Acta Oto-Laryngologica
ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas Maria E. Christensen, Marianne H. Therkildsen, Bente L. Hansen, Georg N. Hansen & Poul Bretlau To cite this article: Maria E. Christensen, Marianne H. Therkildsen, Bente L. Hansen, Georg N. Hansen & Poul Bretlau (1992) Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas, Acta Oto-Laryngologica, 112:4, 734-738, DOI: 10.3109/00016489209137467 To link to this article: http://dx.doi.org/10.3109/00016489209137467
Published online: 08 Jul 2009.
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Date: 21 April 2016, At: 00:20
Acta Otolaryngol (Stockh) 1992; 1 1 2 734-738
Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas MARIA E. CHRISTENSEN,' MARIANNE H. THERKILDSEN: BENTE L. HANSEN: GEORG N. HANSEN4 and FQUL BRETLAU' From the Departments of 'Oto-Loryngology and Head I Neck Surgery, and 2Pathology, Rigshospitalet, University of Copenhagen; and Institutes of 'Medical Microbiology and 4CeIl Biology and Anatomy, Unicersity of Copenhagen, Denmark
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Christensen ME,Therkildsen MH, Hansen BL, Hansen GN,Bretlau P. Immunohistochemical detection of epidermal growth Factor receptor in laryngeal squamous cell carcinomas. Acta Otolaryngol (Stockh) 1992; 112 734-738.
Laryngeal squamous cell carcinomas from 15 consecutive preoperatively irradiated patients were investigated for the expression of epidermal growth factor receptor (EGF receptor). The study was performed on frozen sections by means of the 5-layer APAAP technique employing an antibody recognizing the extracellular part of the EGF receptor. In sections from 9 of the patients with laryngeal squamous cell carcinoma, normal differentiated epithelia were included. Sections from 6 of these patients, in addition, contained dysplastic epithelia. Expression of EGF receptor-like material was demonstrated in the basal cell layer of normally differentiated laryngeal epithelia and in dysplastic epithelia. Fourteen of the squamous cell carcinomas proved EGF receptor positive. Nearly all cells in the poorly differentiated carcinomas showed positive staining with the antibodies. In moderately to well differentiated carcinomas a reduction in the extent of staining was seen in certain areas. Especially for the epithelial pearls, the staining reaction was localized to the undifferentiated cells in the periphery. This finding corresponds to the staining pattern observed in the basal cell layers of normal epithelia. The present investigation confirms the expression of EGF receptor-like material in normal laryngeal epithelia, dysplastic epithelia and squamous cell carcinoma. The staining pattern was similar to that observed in oral squamous cell carcinomas, predominantly varying inversely with cellular differentiation. Key word: EGF receptor, epithelia, dyspasia, carcinoma, immunoreactiue, cellular differentiation.
INTRODUCTION Epidermal growth factor (EGF) is a polypeptide stimulating growth and, presumably, differentiation of a variety of mammalian epithelial tissues and cell types (1,2, 3). EGF binds to a specific membrane receptor and thereby activates a tyrosine-specific protein kinase which is part of the intracellular domain of the receptor (4). This in turn leads to a variety of biochemical and physiological events and ultimately to DNA replication and cell division (2). The EGF receptor is detectable on a large variety of cell types and tissues, including the proliferative compartment of epithelia (1, 5, 6). A close similarity between the amino acid sequence from the erb-B oncogene of AEV-H ( a strain of the avian erytroblastosis virus) and the cytoplasmic and transmembrane part of the EGF receptor has been described, suggesting that EGF receptor expression and malignancy might be interconnected (7). The frequent overexpression of EGF receptors in cell lines derived from tumours, in particular -but not exclusively -carcinomas might also suggest an interconnection between EGF receptor and malignancy (8, 9). By use of immunohistochemistry, we recently have demonstrated EGF receptor-like material in s p i m e n s from oral mucosa, dysplastic epithelia and oral squamous cell carcinomas. The staining pattern was shown to vary inversely with cellular differentiation ( 10). The aim of the present investigation was to demonstate the presence of EGF receptor
Acta Otolaryngol (Stockh) 112
EGF receutors in laryngeal carcinomas 735
in laryngeal squamous cell carcinomas. In particular we were interested in detecting a possible correlation with cellular differentiation.
