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EML4-ALK fusion gene in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a malignant tumor with a high morbidity and mortality rate that is a threat to human health. With the development of molecular targeted research, breakthroughs have been made on the molecular mechanism of lung cancer. The echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is one of the most important pathogenic driver genes of NSCLC discovered thus far. Four generations of targeted drugs for EML4-ALK have been developed, with patients benefiting significantly from these drugs. Therefore, EML4-ALK has become a research hotspot in NSCLC. The aim of the present study is to introduce the current research progress of EML4-ALK and its association with NSCLC.


Introduction
According to the latest data released by the International Agency for Research on Cancer, the incidence of lung cancer is increasing annually (1); it is one of the most common malignant tumors, accounting for 15% of global cancer diagnoses, with a 10-year survival rate of just 5% (1)(2)(3). Lung cancer is the second most common cancer type worldwide and the malignant tumor with the highest mortality rate; it is also associated with poor survival following the initial diagnosis (1). Possibly due to the popularization of diagnostic imaging technology and the improvement in the awareness of physical examinations, more patients with lung cancer are being diagnosed at an early stage; however, a number of them are young (4).
With the emergence of technologies such as fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS), tumor diagnosis and treatment have entered the molecular field, and tumor gene screening has become a routine diagnostic and treatment method (5)(6)(7). Non-small cell lung cancer (NSCLC) is the main pathological type of lung cancer; in recent years, breakthroughs have been made in the research on targeted genes for NSCLC (8). Among them, the echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4-ALK) fusion gene is the most important pathogenic gene of NSCLC discovered so far (9). Targeted drug therapy for EML4-ALK has achieved marked curative effects, bringing a glimmer of hope to patients with NSCLC (9,10).

EML4-ALK fusion gene
The EML4-ALK fusion gene was first reported by Soda et al (11), after amplification of a 3926-bp DNA fragment in the tumor tissue of a patient with lung adenocarcinoma, which encoded a protein composed of 1,059 amino acids, the fusion protein EML4-ALK (11). In the follow-up experiments by Soda et al (12), the implantation of the EML4-ALK gene into normal lung cells was shown to induce carcinogenesis, suggesting that EML4-ALK has an oncogenic effect.
Suprenant et al (13) was the first to discover a substance that binds to tubulin and is involved in mediating mitosis, the echinoderms microtubule-associated protein (EMAP; also known as EML). To date, a total of 6 human-expressed EML family members (EML1-6) have been found (14,15), and EML4 is the homologous protein that expresses the most representative EMAP characteristics (16). EML4 is composed of an N-terminal basic domain, a hydrophobic motif in EML proteins (HELP) and a C-terminal tryptophan-aspartic acid repeats (WD) (17). The base domain of the N-terminal is an a-helical-coiled base region that contains a coiled region that promotes trimerization oligomerization, namely that of the trimerization domain (15). The initial study by Soda et al (11) found that the construction of EML4 cells with basal domain deletion did not induce tumorigenesis in nude mice, while HELP and WD deletion did, which indicated that the basal domain in EML4 played a key role in inducing tumorigenesis.
ALK is an insulin receptor subfamily of the receptor tyrosine kinase family (18), originally identified in anaplastic large cell lymphoma (19). ALK is normally expressed during the embryonic period and is involved in the regulation and development of the nervous system (20,21). ALK is mainly composed of a tyrosine kinase domain and a transmembrane domain (22)(23)(24). Under normal circumstances, following the activation of ALK by exogenous ligands, two ALK monomers are phosphorylated to form an ALK dimer with kinase activity to participate in cellular regulation (23)(24)(25).
The EML4 and ALK genes are located on p21 and p23 of human chromosome 2, respectively, and are ~10 Mb apart (26); the orientation of their gene sequences is reversed on the short arm of chromosome 2 (27). The essence of EML4-ALK is a translocation fusion caused by an intra-arm interchange, and one of the genes needs to be reversed during gene fusion (27,28). In the EML4-ALK fusion gene, the EML4 gene fragments all contain the basal domain, and ALK contains the kinase region (29). The fusion gene of EML4 and ALK can encode a fusion protein with tumorigenic activity, namely the EML4-ALK fusion protein (30). The fusion protein can directly form an ALK dimer without the activation of an exogenous ligand, thereby activating ALK and its downstream RAS/ERK/STAT3/mTOR and other signaling pathways. Finally, through the promotion of cell proliferation and invasion, and the inhibition of apoptosis, it leads to the occurrence of NSCLC. The RAS/ERK signaling pathway is associated with cell proliferation, and the mTOR and STAT3 pathways are associated with cell survival and apoptosis (31,32). Studies have shown that the HELP domain on EML4 is necessary for the specific activation of RAS, and the EML4-ALK fusion protein can promote the upregulation of RAS and the phosphorylation of ERK. The EML4-ALK fusion protein activates and upregulates the expression of STAT3, and the overexpression of STAT3 promotes the phosphorylation level of mTOR and promotes the tumor anti-apoptotic ability by activating mTOR signaling ( Fig. 1) (33)(34)(35)(36)(37).
To date, several variants of the EML4-ALK fusion gene have been identified (38), and the differences among variants mainly depend on the different truncation sites in the WD region of EML4, which form EML4 gene cleavage fragments of different lengths (39). In a previous study, these EML4 fragments of different lengths were inserted into exon 20 of the ALK gene to form different fusion genotypes (40). Currently the most common EML4-ALK fusion genotypes in NSCLC are as follows: EML4-ALK V1 (exon 13 of EML4 fused to exon 20 of ALK; 33%), EML4-ALK V2 (exon 20 of EML4 fused to exon 20 of ALK; 10%), EML4-ALK V3 a/b (exon 6 of EML4 is fused to exon 20 of ALK; 29%) ( Fig. 2) (41)(42)(43), and different fusion genotypes have different tyrosine kinase activities (44). Further research on the differences in biological behavior among variants of the EML4-ALK fusion gene is required (45).

