Section 4 -

Targeted Therapies for Lung Cancer - EGFR Inhibitors Wilbur A. Franklin University of Colorado Denver Health Sciences Center Fitzsimons Campus Aurora, Colorado

Introduction EGF was one of the first epithelial cell signaling molecules to be isolated and characterized [1]. In the 45 years since this original observation, what has become the EGFR (or ErbB) pathway has been explored and characterized in minute detail and has been found particularly active in lung. The recent introduction of agents that block EGFR signaling has spurred interest in the pathway. This review will provide a summary of the current understanding of the role of epidermal growth factor receptor (EGFR) and its related signaling pathway in lung cancer biology and treatment. EGFR Structure and Function The EGFR family consists of four homologous tyrosine kinase proteins that are present at the surface of lung cancer cells. These receptors include EGFR, HER2/neu, ErbB-3 and ErbB-4. Each of the proteins with the exception of ErbB3 is composed of three functional domains--extracellular, transmembrane and intracytoplasmic. ErbB3 lacks a functional intracellular domain. Binding to EGF, TGFa or to any of several other ligands activates the receptors. On ligand binding, receptors form either homodimers or heterodimers at the cell surface. The formation of the dimers permits phosphorylation of serine residues of the intracytoplasmic domains of the now adjacent dimer partners (trans autophosphorylation). Phosphorylation can activate several downstream signaling cascades. These include the formation of a complex that includes src2, GRB2, SH3, and SOS complex with activated receptor. The formation of this complex results in the sequential phosphorylation (activation) of ras, raf, MAP kinase, Akt and finally in gene activation. Molecular lesions that could lead to ErbB pathway activation in lung cancer are numerous. EGFR Expression in Lung Carcinogenesis In normal lung tissue EGFR is strongly expressed by basilar cells of the respiratory mucosa and in most cases by pneumocytes lining the peripheral airways. In the earliest histologically recognizable proliferative lesions in the bronchial epithelium, basal cell hyperplasia, increased thickness of the basilar cell layer is associated with corresponding increase in the expression of EGFR. In progressively more advanced lesions ranging from squamous metaplasia through carcinoma in situ there is consistent and heavy overexpression of EGFR. The consistent expression of EGFR in premalignant epithelium suggests that EGFR blocking agents may be useful for chemoprevention in high-risk patients with recognizable lesions in the lower airways but to date no clinical trials have been successfully mounted to test this hypothesis and most attention has been focused on EGFR in invasive lung cancer. EGFR Expression in Lung Cancer For several years it has been known that EGFR is overexpressed in lung carcinoma. The first demonstration of EGFR overexpression in lung cancer in situ was a 1984 study that used a radiolabeled antibody and autoradiography to localize the receptor to tumor cells [2]. Since that initial report there have been many studies documenting expression in situ by immunohistochemical (IHC) methods in both frozen and paraffin sections. Despite variations in detection techniques, there is general agreement that EGFR is expressed at highest levels and most frequently (80%) in squamous cell carcinoma while it is expressed less frequently in adenocarcinomas and large cell carcinomas (about 50%) [3]. The protein is expressed along the plasma membranes and in the cytoplasm of tumor cells. The prognostic significance of EGFR expression is still controversial with some reports indicating a correlation with poor prognosis [4] and others suggesting no effect on prognosis [5]. These variations may be due to inconsistency in testing methods or to variations in the patient populations evaluated [6, 7, 8, 9, 10, 11, 12]. Assessment of prognostic significance of EGFR is complicated by the diverse effects of EGFR and related signaling molecules as described below. Genetic changes in EGFR Although overexpression of EGFR has been observed for some time, it has only been recently that attention has been focused on genetic mechanisms that might explain this overexpression. Changes in expression levels and activation status may be affected by two mechanisms that are often observed in the same tumors: increased gene copy number and mutation. Increase Gene Copy Number (High Polysomy and True Amplification) in Lung Cancers EGFR is encoded on chromosome 7. Increase in EGFR gene copy number has been evaluated by DNA blotting methods such as Southern blot or array CGH and by FISH. Blotting methods have the disadvantage that cellular localization of signal cannot be assessed and quantification of amplification in individual cells is not