Improving survival of cancer patients has been a difficult task for oncologists and all other related medical specialists due to the complexity of this group of diseases. Prevention, early detection and improvement in therapeutic options are the major approaches that can make a difference and have received outstanding attention from cancer physicians, researchers and funding agencies. Much has been learned in the last decade about tumor biology and genetics, and a better understanding of cellular mechanisms underlying the initiation and progression of cancer has enabled the development of innovative therapeutical strategies. Among these are the molecular-based therapies, which address specific cell signaling pathways that are important tumor-drivers.
The molecular targeted therapy field is still in its early stages of exploration. However, exciting results have been reported including examples of dramatic improvement in outcome for neoplasias previously known for their poor prognosis. One of the first validated targeted therapies in oncology involves metastatic breast cancer and the monoclonal antibody trastuzumab (Herceptin, Genentech Inc, San Francisco CA) . In approximately 20% of breast cancers the human epidermal growth factor receptor 2 gene (c-erb-B2, ERRB2 or HER2), a member of the receptor tyrosine kinase 1 (RTK1) family, is amplified and overexpressed at the receptor level and these tumor characteristics are significantly associated with poor clinical outcome . However, women with HER2 overexpressing metastatic breast cancer received a significant benefit from trastuzumab, a recombinant humanized monoclonal antibody launched as a therapeutic option in 1998. The selection of these patients for treatment has been made by evaluating the levels of protein expression in immunohistochemical assays (IHC) and/or the number of copies of the HER2 gene in fluorescence in situ hybridization assays (FISH) [3, 4].
More recently, one international (HERA) and two NCI-sponsored phase III clinical trials (NSABP B31 and NCCTG N9831), which have enrolled more than 6,000 patients, have shown that combining paclitaxel with trastuzumab after adjuvant chemotherapy significantly improved outcome among women with surgically removed HER-2 positive breast cancer [5, 6]. These results expanded the spectrum of breast cancer patients potentially eligible for trastuzumab therapy from metastatic to early stage breast cancer.
Non-small cell lung cancer (NSCLC) is another solid tumor which has seen a favorable impact from targeted therapy. Lung cancer is a significant public health problem in western countries and has long been the most common cause of cancer death . NSCLC is usually diagnosed in advanced stage, when prognosis is poor and options for chemotherapy are limited. Another member of the RTK1 family, the epidermal growth factor receptor (EGFR, HER1), is long known to be overexpressed in a significant fraction of NSCLC . EGFR is a 170 kDa type I growth factor membrane receptor with 1186 amino acids encoded by 28 exons spanning near 190 kb on chromosome 7p11.2. These receptors exist as active monomers but, upon binding to ligands such as the epidermal growth factor (EGF) and the transforming growth factor alpha (TGFα), they undergo conformational changes that facilitate dimerization, either with another EGFR molecule or with HER2, HER3 or HER4 molecules. The dimerization is followed by intermolecular autophosphorylation of key tyrosine residues in the activation loop of catalytic tyrosine kinase domains through the transfer of phosphates from adenosine triphosphate (ATP). EGFR-activated pathways include the mitogen-activated protein kinase (MAPK) pathway, which induces cell proliferation, as well as the AKT and the signal transducer and activator of transcription (STAT) pathways, which contribute to cell survival.
The role of EGFR as an oncogene has been elucidated for many years and the level of EGFR protein expression has been shown to be elevated in multiple cancer types relative to normal tissues . In lung cancer, there are several key mechanisms for EGFR activation, such as overexpression of ligands , gene amplification [11, 12] and activating mutations . The discovery of agents with the ability to antagonize EGFR functions in cancer cells, such as the monoclonal antibody cetuximab (Erbitux, ImClone Systems Inc) and the specific tyrosine kinase inhibitors (TKIs) gefitinib (Iressa, AstraZeneca, UK) and erlotinib (Tarceva, OSI Pharmaceuticals Inc) have heightened the clinical interest in this growth factor receptor (14,15). Gefitinib and erlotinib are orally active EGFR-TKIs that block signal transduction pathways implicated in the proliferation and survival of cancer cells and other host-dependent processes promoting cancer cell growth. These drugs have intracellular mechanisms of action and compete with ATP for binding to EGFR thus directly inhibiting EGFR autophosphorylation. In retrospective studies, patients with advanced NSCLC treated with gefitinib or erlotinib have shown higher rates of objective response and longer survival than non-treated patients [16, 17]. Erlotinib was shown in the NCIC-BR.21 phase III trial to provide significant survival benefit to NSCLC patients compared with placebo  and was approved for clinical use in the US (November 2004) and Europe (June 2005). Gefitinib failed to reach significant improvement in survival in the large randomized ISEL (Iressa Survival Evaluation in Lung Cancer) study . Nevertheless, there was a clear trend towards better outcome in the treated group of patients comparing to placebo, suggesting that there is a subset of patients who benefit from using gefitinib. Interestingly, in both erlotinib and gefitinib NSCLC randomized trials approximately 30% of the patients died within 4 months of treatment time, which strongly suggest that there is a subset of lung cancer patients who receive no clinical benefit from EGFR-TKI therapy.
The availability of molecular targeted therapy agents with significant anti-tumor activity in advanced NSCLC both as a single agent and in combination with chemotherapy and the need to increase the efficiency of these treatments are factors urging the development and validation of laboratory tests for assessment of the EGFR status in lung cancer patients. Research efforts have focused on a large number of clinical and biological factors that may predict which NSCLC patients could benefit from molecular targeted treatments and ultimately aimed to the development of sensitive and specific laboratorial tests to screen patients. Early on, the expression levels of EGFR was tested without a predictive success for response to gefitinib , although more recent studies in larger phase II cohorts have detected a positive association between high EGFR protein levels and sensitivity to both gefitinib and erlotinib [23, 24]. Activating missense mutations and deletions in the tyrosine kinase domain of the EGFR gene were the first molecular changes clearly associated with objective response to gefitinib and erlotinib [25–27]. The impact of these EGFR mutations on overall survival, however, has varied from a strong positive correlation in some populations [28–30] to a much less marked correlation in others [23, 24]. Interestingly, EGFR mutations also conferred superior outcome for chemotherapy in the TRIBUTE trial  and may even be prognostic markers according to recent findings that non-treated patients with mutations have longer survival [24, 32].
Genomic gain for EGFR sequences detected by fluorescence in situ hybridization (FISH) has also been demonstrated as an excellent predictor of response [23, 24, 33] and longer survival [23, 33] in large cohorts of patients treated with gefitinib and erlotinib. More recently, molecular analyses performed in patients included in the ISEL study also have shown that longer survival was correlated with high levels of EGFR genomic gain detected by FISH . Interestingly, if the EGFR genomic abundance in tumors is evaluated by other techniques, such as Southern Blotting or quantitative real-time PCR, the association between increased gene copy numbers and sensitivity to TKI is non-significant or marginally significant (30,35,36). The difference probably reflects the technical characteristics of each of the methodologies. FISH is an in situ method that allows for identification of single cells in the context of the tissue architecture.
Southern Blot and PCR-based techniques are global extraction-based methods that are influenced by the dilutional effects of stroma and infiltration of reactive cells.
Mutations in the tyrosine kinase domain of the HER2 gene which were similar to those described for the EGFR gene were detected at very low frequencies in NSCLC and are probably not clinically relevant [37, 38]. Conversely, high level of genomic gain for the HER2 gene detected by FISH was found to be a significant predictor of outcome to gefitinib and an enhancer of sensitivity to gefitinib in EGFR FISH positive NSCLC patients .