A substantial body of evidence has recently emerged indicating that aberrant signaling and/or unregulated activation of the epidermal growth factor receptor (EGFR) may be a possible key factor in the development and progression of many cancers. ^1,2 Uncontrolled cellular proliferation mediated via dysfunctional EGFR pathways can be found in a wide variety of solid cancers of epithelial origin and data have linked tumor EGFR expression, overexpression and/or dysregulation to advanced disease, metastatic phenotype, resistance to chemotherapy and an overall poorer prognosis. ³ Furthermore, data has also implicated EGFR in increased tumor invasion, inhibition of cellular apoptosis, increased cellular adhesion and angiogenesis. ⁴ However, controversy still exists regarding the degree of EGFR expression and overall prognosis of a patient, and future studies are warranted to evaluate this issue.

Solid Tumors Expressing EGFR ⁵

Prognostic Significance of EGFR statement in Certain Cancers ⁵

The EGFR is a member of a larger family of closely related transmembrane receptors ( erbB receptors), all of which appear to be involved in the regulation of cellular growth, replication and/or differentiation. ErbB includes four receptors, including EGFR ( erbB-1), Her 2 ( erbB-2), Her 3 ( erbB-3) and Her 4 ( erbB-4). All four receptors share similar structure including an extracellular region which consists of glycosylated domains and binds to extracellular ligand, a short helical transmembrane domain secured by a single hydrophobic sequence and an intracellular tyrosine kinase domain that is responsible for initiating and regulating intracellular signaling. (One known exception to this is the erbB-3 receptor, which is activated through other erbB tyrosine kinases, as erbB-3 lacks its own kinase activity). ErbB receptors exist on normal epithelial, mesenchymal and neuronal tissue; however, cellular replication through erbB pathways is strictly regulated and controlled in healthy tissue. ^6,7,8,9

Excessive activation of EGFR occurring through receptor mutation, increased concentration of ligand (extracellular or intracellular), increased receptor number and/or decreased receptor turnover can drive the initiation of uncontrolled cellular growth. ¹⁰ Mutations in any domain of EGFR or components of its pathways may lead to overactive signaling, which may ultimately result in the development and/or metastasis of cancer.

EGFR has demonstrated high affinity to the ligands including, but not necessarily limited to: epidermal growth factor (EGF), transforming growth factor-alpha, amphiregulin, heparin-binding EGF, betacellulin and epiregulin. ¹¹ Once the extracellular domain binds to ligand, the bound receptor forms either a homodimer or heterodimer with a neighboring erbB receptor. Dimerization initiates intramolecular phosphorylation of the EGFR tyrosine kinase and substrates, which generates a downstream cascade of catalytic events that ultimately carries growth signal into the nucleus of the cell. ¹² Following activation of EGFR, the receptor is internalized through endocytosis and either degraded through lysosomal processes or recycled to the cell surface. ¹³

The intracellular biochemical cascade is complex, with many existent pathways through which signal transduction may occur. Research efforts are focused on understanding the steps of various pathways through which cellular replication occurs via EGFR stimulation. ¹⁴ In turn, biological therapies for the treatment of cancer will inevitably continue to focus on disrupting the progression of growth signals at various points along these studied pathways.

On the clinical forefront, several agents that target signaling pathways directly related to EGFR are now being studied in clinical trials. Because these agents are selective and have different mechanisms of action than standard cancer therapies, researchers are hopeful that the addition of EGFR targeted therapies may augment responses to chemotherapy and/or radiation with minimal additional toxicity. EGFR targeted therapies may also provide a treatment option for patients who cannot tolerate toxicities of standard therapies, offering a prolonged survival and/or quality of life benefit. Finally, recent research indicates that EGFR is one part of a complex, pleotropic network in which multiple agents that block signal transduction and cellular proliferation from many angles along EGFR and ErbB pathways may provide optimal results. ¹³

Although the number of agents that selectively inhibit EGFR are growing, Iressa®, Tarceva™ and cetuximab (Erbitux®) are the three agents that are in the final phases of trials. Furthermore, these three agents are being studied in a variety of solid tumors either as single-agent therapy or in combination with chemotherapy and/or radiation. Two broad classes of these agents include the EGFR tyrosine kinase inhibitors and EGFR monoclonal antibodies.