possible. The most accurate method for determining gene copy number at the cellular level is FISH. The quantification of FISH however, has not been well standardized in the past and is complicated by heterogeneity among tumor cells. Dr. Garcia at the University of Colorado has proposed a quantification method in which tumors have been divided into two categories based on the presence or absence of >3 copies in 40% of tumor cells. Using this criterion, we initially found that 25% of NSCLC were FISH positive and that high EGFR protein expression levels were more frequent in FISH positive tumors than in FISH negative tumors [13]. We could detect no prognostic significance from amplification or overexpression. The availability of drugs that block EGFR heightened interest in these results. Several studies have now shown that a positive FISH test as defined above is associated with response to EGFR blockade [14, 15, 16, 17]. Increased copy number appears to drive overexpression of EGFR and tumors that are sensitive to EGFR blockers may be dependent on (addicted to?) this pathway for survival and growth while resistant tumors may be driven by any of myriads of other signaling abnormalities that known to occur in lung cancer. Mutations of the EGFR1 Gene in Lung Cancer In addition to EGFR gene copy number and expression abnormalities, it is now evident that EGFR may be affected by somatic mutations in the EGFR gene. The best defined and probably most frequent mutations are those in exons that code for the tyrosine kinase domain. In 2004, two Boston groups almost simultaneously reported [18, 19] that NSCLC contained in frame deletions in exon 19 and point mutations of exon 21 that cluster around the ATP binding pocket of the tyrosine kinase domain. Tumors that contain these mutations tended to occur in non-smoking Asian females and to be associated with superior response to EGFR blockade. Since these initial reports these results have been verified in many studies. However, few studies, have reported improved overall survival in patients with mutations. One possible explanation for this is that not all mutations may equally affect outcome. It has been recently reported in abstract form that response to EGFR blockade and survival may be significantly better in tumors with exon 19 deletion than in those with point mutation. This is presumably due to enhanced effect on receptor phosphorylation and enhanced downstream signaling in tumors with deletion. An acquired mutation in exon 20 (T790M) that imparts resistance to receptor blockade has also been described [21]. This mutation may not be present in primary tumor and may thus be acquired in response to EGFR blocking treatment. Finally, it should be noted that EGFR mutations are not the only mutations that may drive tumor cells and that Ki-ras mutations occur tumors from smokers and may drive part of the same downstream pathway activated EGFR itself. EGFR and Ki-ras mutations have been reported to occur separately with little if any overlap in individual tumors and demographic groups. A small group of lung carcinomas may also be HER2/neu mutated [22] and/or amplified [23]. Taken together these results suggest that complex and inconsistent genetic rearrangements may be responsible for the malignant phenotype of lung cancer. Currently the precise role for mutational analysis as a predictor of response to EGFR blockers is unsettled. One problem is selection of the optimal method for mutation testing. Accurate assessment of mutation in clinical samples can be technically difficult. In most institutions, frozen samples of tumor are not routinely available and this has lead to reliance on DNA from fixed tissue. Several testing variables may affect results of testing DNA from formalin-fixed, paraffin-embedded material including the amount of tumor sample, the use of microdissection to isolate tumor cells from stroma, deamination of adenine and cytosine in formalin fixed DNA [20], the particular Taq polymerase used for sequencing and the primer design employed for PCR in cases where only small amounts of DNA are available. Standards for EGFR mutational testing will have to be adopted for meaningful comparisons to be made across studies. Correlations Among Markers It has been found that 8 of 9 EGFR mutant cell lines also have high gene copy (either high polysomy or amplification). There is thus reason to suspect that there may be considerable overlap in marker status. Several prognostic studies have shown that the effect of mutation, gene copy number and overexpression by IHC are additive [14]. These studies suggest that high gene copy number and protein overexpression enhance the effect of mutation alone. The constellation of mutational status, gene copy number and protein overexpression may have the optimal predictive power for predicting response to EGFR blockers. In future clinical trials it