Skip to main content
PMC home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1995 Dec;72(6):1487–1493. doi: 10.1038/bjc.1995.534

A sensitive enzyme-linked immunosorbent assay used for quantitation of epidermal growth factor receptor protein in head and neck carcinomas: evaluation, interpretations and limitations.

M E Christensen 1, F Engbaek 1, M H Therkildsen 1, P Bretlau 1, E Nexø 1
PMCID: PMC2034104  PMID: 8519664

Abstract

The EGF receptor is a transmembrane glycoprotein exerting mitogenic effects on epithelial cells. The purpose of the present study was to develop a sensitive enzyme-linked immunosorbent assay (ELISA) for determination of the epidermal growth factor receptor (EGFR) protein to examine whether the receptor was overexpressed in head and neck squamous cell carcinomas compared with the normal counterpart, and to establish whether clinicopathological correlations were present by investigating a broad spectrum of parameters (tumour size, clinical stage, positive lymph nodes, tumour site, histological grade, keratinisation, preoperative irradiation and clinical outcome). The assay employs two commercially available monoclonal antibodies, both detecting protein epitopes. The material comprises 60 head and neck carcinomas, corresponding normal tissue and normal oral mucosa from healthy individuals. The study demonstrates significantly higher receptor levels in tumours compared with normal tissue (P < 0.002) and a range in tumours and normal tissues of 0.4-10.5 and 0.1-4.3 nmol g-1 membrane protein respectively. Quantitation of receptors in normal mucosa emphasises the importance of using the patients' corresponding normal tissue, because using the patients' mucosa resulted in 83% overexpression, while using normal mucosa from healthy individuals only demonstrated overexpression in 50% of cases. No significant clinicopathological correlations could be established, although the mean values for EGFR increased with tumour size and advanced clinical stage. Furthermore, the prognostic value concerning disease-free survival, recurrence and the time interval for recurrence were investigated but no significance could be demonstrated. In conclusion, the investigation supports the theory of overexpression of EGFR protein as a common motif for malignant epithelial tumours, but limitations in interpretations are demonstrated and discussed further.

