2 kinase inhibitors (TKIs) and ALK inhibitors, respectively. Furthermore, we comment on assays for accurate and spe- ciﬁc histologic classiﬁcation of NSCLC which are necessary for appropriate use of pemetrexed and bevacizumab treat- ment. 2. EGFR EGFR is a transmembrane receptor present in the majority of patients with NSCLC [7]. The receptor mediates cellular response to various extracellular signals. It is encoded by the proto-oncogene EGFR and demonstrates diverse function in the biology of NSCLC. EGFR activates two major downstream pathways, medi- ated by Kirsten rat sarcoma (KRAS) and phosphatidylinositol 3-kinase (PI3K) proteins, respectively. KRAS is a protein with GTPase activity which activates BRAF and ﬁnally ERK as part of the mitogen-activated protein kinase (MAPK) signaling cascade. On the other hand, PI3K activates phospholipase C (PLC), protein kinase beta (PKB/AKT), and the mammalian target of rapamycin (mTOR) complex. A set of interactions between these two pathways, as well as positive and negative feedback loops, compose a complicated network which mediates the impact of EGFR and other transmembrane receptors on cell proliferation, inhibition of apoptosis, tumor growth, and invasiveness. 2.1. EGFR Mutations. The receptor carries activating muta- tions in its tyrosine kinase (exons 18–21) domain in a subset of the NSCLC population [8–10]. Patients with certain epidemiologic characteristics (adenocarcinoma histology, never smoking status, female gender, and Asian ethnicity) are more likely to harbor the mutations [8–10]. Most of the mutations are in frame deletions in exon 19 and a point mutation in exon 21 (L858R) [11]. A deletion between codons 746 and 750 accounts for 65–75.5% of the deletions in exon 19 [12]. Erlotinib and geﬁtinib are compounds that reversibly inhibit the tyrosine kinase activity of EGFR and have been employed in the treatment of patients with NSCLC. Clinical beneﬁt is modest in the unselected population with the disease [13]. However, tumors which harbor activating mutations of the tyrosine kinase domain of EGFR tend to respond to TKIs [8–10]. Patients with the mutations have a prolonged time to progression when treated with geﬁtinib as a ﬁrst-line regimen compared to chemotherapy, whereas the opposite is true at the absence of a mutation [14–16]. The results of such clinical trials led to the consensus that an eﬀort should be made to deﬁne the mutational status in every newly diagnosed patient with NSCLC in order to decide appropriately on the use of TKIs in the ﬁrst-line setting [17]. 2.1.1. Consensus on EGFR Mutations Testing. The recent clinical trials that have established TKIs treatment in the ﬁrst-line setting for NSCLC patients with activating EGFR mutations have employed a variety of methods for mutation detection (Table 1). Furthermore, a number of diﬀerent methods have been described in other studies outside the Pathology Research International Table 1: Diﬀerent assays for detection of EGFR mutation that have been used before TKI treatment in phase III trials in the ﬁrst-line setting. Study Maemondo et al. [15] Mitsudomi et al. [16] Mok et al. [14] Assay Peptide nucleic acid-locked nucleic acid PCR clamp Fragment analysis, Cycleave method, direct sequencing, peptide nucleic acid-locked nucleic acid PCR, PCR invader Ampliﬁcation refractory mutation system (ARMS) context of clinical trials [18, 19]. Each of these methods has been validated by comparison to direct sequencing in order to estimate its sensitivity and speciﬁcity. However, many of the validation studies have been performed in small cohorts and by a limited number of research groups for each method [20–22]. Taken together, the lack of a common protocol for testing tumors for EGFR mutations and the need for more solid validation of such protocols and procedures in large studies by independent groups indicate the type of studies that will support a consensus on testing for the presence of EGFR mutations. The most commonly used mutation detection method is direct DNA sequencing following extraction of DNA from a tumor sample. DNA sequencing is, however, a challenging task when a small biopsy or even a cytology block is the only sample available for companion diagnostics. This is very often the case in the metastatic setting for NSCLC patients. Another limitation is that genotyping methods based on DNA extraction are subject to sample contamination with non-tumor DNA derived from normal or stromal cells. When the percentage of non-tumor to tumor DNA exceeds a certain threshold, it is likely that a mutation might not be detected by direct sequencing as the mutation-speciﬁc signal will not surpass the background. Other methods have been developed and reported to have superior sensitivities [23–25]. In addition, the number of malignant cells that need to be present in a sample and the number of diﬀerent cores or blocks that need to be tested before providing a reliable result on EGFR mutation status are not known. It is largely dependent on the heterogeneity of mutated EGFR expression within a core, across diﬀerent cores and diﬀerent tissue blocks. Although EGFR mutations occur early in the tumorigenesis process and one would, therefore, expect them to be homogeneous, tumor heterogeneity of EGFR mutations has not been assessed thoroughly so far. In fact, it has been reported that there is discrepancy in the mutational status between the primary tumors and their metastases [26, 27], as well as a level of heterogeneity within a tissue block [28, 29]. Thus, it is imperative to determine the level of heterogeneity of mutated EGFR in order to propose a standard protocol for reliable mutation detection. Besides genotyping, immunohistochemistry (IHC) has become available for the most common mutations [19]. Antibodies have been designed to detect speciﬁcally in frame deletions in exon 19 and point mutations in exon 21. Despite