Downloaded by [UNSW Library] at 00:20 21 April 2016
MATERIAL AND METHODS Tissue samples were obtained from 15 consecutive cases (1 woman and 14 men) of squamous cell carcinomas. In sections from 9 patients with squamous cell carcinoma, normal differentiated laryngeal epithelia were included. Sections from 6 patients with squamous cell carcinoma contained, in addition, dysplastic epithelia. All patients were staged according to the UICC TNM classification (1 1) and had received radiotherapy preoperatively (6266 Gray). Heamatoxylin-eosine staining was carried out on parallel froz n sections obtained and cut from the same tumour specimens as those on which the immunohistochemical investigation was performed. The squamous cell carcinomas were graded as poorly, moderately or well differentiated based on: 1) Evidence of differentation (intercellular bridges, keratinization and epithelial pearls) and 2) evidence of rapid, abnormal proliferation (mitoses per highpower field, atypical mitoses, nuclear and cellular pleomorphism, multinucleated and tumour giant cells) (12). The majority of the tumour specimens were moderately differentiated. Tissue specimens for EGF receptor identification were cut from the tumour immediately after removal and rapidly frozen in pre-cooled isopentane at -880°C. Five-micron sections were cut, placed on glass slides and airdried overnight at room temperature. The sections were then post-hed in pre-cooled acetone at -20°C for 10 min and stored at -80°C until immunohistochemical screening. Indirect immunohistochemical staining was performed with mouse monoclonal anti-alkaline phosphatase (APAAP) 5-layer technique modified from Cordell (13). Sections were treated at room temperature unless otherwise mentioned. Before application of primary antibodies sections were washed for 3 x 5 min with 0.5M Tris-HC1 buffer pH 7.2 supplemented with 0.5M NaCl and 0.1% Triton X-100.Nonspecific binding of proteins to sections was reduced by incubation (30min) with normal rabbit serum (Dakopatts a/s, Denmark; code No. X902) diluted 1:30 in Tris-HC1 supplemented with 3% bovine serum albumin (BSA, Sigma). The sections were then covered (for 24 h in a humid chamber at 4°C) with a 1:40 dilution of mouse monoclonal antibody (primary antibodies, clone EGFRl code RPN.513, Amersham) directed against the external segment of the EGF receptor (14). All antibodies were diluted in Tris-HC1-BSA. Before application of secondary layers the sections were equilibrated to room temperature for 30min and then washed for 3 x 5 min as described above. The secondary antibodies (application time 30 min) were rabbit anti-mouse (Dakopatts; code No, 2259) diluted 1:20. A third layer consisted of APAAP complexes (Dakopatts; code No. D651) diluted 1:25. The steps with secondary antibodies and APAAP complexes were repeated. After the final incubation with APAAP complexes, sections were washed for 2 x 5 min in tris HCI pH 7.2 and then equilibrated in Tris-HC1 pH 8.2 without triton X-100 for 5min. Enzyme reaction (25min) was visualized with Naphthol Asmix Phosphate (Sigma; code No. N-4875) and fast red TR Salt (Sigma; code No. F-1500) containing 1 mM levamisole (Sigma; code No. L-9756) to block endogenous alkaline phosphatase. Sections were counterstained with Mayer’s haematoxylin for 90 s, washed in cold water for 10 min and then mounted with Glycergel (Dakopatts; code No. C563). Positive controls were sections of normal human placenta known to have abundant EGF receptor expression, normal human skin and normal oral mucosa (1). Methodological controls consisted of the replacement of primary antibodies with a matched IgG subclass of unrelated specificity (Dakopatts; code No. M759). Positive controls
736 M. E. Christensen er d.
~olpryngol(Stoekh) 112
were included in each staining process to ensure minimal day to day methodological variation. Specificity controls consisted of absorption of the primary antibodies on a monolayer of A431 cells ( 14). Staining results were evaluated by two independent observers. The sections were graded as positive only if more than 5% of the tumour cells were stained.
