The EGFR is a 170-kDa plasma membrane glycoprotein composed by an extracellular region, a single transmembrane domain, and an intracellular domain with tyrosine kinase activity and a C-terminal tail. Activation of the EGFR receptor via phosphorylation produces downstream signals to the phosphatidylinositol 3-kinase (PI3K)/AKT and RAS/RAF/mitogen-activated protein kinase (MAPK) pathways, which are responsible for the normal regulation of cellular proliferation and apoptosis. These intracellular signaling pathways are modified by neoplastic cells with EGFR mutations [13].

The Asia-Pacific patients with NSCLC adenocarcinoma (ADC) subgroup had the highest EGFR mutation frequency at 47%. EGFR-mutated lung cancers represent around 15–20% of all lung ADCs diagnosed in the United States [14], 6–41% in Europe (mean 15%) [15], and 36% (range 14–51%) [16] in Latin America, where there is important heterogeneity.

In the Caucasian population, a mutation of the EGFR gene is more frequent in patients with adenocarcinoma NSCLC with an acinar or papillary pattern (found in 27% of patients); the mucinous adenocarcinoma is usually negative.

Beyond the unequivocal influence of Asian ethnicity, prevalence of EGFR in different ethnic groups is unclear. Some studies have shown no significant difference in EGFR mutation rates between African-American (19%) and white patients (13%), while others suggest a lower frequency of EGFR in African-Americans [17]. Worldwide literature shows that EGFR mutation frequency in lung adenocarcinoma is higher in women than men and in never-smokers versus smokers (66% vs. 22%) [18].

However, the substantial lack of data from several geographic regions of the world (mainly Africa, Middle East, Central Asia, and Central America) and ethnic subgroups limits interpretation.

EGFR 6 (also known as HER-1 or Erb1) belongs to the ErbB receptor tyrosine kinase (RTK) family. This group also includes HER-2/neu (ErbB2), HER-3 (ErbB3), and HER-4 (ErbB4). These transmembrane receptor tyrosine kinases involved in signal transduction pathways regulate proliferation and apoptosis. Known EGFR mutations have been detected in the tyrosine kinase domain of the EGFR gene, which spans exons 18–24. Most mutations are deletions of exon 19 that affect a three-amino acid (LRE) sequence or a point mutation L858R on exon 21 [19]. On the other hand, mutations affecting exon 20, mainly small insertions in T790M (that explain 3–5% of all EGFR mutations), are related with primary resistance to EGFR TKIs. In contrast to the reported higher response rates, better quality of life (QoL), and longer PFS in the whole group of EGFR-mutated ADC compared to wild-type subgroup, a retrospective study in Korean patients with exon 20 insertion reported objective response in only 25% of patients treated with gefitinib [20]. Patients with exon 19 deletion-type EGFR mutation have also shown more significant benefit than patients with L858R mutation. Increased OS (38 months vs. 17 months) has been reported in patients with EGFR exon 19 deletion, compared with patients with L858R mutation [21]. The low frequency of the less common types of EGFR mutations involving exon 18 or 20 has not allowed the definition of the prognostic impact of these genetic alterations.

Based on the available clinical trial data, current guidelines indicate testing all patients with metastatic NSCLC adenocarcinoma for the presence of activating EGFR mutations and recommend first-line EGFR-TKI treatment for patients with adenocarcinoma and a known EGFR mutation [14]. Gefitinib (since 2009 in Europe/Asia and 2015 in the United States), erlotinib (since 2011 in the United States), and afatinib (since 2013 in the United States) have been approved with the condition that, in first-line setting, their use should be restricted to the treatment of lung ADCs with sensitizing EGFR mutations as exon 19 deletions or L858R mutations.

Secondary mutations in EGFR may appear in patients that develop resistance to EGFR TKIs. The most common resistance mutation is the T790M activating point mutation in exon 20 thus interfering with binding of reversible TKIs. Almost 50% of tumors from patients who develop acquired TKI resistance show a T790M, but they may also be found in patients who have never received TKI treatment.

KRAS is a commonly detected mutation in NSCLC, harbored by approximately 20–25% of lung adenocarcinomas in the United States [22] and 14% in France [23]. Most frequently, these mutations involve codon 12 or 13 and, rarely, codon 6 [24].

KRAS mutations are more common in tumors with adenocarcinoma histology than in squamous type NSCLC. KRAS mutations are less common among patients of Asian ethnicity and more common in smokers (43% vs. 0%) [25]. That strong link between cigarette smoking and KRAS mutations in adenocarcinoma of the lung supports some pathogenic role of this mutation in the carcinogenetic properties of tobacco in NSCLA. In small studies, the occurrence of KRAS and EGFR mutations seems to be mutually exclusive [26].