EGFR Tyrosine Kinase Inhibitors:

IRESSA® (ZD 1839): Iressa® is an oral derivative of the quinazoline family and inhibits EGFR tyrosine kinase function. Iressa® reversibly binds to the ATP binding site in a hydrophobic region of the kinase. Data have indicated that overexpression of EGFR is not necessary for tumor response to Iressa®. Furthermore, Iressa® appears to augment activity of some chemotherapy agents and radiation. ⁵

Two recent clinical trials evaluating Iressa® in refractory NSCLC were presented at the 38th Annual Meeting of the American Society of Clinical Oncology. The first trial, referred to as IDEAL 1, included 208 patients with recurrent NSCLC who were treated with either 250 mg or 500 mg of Iressa® daily. Partial responses, progression-free survival and overall survival were not significantly different between the two doses. PR was approximately 19%, the average time to progression was approximately 2.7 months and the average duration of survival was approximately 8 months. There were no differences in these results between patients who had received one or two previous chemotherapy regimens. An improvement in cancer symptoms including dyspnea, anorexia and cough was reported in approximately 40% of patients. The majority of side effects in this trial were mild to moderate, with the most common being rash, diarrhea and pruritis. Fewer patients receiving 250 mg of Iressa® experienced severe side effects than patients receiving 500 mg. ^15,16

The second clinical trial, referred to as IDEAL 2, included 216 patients who recurred following at least two prior chemotherapy regimens, with 25% of patients having received four or more previous regimens. In this trial, 102 patients received 250 mg of Iressa® daily and 114 received 500 mg daily. In the group of patients receiving 250 mg, nearly 12% had a partial response and 43% reported a reduction in symptoms including dyspnea, anorexia and cough. In the group of patients receiving 500 mg of Iressa®, nearly 9% of patients had a partial response and 35% reported reductions in the symptoms. Of patients who demonstrated a response to Iressa®, the average duration of survival was 8.1 months, versus only 3.7 months in patients who did not respond. The majority of side effects were mild to moderate and consisted of diarrhea and skin rash. ^17,18

TARCEVA™ (OSI-774, erlotinib): Tarceva™ is also an oral derivative of the quinazoline family, providing a very similar mechanism of action to Iressa® by competitively and reversibly binding to ATP-binding sites of the EGFR-TK. ⁵

A recent clinical trial evaluating Tarceva™ in recurrent NSCLC was presented at the 38th annual ASCO meeting. This trial involved 57 patients who were EGFR positive and had progressed following platinum therapy. Patients were treated with Tarceva™ 150mg/day. One patient had a complete response, six patients had partial responses, 20 patients had disease stabilization and 30 patients progressed while on treatment. The median survival was 36 weeks and one-year survival was 40%. The number of previous chemotherapy regimens did not affect response rates. In addition, different levels of EGFR expression did not affect response rates to Tarceva™. Approximately 75% of patients had reversible rash and/or mild diarrhea. Only 1.8% of patients had grade 3 or 4 rash which disappeared even through Tarceva™ continuation. ¹³

Currently, Tarceva™ is in two phase III clinical trials in which previously untreated NSCLC patients are treated with Tarceva™ or placebo in combination with paclitaxel/Paraplatin® in one trial and Gemzar®/Platinol® in the second trial.