Downloaded by [UNSW Library] at 00:20 21 April 2016
RESULTS Fourteen of the I5 carcinomas showed positive staining reaction with the anti-EGF receptor antibodies. The immunoreaction was predominantly localized to the cellular surface membrane. In sections including normal differentiated laryngeal epithelia the basal cell layer was positively stained. One section showed positive staining reaction in the basal cell layer as well as in the spinous cells. In sections containing dysplastic epithelia nearly all cells showed positive staining. However, in one dysplastic section, staining reaction was observed only in the basal cell layer and in the spinous cells. In the squamous cell carcinomas intra- and intersection variability in staining intensity was observed. In poorly differentiated carcinomas nearly all cells were EGF receptor positive. In moderately and well differentiated carcinomas a reduction in the extent of stained areas was seen, paralleling the situation observed in the differentiated upper layers of nonnal oral mucosa. In the epithelial pearls the EGF receptor positive cells were localized to the mdifierentiated cells in the periphery (Fig. 1). Methodological controls with antibodies of unrelated specificity revealed negative staining results. The included placental sections, normal human skin and normal oral mucosa were
Fig. 1. EGF -tor staining of laryngeal squamous cell carcinoma, well differentiated. Note, in the epithelial pear4 the staining reaction is localized to the und8erentiated ells in the periphery (asterisks). APAAP with fut red and h t o x y l i n e ( x 250).
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EGF receptors in laryngeal carcinomas 131
always EGF receptor positive. Specificity controls with absorption of the primary antibodies with A431 cells showed negative staining results on placenta and the laryngeal carcinomas.
Downloaded by [UNSW Library] at 00:20 21 April 2016
DISCUSSION The results of the present study elucidate the relationship between expression of EGF receptors (as determined by the monoclonal antibody) and cellular differentiation in laryngeal squamous cell carcinomas. In the material studied, expression of the EGF receptor-like material was widely distributed but with a variable staining intensity. In general, the EGF receptor was expressed in the basal cell layer of normal differentiated laryngeal epithelia and in dysplastic epithelia. Our results are in accordance with results from other studies comprising normal epithelia, including oral mucosa. In these studies EGF receptors have been proved in the basal cell layer, which is the proliferative compartment of all epithelia (1, 5, 6, 16). In dysplastic epithelia nearly all cells showed positive staining, which is in agreement with other investigations concerning oral dysplastic epithelia (10). Nevertheless, other authors could not confirm this pattern of EGF receptor expression by immunohistochemical investigation on formalin fixed specimens embedded in paraffin (17). This discrepancy could, however, well be explained by the use of frozen sections versus formalin fixed paraffin embedded sections. Another study, concerning severe laryngeal dysplasia, revealed anti-EGF receptor positive cells ( 18) possibly indicating a connection between histological grade and EGF receptor expression. In our study, grading of dysplasia was not possible because the investigation was performed on frozen sections. The presence of EGF receptor in dysplastic epithelia might reflect the requirement of the receptor in the process of dedifferentiation (10, 18). The receptor has also been related to the benign human skin disease, psoriasis vulgaris (19). In that study the EGF receptor was demonstrated