Meta-analyses have shown that patients with KRAS mutations have a poorer response to EGFR-TKIs [27]. Most of the trials were small and this presumed negative association has not been indicated by larger studies [28]. On the other hand, some studies have shown that KRAS mutations have little impact on PFS in contradiction with the presumed nonresponse of KRAS-mutated patients to TKI therapy [29]. According to Cadranel et al., EGFR and KRAS status independently impacts outcomes in advanced NSCLC patients under EGFR-TKI therapy, but EGFR status impacts both PFS and OS while KRAS only impacts OS [23].

An association between KRAS mutational status and benefit of anti-EGFR monoclonal antibodies has not been demonstrated in NSCLC, and no trials can demonstrate the potential value of testing for KRAS mutations and according tailored therapy [30]. As a consequence, the role of KRAS mutations remains unclear. The results of ongoing clinical trials in KRAS-mutant NSCLC will establish if KRAS mutational status should or should not be included in the selection of treatment for NSCLC.

The oncogenic activity of ALK in anaplastic large cell lymphoma was reported in the 1990s [31]. ALK is a receptor tyrosine kinase normally expressed in several tissues like the small intestine, testes, and the nervous system. In 2007, a translocation in the gene encoding the receptor tyrosine kinase anaplastic lymphoma kinase (ALK), leading to the expression of ALK fusion proteins, was identified as an oncogenic driver in a subset of patients with NSCLC [32]. The genes encoding echinoderm microtubule-associated protein-like 4 (EML4) and ALK are both located on the short arm of chromosome 2 (2p21 and 2p23) [32]. The ALK gene rearrangement consists of a small inversion in the short arm of chromosome 2 between exon 20 of the ALK gene and different exons of the echinoderm microtubule-associated protein-like 4 (EML4) gene, resulting in the abnormal expression and activation of this tyrosine kinase in the cytoplasm of cancer cells. This translocation leads to a chimeric protein with constitutive activation of ALK that presents oncogenic activity demonstrated both in vitro and in vivo. This rearrangement occurs in 2–5% of NSCLC, predominantly in young (50 years or younger), never or light smokers with adenocarcinoma [33] without other genetic disorders, such as mutations of the epidermal growth factor receptor gene [34]. However, clinical characteristics are not as precise as predictive biomarkers to select patients for ALK-targeted therapies. Since the discovery of the ALK-EML4 fusion protein, at least 11 different ALK fusion variants have been identified [35]. Thus, the current guidelines of the International Association for the Study of Lung Cancer (IASLC) and the European Society for Medical Oncology (ESMO) recommend that all patients with advanced-stage lung adenocarcinoma or tumors with an adenocarcinoma component should be tested for ALK, regardless of clinical characteristics.

The presence of alterations in the ALK gene carries a poorer prognosis. It has been reported that patients with ALK translocation have an increased risk of brain and liver metastases (40% vs. 21%) and a greater number of metastatic sites [36] as well as a higher risk of disease progression in comparison with ALK negative patients [37].

Most importantly, ALK gene translocation has been shown to be a predictive biomarker of the efficacy of different agents targeting the ALK kinase activity like crizotinib , ceritinib, and alectinib. Preclinical and single-arm phase I studies have demonstrated that patients with ALK-rearranged NSCLC can be successfully treated with crizotinib. The first published human study of ALK inhibition used the dual ALK/c-MET inhibitor, crizotinib , in patients with ALK-translocated, advanced lung carcinoma [38]. The results of the PROFILE 1007 phase III trial confirmed significantly higher response rates (65 vs. 20%) and longer progression-free survival (7.7 months vs. 3.0 months) compared with chemotherapy as second-line treatment for ALK+ NSCLC [4] and proved better results than standard first-line platinum/pemetrexed chemotherapy in untreated advanced ALK+ NSCLC. Despite the efficacy of crizotinib therapy in patients with ALK-positive lung cancer, most patients develop resistance to crizotinib within the first 12 months [39]. Second-generation ALK inhibitors as ceritinib, alectinib, and brigatinib (AP26113) were developed to overcome crizotinib-resistant mutations. Ceritinib is 5–20 times more potent than crizotinib and has achieved a response rate of 58% and a median PFS of 7 months when tested in ALK-positive patients with a very acceptable profile of adverse effects [40]. These and other research results led to the accelerated approval of ceritinib by the FDA in April 2014 for the treatment of patients with ALK+ metastatic NSCLC with disease progression or who present intolerance to crizotinib. In February 2015, the EMA Committee for Medicinal Products for Human Use (CHMP) adopted a positive opinion, authorizing conditional marketing for ceritinib for patients with advanced ALK+ NSCLC previously treated with crizotinib [41]. Other new-generation ALK kinase inhibitors such as alectinib with potent in vitro activity are showing promising clinical efficacy at extracranial and intracranial sites in ALK-positive NSCLC.

The c-ros oncogene 1 (ROS1) is a member of the insulin receptor family controlling cell cycling and proliferation. It has been identified as a driver mutation of lung cancer cells and primary tumor tissue and has recently been found rearranged in 2% of patients with NSCLC [42]. Multiple ROS1 fusion proteins have been described. ROS1 rearrangements are rarely accompanied by other mutations such as EGFR, ALK, or KRAS. Patients with ROS1 rearrangement have a very similar profile to those with ALK rearrangement, including a good response to crizotinib. Resistance to crizotinib has already been observed in patients with ROS1(+) NSCLC, and newer agents such as foretinib (GSK1363089) are being studied with initial promising results [43].

New molecular targets are being described. In October 2015, pembrolizumab was approved for the treatment of PD-L1-positive NSCLC, and the relevance of BRAF mutations in NSCLC by applying BRAF-targeted treatment is being studied. HER-2, human epithelial receptor 2 (HER-2/ErbB2), is a member of the HER group which is activated by homo- or heterodimerization with ErbB1–4 leading to the activation of the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway. There are several ongoing clinical trials exploring newer-generation kinase inhibitors such as dacomitinib, afatinib, and neratinib for HER-2(+) lung cancer.

BRAF mutation —BRAF is a downstream signaling mediator of KRAS which activates the MAP kinase pathway. Small studies have shown that BRAF inhibition with selected TKIs (vemurafenib and dabrafenib) may be effective.

MET abnormalities are a tyrosine kinase receptor for hepatocyte growth factor (HGF). Increased MET expression may predict response to MET-targeted drugs and also appears to be associated with a worse prognosis. Initial studies show that a potential response to crizotinib and capmatinib (an investigational MET inhibitor) may be effective in patients with NSCLC and MET mutations.

Due to the widespread use of next-generation sequencing (NGS) , additional numerous potential targets will be developed in the next years, but only clinical trials will show which will be appropriate for clinical application.

Most lung cancer patients are diagnosed after examination of a small biopsy (transbronchial or endobronchial) or cytology (fine-needle aspiration or body fluid) specimen. Specimens are processed for diagnosis, staging, and molecular marker analysis. As personalized treatment is now the standard of care for advanced lung cancer, current guidelines recommend that rapid EGFR and ALK mutation testing be performed at diagnosis in all patients with advanced adenocarcinoma regardless of gender, race, smoking history, or other risk factors. Testing is not recommended for tumors without an adenocarcinoma component. However, it should also be considered in large cell carcinoma or poorly differentiated carcinoma because limited sampling does not effectively exclude an adenocarcinomatous component [44].

Molecular testing is essential for patients with unresectable advanced lung cancer who will be offered targeted therapies. Patients with a potential surgical cure may never need antineoplastic drugs. The initial surgical specimen at resection is a high-quality tissue sample and may avoid new biopsies in case of relapse. However, the cost-benefit ratio of potential unnecessary testing must be considered.

If molecular testing of lung cancer is easily available and adequately reimbursed, shortening the waiting time for treatment selection seems reasonable. The sequential performance of molecular testing may be justified in the case of financial constraints that exist in molecular testing. In those cases, starting with KRAS in Caucasian and EGFR in Asian patients may be reasonable. Alternatively, IHS screening for ALK- or ROS1-negative lung cancer may be performed to detect cases that require FISH testing for ALK or ROS1 translocations. Sequential molecular testing of lung cancer can reduce the volume and cost of analysis by roughly 30%; however, this must be offset with the delay in obtaining the necessary information for the right treatment choices.

Early studies [45] have shown discordance in the results of mutational analysis between primary tumor and metastatic disease. However, the current consensus is that discordance in the rates of mutation detection between primary or metastatic tumors is infrequent and consequently the primary or metastatic site can be tested [46].

On the other hand, there is no evidence of intratumoral heterogeneity of mutations in NSCLC, and current data suggest that it is not necessary to test multiple areas of the same tumor. Synchronous primary tumors should be tested independently as different tumors. Molecular analysis of synchronous lesions may contribute to define if lesions are likely to be clonally related.

Despite the multiple biomarkers mentioned above, tissue should be prioritized for EGFR and ALK testing. The cautious use of material should be taken into consideration to avoid new invasive diagnostic procedures.