Clinical features of EML4-ALK in NSCLC
Lung cancer is classified into two histological groups: SCLC (~17.3%) and NSCLC (~82.7%) (46)(47)(48), of which lung adenocarcinoma and lung squamous cell carcinoma are the major subgroups (48). Since the EML4-ALK fusion gene was discovered in lung adenocarcinoma in 2007, it has been considered to be a characteristic gene of lung cancer; however, in recent years, it has gradually been detected in other types of cancer, such as thyroid cancer, gastric stromal tumors and leiomyoma (11,(49)(50)(51). According to a previous study, the oncogenic fusion of EML4-ALK is present in 3-5% of NSCLC (27). One study found that EML4-ALK fusion gene positivity occurred mostly in female patients with NSCLC who did not smoke or smoked infrequently (52), and the positive detection rate was higher in patients with NSCLC without an epidermal growth factor receptor (EGFR) or KRAS gene mutation (53). Male patients with lung cancer with a long-term history of smoking exhibited a particularly low detection rate for the EML4-ALK fusion gene (54). At present, the EML4-ALK fusion gene is a routine gene mutation test for patients with NSCLC.

EML4-ALK detection method
At present, the commonly used EML4-ALK fusion gene detection methods in clinical diagnosis and treatment, as well  as laboratory research, mainly include immunohistochemistry (IHC), FISH, reverse transcription PCR (RT-PCR) and NGS (55). IHC is the simplest, cheapest and most commonly used detection method, and is widely used in hospitals and laboratories (56). IHC uses antigen-antibody reactions to detect whether the EML4-ALK fusion protein is produced in tumor tissues (56). However, due to the low expression level of the EML4-ALK fusion protein in NSCLC lung tissue, this method has low sensitivity and cannot distinguish between fusion types (57).
FISH is a relatively specific and sensitive method that uses fluorescence-labeled specific nucleic acid probes to hybridize with targeted DNA or RNA in cells to generate fluorescent signals (58,59). EML4-ALK fusion gene detection is performed through the fluorescent labeling of EML4 and ALK, and subsequent observation of the positional relationship of the two fluorescent signals to determine whether the chromosome is translocated, so as to determine whether the EML4-ALK fusion gene exists in the tumor tissue; however, this method also fails to distinguish between fusion variant types (60). RT-PCR can distinguish between different types of EML4-ALK fusion genes by designing primers for different fusion variants (60). RT-PCR is characterized by rapid diagnosis and high sensitivity. The quality requirement for the extracted RNA and the positive detection rate of EML4-ALK for fresh tumor specimens are high, but most tumor specimens are fixed in neutral formaldehyde, resulting in RNA degradation and reduced sensitivity (60,61). NGS has revolutionized traditional sequencing, as it can sequence hundreds of thousands to millions of DNA molecules at once, which renders the detailed and comprehensive analysis of the transcriptome and genome of a species possible (62). NGS has a high degree of specificity and sensitivity, and can detect various known and unknown fusion gene types, but the procedure is complicated, the technical difficulty is high and the detection standards are not uniform (63,64) (Table I).