at sites of an abnormally increased proliferative capacity and abnormally differentiated state. The section negative for EGF receptor-like material was taken from a moderately differentiated tumour. One possible explanation might be that the carcinoma expresses exclusively truncated receptors (7). Another explanation of the EGF receptor negative section could be that the tumour cells at this site were engaged in paracrine production of EGF/TGF-u (unpublished data). TGF-a is a polypeptide which shares close amino sequence similarity with EGF (20). The effects of TGF-a are mediated through the EGF receptor resulting in the same biological response (21). TGF-a is known to induce non-neoplastic cells to lose contact inhibition and undergo anchorage-independent growth (22). Recently TGF-a has been demonstrated in human colon, lung and pancreatic carcinomas (23, 24). The results of the present study revealed that nearly all cells in the poorly differentiated carcinomas showed positive anti-EGF staining. For the moderately to well differentiated carcinomas, a reduction in the extent of positive areas was observed. Furthermore, for the epithelial pearls EGF receptor positive cells were characteristically localized to the undifferentiated cells in the periphery. These results indicate that the receptor is always more or less expressed in undifferentiated cells, but when the malignant cells differentiate, as seen in moderately and well differentiated carcinomas, as well as in the epithelial pearls, the EGF receptor is not expressed. Our present investigation verified the presence of EGF receptor in laryngeal squamous cell carcinomas. The staining pattern was similar to that seen in oral squamous cell carcinomas with a correlation to cellular differentiation (10). Whether or not the EGF receptor is overexpressed in head and neck carcinomas remains to be determined in a quantitative study.
738 M. E. Christensen el al.
A a a Otolaryngol (Stockh) 112
ACKNOWLEDGEMENTS The authors are indebted to Mrs. Bente Christensen for skilful technical assistance.
REFERENCES
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1. Gusterson B, Cowley G, Smith JA. Ozanne B. Cellular location of human epidermal growth factor receptor. Cell Biol Int Rep 1984; 8: 649-58.
2. Carpenter C, Cohen S. Epidermal growth factor. Ann Rev Biochem 1979; 48: 193-216. 3. Cohen S. The stimulation of epidermal proliferation by a specific protein (EGF). Dev Biol 1965; 12: 394-407. 4. Ushiro H, Cohen S. Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A431 cell membranes. J Biol Chem 1980 255: 8363-5. 5. Green MR, Basketter DA, Couchman JR, Rees PA. Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev Biol 1983; 100: 506-12. 6. Nanney LB, Mckanna JA, Stoscheck CM, Carpenter G, King LE. Visualization of epidermal growth factor receptors in human epidermis. J Invest Dermatol 1984; 8 2 165-9. 7. Downward J, Yarden Y, Mayes E et al. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature 1984; 307: 521-7. 8. Gill GN, Lazar CS. Increased phosphotyrosine content and inhibition of proliferation in EGFtreated A431 cells. Nature 1981; 293: 305-7. 9. Kamata N, Chida K, Rikimaru K, Horikoshi M, Enomoto S, Kuroki T. Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture. Cancer Res 1986; 46: 1648-53. 10. Christensen ME, Thirkildsen MH, Hansen BL, Albeck H, Hansen GN, Bretlau P. Epidermal growth factor receptor expression on oral mucosa, dysplastic epithelia and squamous cell carcinomas. Eur Arch Otorhinolaryngol (in press). 11. Hermanek P, Sobin LH. UICC TNM Classification of malignant tumors. 4th ed. Berlin: Springer, 1987. 12. Kissane JM. Andersson’s pathology. St Louis: The CV Mosby Company, 1985; 1: 539. 13. Cordell JL, Falini B, Erber WN et al. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexa). J Histochem Cytochem 1984; 32. 219-24. 14. Waterfield MD, Mayes EL, Stroobant P et al. A monoclonal antibody to the human epidermal growth factor receptor. J Cell Biochem 1982; 20: 149-61. 15. Wrann MM, Fox CF. Identification of epidermal growth factor receptors in a hyperproducing human epidermoid carcinoma cell line. J Biol Chem. 1979; 254: 8083-6. 16. Partridge M, Gullick WJ, Langdon JD, Sherriff M. Expression of epidermal growth factor receptor on oral squamous cell carcinomas. Br J Oral Maxillofac Surg 1988; 2 6 381-9. 17. Miyaguchi M, Olofsson J, Hellquist HB. Expression of epidermal growth factor receptor in laryngeal dysplasia and carcinoma. Acta Otolaryngol (Stockh) 1990; 110: 309-13. 18. Miyaguchi M, Olofsson J, Hellquist HB. Immunohistochemical study of epidermal growth factor receptor in severe dysplasia and carcinoma in situ of the vocal cords. Acta Otolaryngol (Stockh) 1991; 11: 149-52. 19. Namey LB, Stoschek CM, Magid M,King LE. Altered [Iz5I]epidermal growth factor binding - and receptor distribution in psoriasis. J Invest Dermatol 1986; 8 6 260-5. 20. Derynck R, Roberts AB, Winkler ME, Chen EY, Goeddel DV. Human transforming growth factor-a: Precursor structure and expression in E. coli. Cell 1984; 38: 287-97. 21. Reynolds FH,Todaro GJ, Fryling C, Stephenson JR. Human transforming growth factor induce tyrosine phosphorylation of EGF-receptors. Nature 1981; 292 259-62. 22. De Larco J, Todaro G. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci 1978; 75: 4001-5. 23. Liu C, Woo A, Tsao MS. Expression of transforming growth factor-alpha in primary human colon and lung cucinomas. Br J Cancer 1990; 6 2 425-9. 24. Barton CM, Hall PA, Hughes CM, Gullick WJ, Lemoine NR. Transforming growth factor alpha and epidermal growth factor in human pancreatic cancer. J Pathol 1991; 163: 111-6. Manuscript received October 17, 1991; accepted February 7, 1992 Address for correspondence: Maria Elisabeth Christensen, Department of Oto-Laryngology and Head & Neck Surgery, Righospitalet, Blegdamsvej 9, .DK-2100 Copenhagen, Denmark
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ISSN: 0001-6489 (Print) 1651-2251 (Online) Journal homepage: http://www.tandfonline.com/loi/ioto20
Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas Maria E. Christensen, Marianne H. Therkildsen, Bente L. Hansen, Georg N. Hansen & Poul Bretlau To cite this article: Maria E. Christensen, Marianne H. Therkildsen, Bente L. Hansen, Georg N. Hansen & Poul Bretlau (1992) Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas, Acta Oto-Laryngologica, 112:4, 734-738, DOI: 10.3109/00016489209137467 To link to this article: http://dx.doi.org/10.3109/00016489209137467
Published online: 08 Jul 2009.
Submit your article to this journal
Article views: 2
View related articles
Citing articles: 3 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ioto20 Download by: [UNSW Library]
Date: 21 April 2016, At: 00:20
Acta Otolaryngol (Stockh) 1992; 1 1 2 734-738
Immunohistochemical Detection of Epidermal Growth Factor Receptor in Laryngeal Squamous Cell Carcinomas MARIA E. CHRISTENSEN,' MARIANNE H. THERKILDSEN: BENTE L. HANSEN: GEORG N. HANSEN4 and FQUL BRETLAU' From the Departments of 'Oto-Loryngology and Head I Neck Surgery, and 2Pathology, Rigshospitalet, University of Copenhagen; and Institutes of 'Medical Microbiology and 4CeIl Biology and Anatomy, Unicersity of Copenhagen, Denmark
Downloaded by [UNSW Library] at 00:20 21 April 2016
Christensen ME,Therkildsen MH, Hansen BL, Hansen GN,Bretlau P. Immunohistochemical detection of epidermal growth Factor receptor in laryngeal squamous cell carcinomas. Acta Otolaryngol (Stockh) 1992; 112 734-738.