EGFR Monoclonal Antibodies:

Cetuximab (Erbitux^®  )(IMC-225): Cetuximab is a monoclonal antibody that binds to the extracellular domain of EGFR, competitively inhibiting the binding of extracellular ligand. In addition, when cetuximab is bound, it appears to stimulate internalization of EGFR. ⁵

Results from a multi-institutional clinical trial were presented at the 38th Annual ASCO Meeting that evaluated cetuximab as a single agent in 57 patients with colorectal cancer refractory to both Camptosar® and 5-FU. Patients were EGFR positive by immunohistochemistry. Six patients (11%) achieved a partial response and 13 patients achieved a minor response or disease stabilization. At a median follow-up of four months, the median survival had not yet been reached. The most common adverse events were an acne-like rash, experienced by 86% of patients (16% grade 3) and asthenia, experienced by 53% of patients (7% grade 3). Two patients (3.5%) had to discontinue treatment due to grade 3 allergic-type reactions. ¹⁹

Testing for EGFR

Since aberrant signaling may be caused by mutations in any domain of the receptor, differing methods of laboratory testing may produce variability in results of EGFR statement.

EGFR Testing Methods ⁵

Immunohistochemical staining (IHC): Primary antibodies against antigens of interest are incubated with tumor specimen. Secondary antibodies targeted against primary antibodies are then added, which react with added reagents. The reaction produces color and/or fluorescence that can be detected. ²⁰

Fluorescence in-situ hybridization (FISH): Specific single-stranded DNA sequence complementary to DNA sequence of interest is tagged with fluorescence (probe). If the DNA anneals to the DNA probe and fluorescence can be visualized, the sequence of interest exists in the sample. ²¹

Southern Blot: DNA from the specimen is cut by restriction enzymes and separated by size elecrophoretically on agarose gel. The separated DNA is transferred to a filter and mixed with a radioactive probe(s) complementary to DNA sequence of interest. If the DNA sequence of interest exists in the sample, it hybridizes to the complimentary DNA probe and remains on the filter while unbound probe is washed away. The position of the probe is determined by exposing the filter to x-ray film, where a band on the film represents the location of a probe, which is representative of the DNA sequence of interest. ²²

Polymerase Chain Reaction (PCR): Specific DNA sequence of interest is amplified so that only a minute amount of DNA from the specimen is needed for detection. First, DNA from specimen is heated to create single-stranded DNA (ssDNA). Oligonucleotide primers complimentary to the desired starting points of amplification are added, along with DNA polymerase, to initiate synthesis at the point where the primer and DNA anneal. DNA synthesis occurs to create double-stranded DNA (dsDNA), which is again heated to create ssDNA and the process continues. ²³

Real-Time Polymerase Chain Reaction: This technique can utilize cDNA, which is copied through reverse transcription from mRNA of specimen. It not only tests for the presence of a specific sequence as in PCR, but can determine the amount of amplicons produced at each successive amplification cycle. ²⁴

Serum/Tumor Enzyme-Linked Immunosorbant Assay (ELISA): Antibodies against the targeted antigen induce a color change with added reagent that can be visualized if the specific antigen is present in specimen. ²⁵

References:

1. Woodburn Jr. The epidermal growth factor receptor and its inhibition in cancer. Pharmacology and Therapeutics. 1999;82:241-250.

2. Raymond E, Faivre S, Armand J. Epidermal growth factor receptor tyrosine kinase as target for anticancer therapy. Drugs. 2000;60 (suppl 1):15-23.

3. Salomon, D, Brandt R, Ciardiello F, et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Critical Reviews in Oncology/Haematology. 1995;19:183-232.

4. Sedlacek HH. Kinase inhibitors in cancer therapy: a look ahead. Drugs. 2000;59:435-576.

5. AstraZeneca Oncology. Proceedings from the Iressa® non-small cell lung cancer advisory board meeting. Los Angeles, CA. March 19-20, 2002.

6. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell. 2000;103:13-15.

7. Simon MA. Receptor tyrosine kinases: specific outcomes from general signals. Cell. 200;103:13-15.

8. Walk RA, The erbB/HER type 1 tyrosine kinase receptor family. Journal of Pathology. 1998;185:234-235.

9. Pinkas-Kramarski R, Alroy I, Yarden Y. ErbB receptors and EGF-like ligands: cell lineage determination and oncogenesis through combinatorial signaling. Journal of Mammary Gland Biology in Neoplasia. 1997;2:97-107.

