatz.

Study supervision: Lazarova, Wong, Hofbauer, Olatz.

Conflict of Interest Disclosures: None reported.

Funding/Support: Dr Dziunycz was supported by the Olga-Mayenfisch Foundation, the René-Touraine-Foundation, and the European Skin Research Foundation.

Role of the Sponsors: The sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript.

Lomas  A, Leonardi-Bee  J, Bath-Hextall  F.  A systematic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol. 2012;166(5):1069-1080.
PubMed   |  Link to Article
Samarasinghe  V, Madan  V, Lear  JT.  Management of high-risk squamous cell carcinoma of the skin. Expert Rev Anticancer Ther. 2011;11(5):763-769.
PubMed   |  Link to Article
US National Institutes of Health. Clinical trials. http://clinicaltrials.gov/ct2/home. Accessed October 22, 2012.
Wollina  U.  Cetuximab in non-melanoma skin cancer. Expert Opin Biol Ther. 2012;12(7):949-956.
PubMed   |  Link to Article
Sok  JC, Coppelli  FM, Thomas  SM,  et al.  Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting. Clin Cancer Res. 2006;12(17):5064-5073.
PubMed   |  Link to Article
Ridd  K, Bastian  BC.  Somatic mutation of epidermal growth factor receptor in a small subset of cutaneous squamous cell carcinoma. J Invest Dermatol. 2010;130(3):901-903.
PubMed   |  Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.
Representative Immunohistochemical Staining of SCC Tumor Samples

Paraffin-embedded tumor samples were stained with mouse anti-epidermal growth factor receptor (EGFR) (31G7) antibodies, which recognize the wild-type EGFR (wtEGFR). Mouse monoclonal IgG1 was used as an isotype control. Immunoreactivity was rated as (A) 1 for weakly positive (<25% of the tumor mass stained), (B) 2 for moderately positive (25%-75% of the tumor mass stained), and (C) 3 for strongly positive (>75% of the tumor mass stained) staining. D, EGFR immunostaining scores of normal skin, in situ, and invasive squamous cell carcinoma (SCC). E, No significant differences in the expression of wtEGFR have been detected when comparing normal skin with SCC or when comparing well, moderately (mod), and poorly differentiated (diff) SCC. Error bars in D and E indicate standard deviations.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.
Representative Immunohistochemical Staining of Cutaneous SCC and SCC Metastasis

Representative Immunohistochemical staining of cutaneous squamous cell carcinoma (SCC) (A-C), and SCC metastasis for type III EGFR deletion mutant (EGFRvIII) and wild-type EGFR (D-F). (EGFR indicates epidermal growth factor receptor.) Metastatic SCCs were collected (n = 6 from 4 patients), paraffin embedded, and stained with hematoxylin-eosin (D), mouse anti-EGFR (31G7) (E), and mouse anti-EGFRvIII (L8A4) antibodies (F). Mouse monoclonal IgG1 was used as isotype control. None of the specimens studied expressed EGFRvIII. NR6M cells transduced with EGFRvIII served as a positive control.

Graphic Jump Location

Tables

References

Lomas  A, Leonardi-Bee  J, Bath-Hextall  F.  A systematic review of worldwide incidence of nonmelanoma skin cancer. Br J Dermatol. 2012;166(5):1069-1080.
PubMed   |  Link to Article
Samarasinghe  V, Madan  V, Lear  JT.  Management of high-risk squamous cell carcinoma of the skin. Expert Rev Anticancer Ther. 2011;11(5):763-769.
PubMed   |  Link to Article
US National Institutes of Health. Clinical trials. http://clinicaltrials.gov/ct2/home. Accessed October 22, 2012.
Wollina  U.  Cetuximab in non-melanoma skin cancer. Expert Opin Biol Ther. 2012;12(7):949-956.
PubMed   |  Link to Article
Sok  JC, Coppelli  FM, Thomas  SM,  et al.  Mutant epidermal growth factor receptor (EGFRvIII) contributes to head and neck cancer growth and resistance to EGFR targeting. Clin Cancer Res. 2006;12(17):5064-5073.
PubMed   |  Link to Article
Ridd  K, Bastian  BC.  Somatic mutation of epidermal growth factor receptor in a small subset of cutaneous squamous cell carcinoma. J Invest Dermatol. 2010;130(3):901-903.
PubMed   |  Link to Article

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