Laryngeal squamous cell carcinomas from 15 consecutive preoperatively irradiated patients were investigated for the expression of epidermal growth factor receptor (EGF receptor). The study was performed on frozen sections by means of the 5-layer APAAP technique employing an antibody recognizing the extracellular part of the EGF receptor. In sections from 9 of the patients with laryngeal squamous cell carcinoma, normal differentiated epithelia were included. Sections from 6 of these patients, in addition, contained dysplastic epithelia. Expression of EGF receptor-like material was demonstrated in the basal cell layer of normally differentiated laryngeal epithelia and in dysplastic epithelia. Fourteen of the squamous cell carcinomas proved EGF receptor positive. Nearly all cells in the poorly differentiated carcinomas showed positive staining with the antibodies. In moderately to well differentiated carcinomas a reduction in the extent of staining was seen in certain areas. Especially for the epithelial pearls, the staining reaction was localized to the undifferentiated cells in the periphery. This finding corresponds to the staining pattern observed in the basal cell layers of normal epithelia. The present investigation confirms the expression of EGF receptor-like material in normal laryngeal epithelia, dysplastic epithelia and squamous cell carcinoma. The staining pattern was similar to that observed in oral squamous cell carcinomas, predominantly varying inversely with cellular differentiation. Key word: EGF receptor, epithelia, dyspasia, carcinoma, immunoreactiue, cellular differentiation.
INTRODUCTION Epidermal growth factor (EGF) is a polypeptide stimulating growth and, presumably, differentiation of a variety of mammalian epithelial tissues and cell types (1,2, 3). EGF binds to a specific membrane receptor and thereby activates a tyrosine-specific protein kinase which is part of the intracellular domain of the receptor (4). This in turn leads to a variety of biochemical and physiological events and ultimately to DNA replication and cell division (2). The EGF receptor is detectable on a large variety of cell types and tissues, including the proliferative compartment of epithelia (1, 5, 6). A close similarity between the amino acid sequence from the erb-B oncogene of AEV-H ( a strain of the avian erytroblastosis virus) and the cytoplasmic and transmembrane part of the EGF receptor has been described, suggesting that EGF receptor expression and malignancy might be interconnected (7). The frequent overexpression of EGF receptors in cell lines derived from tumours, in particular -but not exclusively -carcinomas might also suggest an interconnection between EGF receptor and malignancy (8, 9). By use of immunohistochemistry, we recently have demonstrated EGF receptor-like material in s p i m e n s from oral mucosa, dysplastic epithelia and oral squamous cell carcinomas. The staining pattern was shown to vary inversely with cellular differentiation ( 10). The aim of the present investigation was to demonstate the presence of EGF receptor
Acta Otolaryngol (Stockh) 112
EGF receutors in laryngeal carcinomas 735
in laryngeal squamous cell carcinomas. In particular we were interested in detecting a possible correlation with cellular differentiation.
Downloaded by [UNSW Library] at 00:20 21 April 2016
MATERIAL AND METHODS Tissue samples were obtained from 15 consecutive cases (1 woman and 14 men) of squamous cell carcinomas. In sections from 9 patients with squamous cell carcinoma, normal differentiated laryngeal epithelia were included. Sections from 6 patients with squamous cell carcinoma contained, in addition, dysplastic epithelia. All patients were staged according to the UICC TNM classification (1 1) and had received radiotherapy preoperatively (6266 Gray). Heamatoxylin-eosine staining was carried out on parallel froz n sections obtained and cut from the same tumour specimens as those on which the immunohistochemical investigation was performed. The squamous cell carcinomas were graded as poorly, moderately or well differentiated based on: 1) Evidence of differentation (intercellular bridges, keratinization and epithelial pearls) and 2) evidence of rapid, abnormal proliferation (mitoses per highpower field, atypical mitoses, nuclear and cellular pleomorphism, multinucleated and tumour giant cells) (12). The majority of the tumour specimens were moderately differentiated. Tissue specimens for EGF receptor identification were cut from the tumour