will be important to collect data on all three these EGFR biomarkers to precisely identify patients who might benefit from EGFR blockade. EGFR Genetic Changes in Premalignancy Chromosomal gain is frequently present in premalignant bronchial epithelium. Recent studies have confirmed that chromosome 7 is of one of the most frequently gained chromosomes in central airway premalignancy. The functional consequences of this gain are not known. To date, the presence of chromosome 7 gain has not been shown to correlate with level of protein expression in premalignant bronchial mucosa. To exert a strong influence on overall cellular physiology, it may be necessary that multiple genetic events occur that would effectively transform the cell. That mutation may be one of these events is suggested by the work of Wistuba who found EGFR mutation in benign epithelium of patients with mutant tumor elsewhere in the lung [24]. That the multihit phenomenon is at work here is suggested by a recently encountered patient with Li-Fraumeni syndrome (hit 1) who at the age of 34 developed an EGFR-mutant (hit 2) lung carcinoma. The amount of available information on EGFR genetic status is still small and anecdotal. In view of the magnitude of lung cancer problem, it will be of considerable importance to better define the role of the EGFR pathway in subjects without invasive carcinoma but at risk who may benefit from EGFR blockade. Conclusions The clinical testing of bioactive compounds targeting specific cell signaling pathways has stimulated the need for more accurate information regarding the identity and level of expression of the specific targets as well as intermediates that may be affected by the targets. Current interest is focused primarily on RTK surface proteins. Experience gained in the assessment of this pathway may be applicable to a number of other pathways that are being rapidly defined through application of tumor profiling methods made possible by high throughput genomic and proteomic technologies. In the next few years, these methods can be expected to result in accelerated identification of potentially useful molecular targets in both malignant and premalignant epithelium. Bioactive drugs aimed at these targets hold the promise of reducing the terrible morbidity and mortality from this most lethal of tumors and for permitting a shift in the focus of treatment from advanced tumors to early invasive or preinvasive lesions. Reference List Cohen, S. Isolation of mouse submaxillary gland protein accelerating incisor eruption and eylid opening in the new-born animal. J Biol Chem 237(5), 1555-1562. 62. Hendler, F. J. and Ozanne, B. W. Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest, 74: 647-51, 1984. Franklin, W. A., Veve, R., Hirsch, F. R., Helfrich, B. A., and Bunn, P. A. Jr Epidermal growth factor receptor family in lung cancer and premalignancy. Semin Oncol, 29: 3-14, 2002. Volm, M., Rittgen, W., and Drings, P. Prognostic value of ERBB-1, VEGF, cyclin A, FOS, JUN and MYC in patients with squamous cell lung carcinomas [published erratum appears in Br J Cancer 1998 Apr;77(7):1198]. Br.J.Cancer, 77: 663-669, 1998. Pfeiffer, P., Clausen, P. P., Andersen, K., and Rose, C. Lack of prognostic significance of epidermal growth factor receptor and the oncoprotein p185HER-2 in patients with systemically untreated non-small-cell lung cancer: an immunohistochemical study on cryosections. Br.J.Cancer, 74: 86-91, 1996. Ohsaki, Y., Tanno, S., Fujita, Y., Toyoshima, E., Fujiuchi, S., Nishigaki, Y., Ishida, S., Nagase, A., Miyokawa, N., Hirata, S., and Kikuchi, K. Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol Rep, 7: 603-7, 2000. Cox, G., Jones, J. L., and O'Byrne, K. J. Matrix metalloproteinase 9 and the epidermal growth factor signal pathway in operable non-small cell lung cancer. Clin Cancer Res, 6: 2349-55, 2000. Pastorino, U., Andreola, S., Tagliabue, E., Pezzella, F., Incarbone, M., Sozzi, G., Buyse, M., Menard, S., Pierotti, M., and Rilke, F. Immunocytochemical markers in stage I lung cancer: relevance to prognosis. J Clin Oncol, 15: 2858-65, 1997. Rusch, V., Klimstra, D., Venkatraman, E., Pisters, P. W., Langenfeld, J., and Dmitrovsky, E. Overexpression of the epidermal growth factor receptor and its ligand transforming growth factor alpha is frequent in resectable non-small cell lung cancer but does not predict tumor progression. Clin Cancer Res, 3: 515-22, 1997. Fontanini, G., De Laurentiis, M., Vignati, S., Chine, S., Lucchi, M., Silvestri, V., Mussi, A., De Placido, S., Tortora, G., Bianco, A. R., Gullick, W., Angeletti, C. A., Bevilacqua, G., and Ciardiello, F. Evaluation of epidermal growth factor-related growth factors and receptors and of neoangiogenesis in completely resected stage I-IIIA non-small-cell lung cancer: amphiregulin and microvessel count are independent prognostic indicators of survival. Clinical Cancer Research, 4: 241-249, 1998. Greatens, T. M., Niehans, G. A., Rubins, J. B., Jessurun, J., Kratzke, R. A., Maddaus, M. A., and Niewoehner, D. E. Do molecular markers predict survival in non-small-cell lung cancer? American Journal of Respiratory & Critical Care Medicine, 157: 1093-1097, 1998. D'Amico, T. A., Massey, M., Herndon, J. E. 2nd, Moore, M. B., and Harpole, D. H. Jr A biologic risk model for stage I lung cancer: immunohistochemical analysis of 408 patients with the use of ten molecular markers. J Thorac Cardiovasc Surg, 117: 736-43, 1999. Hirsch, F. R., Varella-Garcia, M., Bunn, P. A. Jr, Di Maria, M. V., Veve, R., Bremmes, R. M., Baron, A. E., Zeng, C., and Franklin, W. A. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol, 21: 3798-807, 2003. Cappuzzo, F., Hirsch, F. R., Rossi, E., Bartolini, S., Ceresoli, G. L., Bemis, L., Haney, J., Witta, S., Danenberg, K., Domenichini, I., Ludovini, V., Magrini, E., Gregorc, V., Doglioni, C., Sidoni, A., Tonato, M., Franklin, W. A., Crino, L., Bunn, P. A. Jr, and Varella-Garcia, M. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small-cell lung cancer. J Natl Cancer Inst, 97: 643-55, 2005. Hirsch, F. R., Varella-Garcia, M., McCoy, K., , West H., Xavier, A. C., Gumerlock, P., Bunn, P. A. Jr, Franklin, W. A., Crowley, J., and Gandara, D. R. Increased EGFR gene copy number detected by FISH associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes. J Clin Oncol . 2005. Travis, W. D., Garg, K., Franklin, W. A., Wistuba, I. I., Sabloff, B., Noguchi, M., Kakinuma, R., Zakowski, M., Ginsberg, M., Padera, R., Jacobson, F., Johnson, B. E., Hirsch, F., Brambilla, E., Flieder, D. B., Geisinger, K. R., Thunnisen, F., Kerr, K., Yankelevitz, D., Franks, T. J., Galvin, J. R., Henderson, D. W., Nicholson, A. G., Hasleton, P. S., Roggli, V., Tsao, M. S., Cappuzzo, F., and Vazquez, M. Evolving concepts in the pathology and computed tomography imaging of lung adenocarcinoma and bronchioloalveolar carcinoma. J Clin Oncol, 23: 3279-87, 2005. Tsao, M. S., Sakurada, A., Cutz, J. C., Zhu, C. Q., Kamel-Reid, S., Squire, J., Lorimer, I., Zhang, T., Liu, N., Daneshmand, M., Marrano, P., da Cunha Santos, G., Lagarde, A., Richardson, F., Seymour, L., Whitehead, M., Ding, K., Pater, J., and Shepherd, F. A. Erlotinib in lung cancer - molecular and clinical predictors of outcome. N Engl J Med, 353: 133-44, 2005. Lynch, T. J., Bell, D. W., Sordella, R., Gurubhagavatula, S., Okimoto, R. A., Brannigan, B. W., Harris, P. L., Haserlat, S. M., Supko, J. G., Haluska, F. G., Louis, D. N., Christiani, D. C., Settleman, J., and Haber, D. A. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med, 350: 2129-39, 2004. Paez, J. G., Janne, P. A., Lee, J. C., Tracy, S., Greulich, H., Gabriel, S., Herman, P., Kaye, F. J., Lindeman, N., Boggon, T. J., Naoki, K., Sasaki, H., Fujii, Y., Eck, M. J., Sellers, W. R., Johnson, B. E., and Meyerson, M. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science, 304: 1497-500, 2004. Marchetti, A., Felicioni, L., and Buttitta, F. Assessing EGFR mutations. N Engl J Med, 354: 526-8; author reply 526-8, 2006. Pao, W., Miller, V. A., Politi, K. A., Riely, G. J., Somwar, R., Zakowski, M. F., Kris, M. G., and Varmus, H. Acquired Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib Is Associated with a Second Mutation in the EGFR Kinase Domain. PLoS Med, 2: e73, 2005. Stephens, P., Hunter, C., Bignell, G., Edkins, S., Davies, H., Teague, J., Stevens, C., O'Meara, S., Smith, R., Parker, A., Barthorpe, A., Blow, M., Brackenbury, L., Butler, A., Clarke, O., Cole, J., Dicks, E., Dike, A., Drozd, A., Edwards, K., Forbes, S., Foster, R., Gray, K., Greenman, C., Halliday, K., Hills, K., Kosmidou, V., Lugg, R., Menzies, A., Perry, J., Petty, R., Raine, K., Ratford, L., Shepherd, R., Small, A., Stephens, Y., Tofts, C., Varian, J., West, S., Widaa, S., Yates, A., Brasseur, F., Cooper, C. S., Flanagan, A. M., Knowles, M., Leung, S. Y., Louis, D. N., Looijenga, L. H., Malkowicz, B., Pierotti, M. A., Teh, B., Chenevix-Trench, G., Weber, B. L., Yuen, S. T., Harris, G., Goldstraw, P., Nicholson, A. G., Futreal, P. A., Wooster, R., and Stratton, M. R. Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature, 431: 525-6, 2004. Cappuzzo, F., Varella-Garcia, M., Shigematsu, H., Domenichini, I., Bartolini, S., Ceresoli, G. L., Rossi, E., Ludovini, V., Gregorc, V., Toschi, L., Franklin, W. A., Crino, L., Gazdar, A. F., Bunn, P. A. Jr, and Hirsch, F. R. Increased HER2 Gene Copy Number Is Associated With Response to Gefitinib Therapy in Epidermal Growth Factor Receptor-Positive Non-Small-Cell Lung Cancer Patients. J Clin Oncol, 23: 5007-18, 2005. Tang, X., Shigematsu, H., Bekele, B. N., Roth, J. A., Minna, J. D., Hong, W. K., Gazdar, A. F., and Wistuba, I. I. EGFR tyrosine kinase domain mutations are detected in histologically normal respiratory epithelium in lung cancer patients. Cancer Res, 65: 7568-72, 2005.