Full text

PDF
1487

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrandon Y., Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell. 1987 Sep 25;50(7):1131–1137. doi: 10.1016/0092-8674(87)90179-6. [DOI] [PubMed] [Google Scholar]
  2. Bayer E. A., Wilchek M. The use of the avidin-biotin complex as a tool in molecular biology. Methods Biochem Anal. 1980;26:1–45. doi: 10.1002/9780470110461.ch1. [DOI] [PubMed] [Google Scholar]
  3. Chen W. S., Lazar C. S., Poenie M., Tsien R. Y., Gill G. N., Rosenfeld M. G. Requirement for intrinsic protein tyrosine kinase in the immediate and late actions of the EGF receptor. 1987 Aug 27-Sep 2Nature. 328(6133):820–823. doi: 10.1038/328820a0. [DOI] [PubMed] [Google Scholar]
  4. Christensen M. E., Therkildsen M. H., Hansen B. L., Albeck H., Hansen G. N., Bretlau P. Epidermal growth factor receptor expression on oral mucosa dysplastic epithelia and squamous cell carcinomas. Eur Arch Otorhinolaryngol. 1992;249(5):243–247. doi: 10.1007/BF00714485. [DOI] [PubMed] [Google Scholar]
  5. Christensen M. E., Therkildsen M. H., Hansen B. L., Hansen G. N., Bretlau P. Immunohistochemical detection of epidermal growth factor receptor in laryngeal squamous cell carcinomas. Acta Otolaryngol. 1992;112(4):734–738. doi: 10.3109/00016489209137467. [DOI] [PubMed] [Google Scholar]
  6. Cross M., Dexter T. M. Growth factors in development, transformation, and tumorigenesis. Cell. 1991 Jan 25;64(2):271–280. doi: 10.1016/0092-8674(91)90638-f. [DOI] [PubMed] [Google Scholar]
  7. Di Marco E., Pierce J. H., Fleming T. P., Kraus M. H., Molloy C. J., Aaronson S. A., Di Fiore P. P. Autocrine interaction between TGF alpha and the EGF-receptor: quantitative requirements for induction of the malignant phenotype. Oncogene. 1989 Jul;4(7):831–838. [PubMed] [Google Scholar]
  8. Downward J., Yarden Y., Mayes E., Scrace G., Totty N., Stockwell P., Ullrich A., Schlessinger J., Waterfield M. D. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 1984 Feb 9;307(5951):521–527. doi: 10.1038/307521a0. [DOI] [PubMed] [Google Scholar]
  9. Eisbruch A., Blick M., Lee J. S., Sacks P. G., Gutterman J. Analysis of the epidermal growth factor receptor gene in fresh human head and neck tumors. Cancer Res. 1987 Jul 1;47(13):3603–3605. [PubMed] [Google Scholar]
  10. Field J. K., Spandidos D. A., Yiagnisis M., Gosney J. R., Papadimitriou K., Stell P. M. C-erbB-2 expression in squamous cell carcinoma of the head and neck. Anticancer Res. 1992 May-Jun;12(3):613–619. [PubMed] [Google Scholar]
  11. Furuta Y., Takasu T., Asai T., Yoshimura S., Tokuchi F., Shinohara T., Nagashima K., Inuyama Y. Clinical significance of the epidermal growth factor receptor gene in squamous cell carcinomas of the nasal cavities and paranasal sinuses. Cancer. 1992 Jan 15;69(2):358–362. doi: 10.1002/1097-0142(19920115)69:2<358::aid-cncr2820690214>3.0.co;2-1. [DOI] [PubMed] [Google Scholar]
  12. Gill G. N., Kawamoto T., Cochet C., Le A., Sato J. D., Masui H., McLeod C., Mendelsohn J. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem. 1984 Jun 25;259(12):7755–7760. [PubMed] [Google Scholar]
  13. Grandis J. R., Tweardy D. J. The role of peptide growth factors in head and neck carcinoma. Otolaryngol Clin North Am. 1992 Oct;25(5):1105–1115. [PubMed] [Google Scholar]
  14. Grimaux M., Mady E., Remvikos Y., Laine-Bidron C., Magdelenat H. A simplified immuno-enzymetric assay of the epidermal growth factor receptor in breast tumors: evaluation in 282 cases. Int J Cancer. 1990 Feb 15;45(2):255–262. doi: 10.1002/ijc.2910450209. [DOI] [PubMed] [Google Scholar]
  15. Gullick W. J. Prevalence of aberrant expression of the epidermal growth factor receptor in human cancers. Br Med Bull. 1991 Jan;47(1):87–98. doi: 10.1093/oxfordjournals.bmb.a072464. [DOI] [PubMed] [Google Scholar]
  16. Gusterson B., Cowley G., Smith J. A., Ozanne B. Cellular localisation of human epidermal growth factor receptor. Cell Biol Int Rep. 1984 Aug;8(8):649–658. doi: 10.1016/0309-1651(84)90045-6. [DOI] [PubMed] [Google Scholar]
  17. Higashiyama S., Abraham J. A., Miller J., Fiddes J. C., Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991 Feb 22;251(4996):936–939. doi: 10.1126/science.1840698. [DOI] [PubMed] [Google Scholar]
  18. Hock R. A., Nexø E., Hollenberg M. D. Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone. J Biol Chem. 1980 Nov 25;255(22):10737–10743. [PubMed] [Google Scholar]
  19. Huang S., Trujillo J. M., Chakrabarty S. Proliferation of human colon cancer cells: role of epidermal growth factor and transforming growth factor alpha. Int J Cancer. 1992 Dec 2;52(6):978–986. doi: 10.1002/ijc.2910520625. [DOI] [PubMed] [Google Scholar]