immediately after removal and rapidly frozen in pre-cooled isopentane at -880°C. Five-micron sections were cut, placed on glass slides and airdried overnight at room temperature. The sections were then post-hed in pre-cooled acetone at -20°C for 10 min and stored at -80°C until immunohistochemical screening. Indirect immunohistochemical staining was performed with mouse monoclonal anti-alkaline phosphatase (APAAP) 5-layer technique modified from Cordell (13). Sections were treated at room temperature unless otherwise mentioned. Before application of primary antibodies sections were washed for 3 x 5 min with 0.5M Tris-HC1 buffer pH 7.2 supplemented with 0.5M NaCl and 0.1% Triton X-100.Nonspecific binding of proteins to sections was reduced by incubation (30min) with normal rabbit serum (Dakopatts a/s, Denmark; code No. X902) diluted 1:30 in Tris-HC1 supplemented with 3% bovine serum albumin (BSA, Sigma). The sections were then covered (for 24 h in a humid chamber at 4°C) with a 1:40 dilution of mouse monoclonal antibody (primary antibodies, clone EGFRl code RPN.513, Amersham) directed against the external segment of the EGF receptor (14). All antibodies were diluted in Tris-HC1-BSA. Before application of secondary layers the sections were equilibrated to room temperature for 30min and then washed for 3 x 5 min as described above. The secondary antibodies (application time 30 min) were rabbit anti-mouse (Dakopatts; code No, 2259) diluted 1:20. A third layer consisted of APAAP complexes (Dakopatts; code No. D651) diluted 1:25. The steps with secondary antibodies and APAAP complexes were repeated. After the final incubation with APAAP complexes, sections were washed for 2 x 5 min in tris HCI pH 7.2 and then equilibrated in Tris-HC1 pH 8.2 without triton X-100 for 5min. Enzyme reaction (25min) was visualized with Naphthol Asmix Phosphate (Sigma; code No. N-4875) and fast red TR Salt (Sigma; code No. F-1500) containing 1 mM levamisole (Sigma; code No. L-9756) to block endogenous alkaline phosphatase. Sections were counterstained with Mayer’s haematoxylin for 90 s, washed in cold water for 10 min and then mounted with Glycergel (Dakopatts; code No. C563). Positive controls were sections of normal human placenta known to have abundant EGF receptor expression, normal human skin and normal oral mucosa (1). Methodological controls consisted of the replacement of primary antibodies with a matched IgG subclass of unrelated specificity (Dakopatts; code No. M759). Positive controls
736 M. E. Christensen er d.
~olpryngol(Stoekh) 112
were included in each staining process to ensure minimal day to day methodological variation. Specificity controls consisted of absorption of the primary antibodies on a monolayer of A431 cells ( 14). Staining results were evaluated by two independent observers. The sections were graded as positive only if more than 5% of the tumour cells were stained.
Downloaded by [UNSW Library] at 00:20 21 April 2016
RESULTS Fourteen of the I5 carcinomas showed positive staining reaction with the anti-EGF receptor antibodies. The immunoreaction was predominantly localized to the cellular surface membrane. In sections including normal differentiated laryngeal epithelia the basal cell layer was positively stained. One section showed positive staining reaction in the basal cell layer as well as in the spinous cells. In sections containing dysplastic epithelia nearly all cells showed positive staining. However, in one dysplastic section, staining reaction was observed only in the basal cell layer and in the spinous cells. In the squamous cell carcinomas intra- and intersection variability in staining intensity was observed. In poorly differentiated carcinomas nearly all cells were EGF receptor positive. In moderately and well differentiated carcinomas a reduction in the extent of stained areas was seen, paralleling the situation observed in the differentiated upper layers of nonnal oral mucosa. In the epithelial pearls the EGF receptor positive cells were localized to the mdifierentiated cells in the periphery (Fig. 1). Methodological controls with antibodies of unrelated specificity revealed negative staining results. The included placental sections, normal human skin and normal oral mucosa were
Fig. 1. EGF -tor staining of laryngeal squamous cell carcinoma, well differentiated. Note, in the epithelial pear4 the staining reaction is localized to the und8erentiated ells in the periphery (asterisks). APAAP with fut red and h t o x y l i n e ( x 250).