10. Wells A. Molecules in focus EGF receptor. International Journal of Biochemistry and Cell Biology. 1999;31:637-643.

11. Fedi P, Kimmelman A, Aarosonson, S. Growth factor signal transduction in cancer. In: Bast RC Jr, Kufe DW, Pollock R, et al, eds. Cancer Medicine. 5th ed. Hamilton, Ontario: B.C. Decker Inc;2000:33-55.

12. Watson J, Gilman M, Witkowski J, et al. Moving signals across membranes. In: Zayatz E, O’Neal B, Simpson J, et al, eds. Recombinant DNA. 2nd ed. Scientific American, Inc. Distributed by W.H. Freeman and Company, New York;1991:313-333.

13. Bonomi, P. Clinical data with OSI-774 (erlotinib), a small molecule inhibitor of EGF receptor tyrosine kinase pathway. Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. Oral presentation. Friday, May 17, 2002.

14. Rowinsky, E. Targeting signal transduction, the erbB receptor family as a target for therapeutic development. Available at http://www.meniscus.com/horizons/2-2pdf. Accessed June 17, 2002.

15. Fukuoka M, Yano S, Giaccone T, et al. Final results from a phase II trial of ZD 1839 (‘Iressa’) for patients with advanced non-small cell lung cancer (IDEAL 1). Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. 2002;21:Abstract 1188.

16. Douillard J, Giaccone G, Horai T, et al. Improvement in disease-related symptoms and quality of life in patients with advanced non-small cell lung cancer (NSCLC) treated with ZD1839 (‘Iressa’) (IDEAL 1). Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. 2002;21: Abstract 1195.

17. Kris M, Natale B, Herbst R, et al. A phase II trial of ZD 1830 (‘Iressa’) in advanced non-small cell lung cancer (NSCLC) patients who had failed platinum- and docetaxel-based regimens (IDEAL 2). Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. 2002;21: Abstract 1166.

18. Natale R, Skarin A, Maddox A-M, et al. Improvements in symptoms and quality of life for advanced non-small cell lung cancer patients receiving ZD 1839 (‘Iressa’) in IDEAL 2. Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. 2002;21: Abstract 1167.

19. Saltz L, Meropol N, Loehrer P, et al. Single agent IMC-C225 (cetuximab) has activity in CPT-11-refractory colorectal cancer (CRC) that expresses the epidermal growth factor receptor (EGFR). Proceedings from the 38th Annual Meeting of the American Society of Clinical Oncology. 2002;21: Abstract 504.

20. Innogex. Immunohistochemistry kits (IHC) for in-situ protein expression analysis. Available at: http://www.innogenex.com/p_ihck.html. Accessed June 17, 2002.

21. Watson J, Gilman M, Witkowski J, et al. The genes behind the functioning of the brain. In: Zayatz E, O’Neal B, Simpson J, et al, eds. Recombinant DNA. 2nd ed. Scientific American, Inc. Distributed by W.H. Freeman and Company, New York;1991: 409-429.

22. Watson J, Gilman M, Witkowski J, et al. The isolation of cloned genes. In: Zayatz E, O’Neal B, Simpson J, et al, eds. Recombinant DNA. 2nd ed. Scientific American, Inc. Distributed by W.H. Freeman and Company, New York;1991:99-131.

23. Watson J, Gilman M, Witkowski J, et al. The polymerase chain reaction. In: Zayatz E, O’Neal B, Simpson J, et al, eds. Recombinant DNA. 2nd ed. Scientific American, Inc. Distributed by W.H. Freeman and Company, New York;1991:79-95.

24. Leutenegger, C. The real-time TaqMan PCR and applications in veterinary medicine. Available at: http://www.vetmed.ucdavis.edu/vme/taqmanservice/pdf/TaqManReviewVST2001.pdf. Accessed June 17, 2002.

25. The University of Arizona Biotech Project. Using the ELISA assay for disease detection. Available at: http://www.biotech.biology.arizona.edu/labs/ELISA_assay_students.html. Accessed June 17, 2002.