4 Pathology Research International Adenocarcinoma Squamous cell carcinoma Large cell carcinoma Other/mixed Not otherwise specified (NOS) NSCLC (all types) Squamous cell carcinomas Adenocarcinomas EGFR KRAS BRAF PI3K Other EGFR ALK KRAS BRAF PI3K HER2 other Figure 1: Summary of histotype classiﬁcation in NSCLC according to the California Cancer Registry [52] and the patterns of mutations seen in squamous cell carcinomas and adenocarcinomas [12, 19, 43, 47, 48, 50]. and which can potentially be switched oﬀ with modern treatment strategies. Reporting of ALK activation in some patients with NSCLC is part of this perspective. ALK is a transmembrane receptor with tyrosine kinase activity that belongs to the insulin growth factor receptor family, encoded on chromosome 2 (2p23). When bound to its ligand or otherwise activated, it transmits antiapoptotic and cell proliferation signals mediated by KRAS and PI3K pathways [53, 54]. Aberrant activation of ALK was ﬁrst described in anaplastic large cell lymphoma (ACLC) [54] and in inﬂammatory myoﬁbroblastic tumor (IMT) [55]. ALK contributes to NSCLC biology after being fused with a number of other genes and most frequently Echinoderm microtubule-associated protein 4 (EML4) [56, 57]. EML4 gene is located on chromosome 2 (2p21) and is reversely oriented with ALK. EML4-ALK fusion might occur after a cleavage of the chromosome at a variable site and chromo- some inversion, giving rise to diﬀerent fusion isoforms [58]. EML4-ALK fusion occurs in a mutually exclusive fashion with EGFR or KRAS mutations and, almost exclusively, in adenocarcinomas [59]. However, there have been rare reports of co-existence of EML4-ALK and EGFR mutations [60] or squamous cell carcinoma histology [61]. The pres- ence of EML4-ALK is more likely in patients with certain demographic characteristics, such as never smoking status or younger age [59]. Although a frequency range of 0.4– 13.5% [62] has been reported, most studies discover this genetic abnormality at a rate of 2–5% [62–64] in the general population of patients with NSCLC. 3.1. Targeting ALK in NSCLC. Tumors that harbor an activated ALK are addicted to the ALK pathway. On the other hand, it appears that these tumors do not respond to EGFR inhibition. Soda et al. showed the oncogenic potential of EML4-ALK in a transgenic mouse model and proved the dependence of the EML4-ALK-positive tumors on the fusion protein [65]. Crizotinib is a dual MET and ALK inhibitor which was already being tested in a clinical trial as a MET inhibitor at the time of ALK discovery. In a phase II trial in 82 patients with activated ALK, crizotinib had a response rate of 57% [66]. A randomized phase III clinical trial in the second- line setting of NSCLC is already recruiting patients with ALK fusion gene. Given the highly promising phase II early results, it is likely that testing for the presence of ALK fusion protein will emerge as a diagnostic guide for ALK inhibition. Thus, standardization of this test is an important goal. 3.2. Testing for ALK Fusion Gene. A number of diﬀerent assays have been used for the detection of ALK fusion in NSCLC. Fluorescent in situ hybridization (FISH) can be used either as a “fusion” or as a “split-signal” assay [67]. In the fusion variant, diﬀerent color ﬂuorescence is used for EML4 and for ALK. When a fusion is present, a third color emerges from the overlay. In the split-signal variant, an ALK break-apart probe with diﬀerent colors (green and red) for the telomeric and the centromeric end of the gene is used. When a fusion is present, 5 and 3 probes are split, or an isolated 3 probe is detected. In the wild- type cells, a merge/yellow signal is obtained. With either variant, a tumor sample is considered to be positive when more than 15% of the tumor cells are positive for the ALK fusion gene. Those techniques have proved feasible in a variety of tumor samples like formalin-ﬁxed and paraﬃn- embedded (FFPE) tissue, pleural eﬀusions, sputum, and small biopsies. FISH has a number of limitations. It requires a level of expertise which is not widely spread in pathology