  20. Hurtt M. R., Moossy J., Donovan-Peluso M., Locker J. Amplification of epidermal growth factor receptor gene in gliomas: histopathology and prognosis. J Neuropathol Exp Neurol. 1992 Jan;51(1):84–90. doi: 10.1097/00005072-199201000-00010. [DOI] [PubMed] [Google Scholar]
  21. Irish J. C., Bernstein A. Oncogenes in head and neck cancer. Laryngoscope. 1993 Jan;103(1 Pt 1):42–52. doi: 10.1288/00005537-199301000-00009. [DOI] [PubMed] [Google Scholar]
  22. Ishitoya J., Toriyama M., Oguchi N., Kitamura K., Ohshima M., Asano K., Yamamoto T. Gene amplification and overexpression of EGF receptor in squamous cell carcinomas of the head and neck. Br J Cancer. 1989 Apr;59(4):559–562. doi: 10.1038/bjc.1989.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jørgensen P. E., Poulsen S. S., Nexø E. Distribution of i.v. administered epidermal growth factor in the rat. Regul Pept. 1988 Nov;23(2):161–169. doi: 10.1016/0167-0115(88)90024-9. [DOI] [PubMed] [Google Scholar]
  24. Kawamoto T., Takahashi K., Nishi M., Kimura T., Matsumura T., Taniguchi S. Quantitative assay of epidermal growth factor receptor in human squamous cell carcinomas of the oral region by an avidin-biotin method. Jpn J Cancer Res. 1991 Apr;82(4):403–410. doi: 10.1111/j.1349-7006.1991.tb01863.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kearsley J. H., Leonard J. H., Walsh M. D., Wright G. R. A comparison of epidermal growth factor receptor (EGFR) and c-erbB-2 oncogene expression in head and neck squamous cell carcinomas. Pathology. 1991 Jul;23(3):189–194. doi: 10.3109/00313029109063564. [DOI] [PubMed] [Google Scholar]
  26. Kraus M. H., Issing W., Miki T., Popescu N. C., Aaronson S. A. Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9193–9197. doi: 10.1073/pnas.86.23.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Krupp M. N., Connolly D. T., Lane M. D. Synthesis, turnover, and down-regulation of epidermal growth factor receptors in human A431 epidermoid carcinoma cells and skin fibroblasts. J Biol Chem. 1982 Oct 10;257(19):11489–11496. [PubMed] [Google Scholar]
  28. Leonard J. H., Kearsley J. H., Chenevix-Trench G., Hayward N. K. Analysis of gene amplification in head-and-neck squamous-cell carcinoma. Int J Cancer. 1991 Jun 19;48(4):511–515. doi: 10.1002/ijc.2910480406. [DOI] [PubMed] [Google Scholar]
  29. Massagué J., Pandiella A. Membrane-anchored growth factors. Annu Rev Biochem. 1993;62:515–541. doi: 10.1146/annurev.bi.62.070193.002503. [DOI] [PubMed] [Google Scholar]
  30. Merritt W. D., Weissler M. C., Turk B. F., Gilmer T. M. Oncogene amplification in squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg. 1990 Dec;116(12):1394–1398. doi: 10.1001/archotol.1990.01870120040005. [DOI] [PubMed] [Google Scholar]
  31. Nanney L. B., McKanna J. A., Stoscheck C. M., Carpenter G., King L. E. Visualization of epidermal growth factor receptors in human epidermis. J Invest Dermatol. 1984 Feb;82(2):165–169. doi: 10.1111/1523-1747.ep12259731. [DOI] [PubMed] [Google Scholar]
  32. Neal D. E., Sharples L., Smith K., Fennelly J., Hall R. R., Harris A. L. The epidermal growth factor receptor and the prognosis of bladder cancer. Cancer. 1990 Apr 1;65(7):1619–1625. doi: 10.1002/1097-0142(19900401)65:7<1619::aid-cncr2820650728>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  33. Nexø E., Hansen H. F. Binding of epidermal growth factor from man, rat and mouse to the human epidermal growth factor receptor. Biochim Biophys Acta. 1985 Nov 22;843(1-2):101–106. doi: 10.1016/0304-4165(85)90055-8. [DOI] [PubMed] [Google Scholar]
  34. Nexł E., Hock R. A., Hollenberg M. D. Lectin-agarose immobilization, a new method for detecting soluble membrane receptors. Application to studies with epidermal growth factor-urogastrone and transcobalamin-II. J Biol Chem. 1979 Sep 25;254(18):8740–8743. [PubMed] [Google Scholar]
  35. Nicholson S., Sainsbury J. R., Needham G. K., Chambers P., Farndon J. R., Harris A. L. Quantitative assays of epidermal growth factor receptor in human breast cancer: cut-off points of clinical relevance. Int J Cancer. 1988 Jul 15;42(1):36–41. doi: 10.1002/ijc.2910420108. [DOI] [PubMed] [Google Scholar]
  36. Ozawa S., Ueda M., Ando N., Shimizu N., Abe O. Prognostic significance of epidermal growth factor receptor in esophageal squamous cell carcinomas. Cancer. 1989 Jun 1;63(11):2169–2173. doi: 10.1002/1097-0142(19890601)63:11<2169::aid-cncr2820631117>3.0.co;2-w. [DOI] [PubMed] [Google Scholar]
