Pathology Research International the high sensitivity and speciﬁcity that was initially reported [12, 19], subsequent studies have shown that the “mutation- speciﬁc” antibodies are able to detect only 80% of the full spectrum of mutations [30, 31]. Kitamura et al. [29] have reported sensitivity as low as 47%, probably because of the high frequency of uncommon mutations in their cohort. Most of the studies, however, conclude that deletion-speciﬁc antibody methodology is ineﬃcient at detecting deletions in exon 19 other than the “classic” in frame deletion between codons 746 and 750. On the other hand, IHC is available in most pathology laboratories and it is more clinically relevant than genotyping as it assesses mutations at the protein level where a TKI’s function occurs. Besides, the assay is highly speciﬁc and can be used as an initial assessment before conﬁrmatory genotyping is performed. While still experimental, this in situ method may become a useful research tool in illuminating topics such as the heterogeneity of mutant EGFR and the dynamic range of mutant EGFR expression. 2.1.2. Prediction of Secondary Resistance to TKIs. Even when an EGFR-mutated tumor is initially controlled with EGFR inhibition, resistance emerges typically after a median of 9–12 months [14–16]. Diﬀerent mechanisms of acquired resistance have been described. About half of the cases become resistant because of a point mutation (T790M) in exon 20 [32, 33] and 20% of the cases because of ampliﬁcation of hepatocyte growth factor receptor (MET) [34]. MET is a transmembrane receptor which can activate EGFR downstream targets via a parallel pathway. Both resistance mechanisms are present in 10% of the patients who become refractory to tyrosine kinase inhibition after initial response [35]. There is evidence to suggest that both MET ampliﬁcation and T790M point mutation might be present in a low number of tumor cells before administration of tyrosine kinase inhibitor treatment. These cells are selectively enriched after EGFR inhibition [36, 37]. Recently, it was shown that resistance can emerge as the result of activation of a loop between TGF beta and IL6 [38], or through derepression of FGFR2 and FGFR3 [39] or even through phosphatase and tensin homolog (PTEN) loss [40]. These three additional modes of acquired resistance have not yet been validated in large cohorts of patients and their importance remains to be determined. Taken together, these data strongly support the notion that patients should be tested for the presence of genetic alterations that might cause resistance after failure of TKIs and treated appropriately on the basis of such diagnostics. There are currently drugs that can overcome T790M-mediated resistance as shown in preclinical reports [41]. Eﬃcient MET inhibitors become relevant in cases of MET ampliﬁcation [42]. Some of these regimens are being tested in ongoing clinical trials. Whether the employment of such modalities should follow the failure of TKIs or, alternatively, should accompany TKIs in the ﬁrst- line setting in order to prevent the emergence of resistance remains an open question. 2.2. Miscellaneous Mutations in the EGFR Pathway. A num- ber of activating mutations have been described in the 3 component molecules of this network among patients with NSCLC. Mutations in KRAS [43] are the most well studied. KRAS mutations cause primary resistance of the tumors to TKIs [43]. However, they are mutually exclusive to EGFR mutations [44] and, at the same time, they are not always present in patients who are resistant to TKIs. Thus, detection of such mutations is inferior to EGFR mutation detection and is not currently recommended as a diagnostic test before EGFR inhibition. In some centers, a strategy of “reﬂex” testing is followed: patients with adenocarcinoma are tested for the presence of EGFR mutations ﬁrst and, if negative, they are tested for the presence of KRAS mutations [45] in an eﬀort to obtain information about the driver mutation while saving time, tissue, and resources. Additional mutations have been identiﬁed in numerous other genes [46]. HER2 [47], BRAF [48], and PI3K [49] mutations are found in fewer than 5% of patients [44, 50]. In contrast to KRAS mutations, PI3K mutations can be found in both EGFR mutant or wild-type tumors and, therefore, they might be responsible for primary resistance to TKIs in patients with activating EGFR mutations. However, they are rather rare and their predictive potential has yet to be determined in the clinical setting. As several molecules of the KRAS and PI3K pathways are targets of speciﬁc inhibitors tested in ongoing clinical trials, such mutations are likely to become clinically relevant in the future. A convenient assay that combines a multiplex PCR step with a single- base extension sequencing step can reliably test for many diﬀerent mutations simultaneously and is already employed by some centers [51]. The separation and identiﬁcation of the diﬀerent alleles in this assay is performed on the basis of diﬀerent ﬂuorescence colors and diﬀerent sizes of allele- speciﬁc probes. Figure 1 summarizes the spectrum of histotype classiﬁ- cation for NSCLC and the frequency of diﬀerent mutations in the two major histotypes. It becomes apparent that the “NSCLC puzzle” has yet to become completed, especially in squamous cell carcinomas. Besides, unraveling the puzzle of genetic alterations, certain treatment strategies are needed in conjunction to novel diagnostics. Since the spectrum of mutations is unique to each histologic type, as shown in Figure 1, it becomes apparent that tumors of diﬀerent histology are expected to behave diﬀerently after exposure to treatments that target speciﬁc genetic alterations. In this regard, tyrosine kinase inhibitors are more clinically relevant for the treatment of patients with adenocarcinoma. Likewise, antiangiogenic compounds, like bevacizumab, are contraindicated in patients with squamous cell carcinoma as they might cause lethal bleeding in those patients. 3. Anaplastic Lymphoma Kinase (ALK) Progress in the understanding of EGFR mutations revealed that some tumors rely on single driving genetic alterations, a phenomenon better known as oncogene addiction. The discovery of an “Achilles’ heel” in a small subset of the NSCLC population has led to the exciting perspective of exploring novel pathways that are vital to cancer existence