Acia Otolaryngol (Stockh) 112
EGF receptors in laryngeal carcinomas 131
always EGF receptor positive. Specificity controls with absorption of the primary antibodies with A431 cells showed negative staining results on placenta and the laryngeal carcinomas.
Downloaded by [UNSW Library] at 00:20 21 April 2016
DISCUSSION The results of the present study elucidate the relationship between expression of EGF receptors (as determined by the monoclonal antibody) and cellular differentiation in laryngeal squamous cell carcinomas. In the material studied, expression of the EGF receptor-like material was widely distributed but with a variable staining intensity. In general, the EGF receptor was expressed in the basal cell layer of normal differentiated laryngeal epithelia and in dysplastic epithelia. Our results are in accordance with results from other studies comprising normal epithelia, including oral mucosa. In these studies EGF receptors have been proved in the basal cell layer, which is the proliferative compartment of all epithelia (1, 5, 6, 16). In dysplastic epithelia nearly all cells showed positive staining, which is in agreement with other investigations concerning oral dysplastic epithelia (10). Nevertheless, other authors could not confirm this pattern of EGF receptor expression by immunohistochemical investigation on formalin fixed specimens embedded in paraffin (17). This discrepancy could, however, well be explained by the use of frozen sections versus formalin fixed paraffin embedded sections. Another study, concerning severe laryngeal dysplasia, revealed anti-EGF receptor positive cells ( 18) possibly indicating a connection between histological grade and EGF receptor expression. In our study, grading of dysplasia was not possible because the investigation was performed on frozen sections. The presence of EGF receptor in dysplastic epithelia might reflect the requirement of the receptor in the process of dedifferentiation (10, 18). The receptor has also been related to the benign human skin disease, psoriasis vulgaris (19). In that study the EGF receptor was demonstrated at sites of an abnormally increased proliferative capacity and abnormally differentiated state. The section negative for EGF receptor-like material was taken from a moderately differentiated tumour. One possible explanation might be that the carcinoma expresses exclusively truncated receptors (7). Another explanation of the EGF receptor negative section could be that the tumour cells at this site were engaged in paracrine production of EGF/TGF-u (unpublished data). TGF-a is a polypeptide which shares close amino sequence similarity with EGF (20). The effects of TGF-a are mediated through the EGF receptor resulting in the same biological response (21). TGF-a is known to induce non-neoplastic cells to lose contact inhibition and undergo anchorage-independent growth (22). Recently TGF-a has been demonstrated in human colon, lung and pancreatic carcinomas (23, 24). The results of the present study revealed that nearly all cells in the poorly differentiated carcinomas showed positive anti-EGF staining. For the moderately to well differentiated carcinomas, a reduction in the extent of positive areas was observed. Furthermore, for the epithelial pearls EGF receptor positive cells were characteristically localized to the undifferentiated cells in the periphery. These results indicate that the receptor is always more or less expressed in undifferentiated cells, but when the malignant cells differentiate, as seen in moderately and well differentiated carcinomas, as well as in the epithelial pearls, the EGF receptor is not expressed. Our present investigation verified the presence of EGF receptor in laryngeal squamous cell carcinomas. The staining pattern was similar to that seen in oral squamous cell carcinomas with a correlation to cellular differentiation (10). Whether or not the EGF receptor is overexpressed in head and neck carcinomas remains to be determined in a quantitative study.
738 M. E. Christensen el al.
A a a Otolaryngol (Stockh) 112
ACKNOWLEDGEMENTS The authors are indebted to Mrs. Bente Christensen for skilful technical assistance.
REFERENCES
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1. Gusterson B, Cowley G, Smith JA. Ozanne B. Cellular location of human epidermal growth factor receptor. Cell Biol Int Rep 1984; 8: 649-58.
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