  37. Plowman G. D., Culouscou J. M., Whitney G. S., Green J. M., Carlton G. W., Foy L., Neubauer M. G., Shoyab M. Ligand-specific activation of HER4/p180erbB4, a fourth member of the epidermal growth factor receptor family. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):1746–1750. doi: 10.1073/pnas.90.5.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Riedel H., Massoglia S., Schlessinger J., Ullrich A. Ligand activation of overexpressed epidermal growth factor receptors transforms NIH 3T3 mouse fibroblasts. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1477–1481. doi: 10.1073/pnas.85.5.1477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. SLAUGHTER D. P., SOUTHWICK H. W., SMEJKAL W. Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin. Cancer. 1953 Sep;6(5):963–968. doi: 10.1002/1097-0142(195309)6:5<963::aid-cncr2820060515>3.0.co;2-q. [DOI] [PubMed] [Google Scholar]
  40. Santini J., Formento J. L., Francoual M., Milano G., Schneider M., Dassonville O., Demard F. Characterization, quantification, and potential clinical value of the epidermal growth factor receptor in head and neck squamous cell carcinomas. Head Neck. 1991 Mar-Apr;13(2):132–139. doi: 10.1002/hed.2880130209. [DOI] [PubMed] [Google Scholar]
  41. Sasada R., Ono Y., Taniyama Y., Shing Y., Folkman J., Igarashi K. Cloning and expression of cDNA encoding human betacellulin, a new member of the EGF family. Biochem Biophys Res Commun. 1993 Feb 15;190(3):1173–1179. doi: 10.1006/bbrc.1993.1173. [DOI] [PubMed] [Google Scholar]
  42. Sato J. D., Kawamoto T., Le A. D., Mendelsohn J., Polikoff J., Sato G. H. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol Biol Med. 1983 Dec;1(5):511–529. [PubMed] [Google Scholar]
  43. Scambia G., Panici P. B., Battaglia F., Ferrandina G., Almadori G., Paludetti G., Maurizi M., Mancuso S. Receptors for epidermal growth factor and steroid hormones in primary laryngeal tumors. Cancer. 1991 Mar 1;67(5):1347–1351. doi: 10.1002/1097-0142(19910301)67:5<1347::aid-cncr2820670513>3.0.co;2-h. [DOI] [PubMed] [Google Scholar]
  44. Schechter A. L., Hung M. C., Vaidyanathan L., Weinberg R. A., Yang-Feng T. L., Francke U., Ullrich A., Coussens L. The neu gene: an erbB-homologous gene distinct from and unlinked to the gene encoding the EGF receptor. Science. 1985 Sep 6;229(4717):976–978. doi: 10.1126/science.2992090. [DOI] [PubMed] [Google Scholar]
  45. Shoyab M., Plowman G. D., McDonald V. L., Bradley J. G., Todaro G. J. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science. 1989 Feb 24;243(4894 Pt 1):1074–1076. doi: 10.1126/science.2466334. [DOI] [PubMed] [Google Scholar]
  46. Spyratos F., Martin P. M., Hacène K., Andrieu C., Romain S., Floiras J. L., Magdelénat H. Prognostic value of a solubilized fraction of EGF receptors in primary breast cancer using an immunoenzymatic assay--a retrospective study. Breast Cancer Res Treat. 1994 Jan;29(1):85–95. doi: 10.1007/BF00666184. [DOI] [PubMed] [Google Scholar]
  47. Stampfer M. R., Pan C. H., Hosoda J., Bartholomew J., Mendelsohn J., Yaswen P. Blockage of EGF receptor signal transduction causes reversible arrest of normal and immortal human mammary epithelial cells with synchronous reentry into the cell cycle. Exp Cell Res. 1993 Sep;208(1):175–188. doi: 10.1006/excr.1993.1236. [DOI] [PubMed] [Google Scholar]
  48. Stroobant P., Rice A. P., Gullick W. J., Cheng D. J., Kerr I. M., Waterfield M. D. Purification and characterization of vaccinia virus growth factor. Cell. 1985 Aug;42(1):383–393. doi: 10.1016/s0092-8674(85)80133-1. [DOI] [PubMed] [Google Scholar]
  49. Waterfield M. D., Mayes E. L., Stroobant P., Bennet P. L., Young S., Goodfellow P. N., Banting G. S., Ozanne B. A monoclonal antibody to the human epidermal growth factor receptor. J Cell Biochem. 1982;20(2):149–161. doi: 10.1002/jcb.240200207. [DOI] [PubMed] [Google Scholar]
  50. Wrann M. M., Fox C. F. Identification of epidermal growth factor receptors in a hyperproducing human epidermoid carcinoma cell line. J Biol Chem. 1979 Sep 10;254(17):8083–8086. [PubMed] [Google Scholar]
  51. Yasui W., Sumiyoshi H., Hata J., Kameda T., Ochiai A., Ito H., Tahara E. Expression of epidermal growth factor receptor in human gastric and colonic carcinomas. Cancer Res. 1988 Jan 1;48(1):137–141. [PubMed] [Google Scholar]
  52. de Larco J. E., Todaro G. J. Growth factors from murine sarcoma virus-transformed cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):4001–4005. doi: 10.1073/pnas.75.8.4001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van Bergen en Henegouwen P. M., den Hartigh J. C., Romeyn P., Verkleij A. J., Boonstra J. The epidermal growth factor receptor is associated with actin filaments. Exp Cell Res. 1992 Mar;199(1):90–97. doi: 10.1016/0014-4827(92)90465-k. [DOI] [PubMed] [Google Scholar]
  54. van Dam P. A., Lowe D. G., Watson J. V., James M., Chard T., Hudson C. N., Shepherd J. H. Multiparameter flow-cytometric quantitation of epidermal growth factor receptor and c-erbB-2 oncoprotein in normal and neoplastic tissues of the female genital tract. Gynecol Oncol. 1991 Sep;42(3):256–264. doi: 10.1016/0090-8258(91)90355-9. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES