In Vitro Diagnostic

HER-2/neu ELISA

Intended Use
The HER-2/neu ELISA is an in vitro, diagnostic device intended for use in the quantitative determination of serum HER-2/neu in women with metastatic breast cancer who have an initial value of 15 ng/ml or greater. HER-2/neu values obtained may be used in the follow-up and monitoring of patients with metastatic breast cancer. HER-2/neu values should be used in conjunction with information available from clinical and other diagnostic procedures in the management of breast cancer. The clinical utility of the serum measurement of HER-2/neu as a prognostic indicator for early recurrence and in the management of patients on immunotherapy regimens has not been fully established.

Background
The HER-2/neu oncogene, also referred to as c-erbB-2, encodes a protein with a molecular weight of 185,000 daltons (p185) and belongs to a family of epithelial growth factor receptors structurally related to the Human Epidermal Growth Factor Receptor [1]. The full length p185 HER-2/neu protein is composed of a cytoplasmic domain with tyrosine kinase activity, a transmembrane domain and an extracellular domain (ECD) that is shed from the surface of breast cancer cells [2,3]. Numerous studies have shown that the shed ECD of HER-2/neu is a glycoprotein with a molecular weight between 97 and 115kDa and designated p105 [3,4]. The ECD can be accurately quantified in serum with an ELISA [4] that uses monoclonal antibodies [5] that are directed to the external epitopes of the HER-2/neu protein. Many publications show that the ECD is shed into the blood of normal individuals and can be elevated in women with metastatic breast cancer [4,6-26]. Many of these serum HER-2/neu studies have confirmed the substantial data from tissue studies that HER-2/neu is a marker of poor prognosis, shorter overall survival and biological aggressiveness.

Clinical Utility
Recent scientific studies suggest that quantitation of the ECD may have several important clinical applications such as monitoring breast cancer patients with metastatic disease [6-10,12,15,17-20,22-26]. These reports have shown that 30-50% of women with positive HER-2/neu tumors at primary diagnosis develop elevated levels of serum HER-2/neu with progression to metastatic breast cancer [4,6-26]. These studies have also illustrated that monitoring serum ECD levels post-surgery correlated with clinical course of disease and that serum HER-2/neu levels were observed to increase with disease progression or to decrease with response to therapy [12,15,19,20,25,26]. Several reports also show that elevated levels of serum HER-2/neu can occur in women with metastatic breast cancer that had primary breast tumors that were negative for HER-2/neu expression by immunohistochemistry [6,11,18,21,27]. According to many immunohistochemistry and serum studies, the HER-2/neu protein is overexpressed in many tumors of epithelial origin including lung [28], prostate [29], pancreatic [30], colon [31], stomach [32], ovarian [33], and hepatocellular cancer [34].

Principle of the Assay
The Serum HER-2/neu ELISA test is a sandwich enzyme immunoassay that utilizes a mouse monoclonal antibody for capture and a different biotinylated mouse monoclonal antibody for the detection of human HER-2/neu protein. Both capture and detector reagents specifically bind to the extracellular domain of HER-2/neu protein. The Capture Antibody has been immobilized on the interior surface of microtiter plate wells. To perform the test, an appropriate volume of specimen is incubated in the coated well to allow binding of the antigen by the Capture Antibody. The immobilized antigen is then reacted with the detector antiserum. The amount of Detector Antibody bound to antigen is measured by binding it with a streptavidin/horseradish peroxidase Conjugate, which then catalyzes the conversion of the chromogenic Substrate o-phenylenediamine (OPD)into a colored product. The colored reaction product is quantitated by spectrophotometry and is related to the amount of HER-2/neu protein in the sample. For instructions, see the Detailed Protocol and Evaluation of Results sections of the assay protocol.

Patents
The following patents have been granted related to quantitation and detection of the ECD p105 domain: (in the US, patent #5,401,638, Canada #2,026,250-8, and Europe #0494135) as well as patents related to the quantitation and detection of the full length p185 molecule (in the US, patent #5,604,107 and Europe patent #0412115).

Selected References

1. Coussens, L., et al., Science, 1985. 230: p. 1132-1139.
2. Brandt-Rauf, P., et al., Critical Reviews in Oncogenesis, 1994. 5(2&3): p. 313-329.
3. Zabrecky, J.R. et al., Journal of Biological Chemistry, 1991. 266(3): p. 1716-1720.
4. Carney, W.P., et al., Journal of Tumor Marker Oncology, 1991. 6(2): p. 53-72.
5. McKenzie, SJ. et al., 1989. Oncogene 4: 543-548
6. Andersen, T.I., et al., Acta Oncologica, 1995. 34(4): p. 499-504.
7. Watanabe, N., et al., Acta Oncologica, 1994. 33(8): p. 901-904.
8. Molina, R., et al., Anticancer Research, 1996. 16(4B): p. 2295-2300.
9. Molina, R., et al., British Journal of Cancer, 1996. 74: p. 1126-1131.
10. Molina, R., et al., Breast Cancer Research & Treatment, 1998. 51: p. 109-119.
11. Fehm, T., et al., Oncology, 1998. 55: p. 33-38.
12. Isola, J.J., et al., Cancer, 1994. 73(3): p. 652-658.
13. Leitzel, K., et al., Journal of Clinical Oncology, 1995. 13(5): p. 1129-1135.
14. Hayes, D.F., et al., Abstract Presented at the 12th Annual San Antonio Breast Cancer Symposium, 1989. (December 8-9, 1989).
15. Kath, R., et al., Annals of Oncology, 1993. 4: p. 585-590.
16. Yamauchi, H., et al., Journal of Clinical Oncology, 1997. 15(7): p. 2518-2525.
17. Mercer, D.W., et al., Clinical Chemistry, Abstract #846, 1995. 41(S6, part 2): p. S226.
18. Kandl, H., et al., British Journal of Cancer, 1994. 70(4): p. 739-742.
19. Narita, T., et al., Breast Cancer Research and Treatment, 1992. 24: p. 97-102.
20. Dia, J., et al., Amer Assoc Clin Chem, 1998. Abstract #208.
21. Krainer, M., et al., Oncology, 1997. 54: p. 475-481.
22. Kynast, B. et al., J. Cancer Res Clin Oncol 119: 249-252, 1993.
23. Takaaski, H. et al. Oncology reports, 1997 Vol 4(2): 349-352.
24. Klein, B. et al., Oncology Report, 1995 Vol 2(5): 759-761.
25. Imoto,S. et al., Jpn J. Clin Oncol, 1999, 29(7): 336-339.
26. Willsher, PC. et al., Breast Cancer Research & Treatment, 1996 40: 251-255.
27. Titus, K., CAP Today, Nov 1999
28. Brandt-Rauf, P. et al., Int. Journal of Cancer, 1994. 56: p. 383-386.
29. Myers, R.B., et al., International Journal Cancer, 1996. 69 (5): p. 398-402.
30. Okada, N., et al., Oncology, 1995. 52: p. 392-396.
31. Wu, J.T., et al., Journal of Clinical Laboratory Analysis, 1993. 7: p. 31-40.
32. Chariyalertsak, S., et al., Tumor Biol, 1994. 15: p. 294-303.
33. Meden, H., et al., Anti Cancer Research, 1997. 17: p. 757-760.
34. Luo, J.-C., et al., Medical Science Research, 1993. 21: p. 305-307.