Targeted therapy for EML4-ALK
In recent years, breakthroughs have been made in targeted therapy technology, and a variety of targeted therapy drugs for EML4-ALK have been developed (65). Crizotinib, approved for marketing in 2011, was the first drug to target the EML4-ALK fusion gene (66). Crizotinib is an orally active aminopyridine-derived small molecule competitive inhibitor (10). The study showed that, in patients with advanced EML4-ALK fusion gene-positive NSCLC, the objective response rate (ORR; 53%) and progression-free survival (PFS) time (8.5 months) of patients receiving crizotinib were significantly higher than those of patients receiving standard platinum-based chemotherapy (67,68). The results showed that targeted therapy with crizotinib was more effective than traditional standard chemotherapy and did not increase the number of serious adverse reactions (69,70). However, when used as a first-line treatment regimen, resistance to crizotinib often develops at varying degrees within 1 year of treatment (71). As a means to overcome resistance to crizotinib, second-generation EML4-ALK-targeted drugs, such as ceritinib (72), brigatinib (73) and alectinib (74), as well as the third-generation targeted drug lorlatinib, have been developed (75), and the fourth-generation targeted drug repotrectinib (TPX-0005) has been undergoing phase I/II clinical trials (76,77). The American Society of Clinical Oncology performed a phase II study on the efficacy of a new generation of targeted drugs in patients with ALK rearrangement-positive advanced NSCLC who progressed after EML4-ALK targeted therapy. The results showed that the new generation of targeted drugs could significantly improve the ORR (77.8%) and PFS time (10.7 months) of patients (78), and at the same time exhibit good efficacy in patients with intracranial metastasis or in other NSCLC patients with mutations in genes such as ROS1 (10,79,80). However, following the long-term use of targeted therapy, acquired resistance inevitably occurs, which affects the therapeutic effect (81). There is currently evidence that different EML4-ALK fusion gene variants have varying degrees of sensitivity to targeted drugs in NSCLC (39). A previous study analyzed 77 tumor biopsies from patients with EML4-ALK V1 and EML4-ALK V3 fusion genes and found that resistance mutations were more common in V3 than in V1 (57 vs. 30%; P=0.023) (82). Therefore, variant typing of the EML4-ALK fusion gene is necessary.
The acquired resistance mechanisms of EML4-ALK discovered in the present study mainly included the following: i) Secondary mutation of the kinase domain (83); the secondary gene mutation in the ALK kinase domain leads to a change in the spatial conformation of the binding region of the kinase and the drug, which increases the binding force of the kinase and ATP, thereby affecting the binding of the drug and the kinase, leading to drug resistance (84). A gene mutation was detected in ~30% of patients with resistance to a first-generation targeted drug, resulting in a point mutation of a glycine residue located in the ATP-binding region to valine. The mutation rate following second-generation drug resistance exceeded 50%, resulting in the mutation of glycine residues to arginine (84). ii) Activation of alternative signaling pathways (85); when the ALK signaling pathway is inhibited by targeted drugs, other tumor-promoting signaling pathway proteins, such as EGFR and KIT, are abnormally activated and continue to promote tumor cell proliferation (86). iii) Epithelial-mesenchymal transition; the transformation of tumor epithelial cells to mesenchymal cells increases the ability of tumor cells to invade and metastasize (87). In patients with NSCLC EML4-ALK-targeted drug resistance, the expression of the mesenchymal marker vimentin was increased, and that of the epithelial marker E-cadherin was decreased, suggesting that epithelial-mesenchymal transition may be involved in the drug resistance response (88)(89)(90). In order to overcome the drug resistance of tumor cells, it is currently possible to strengthen the combination of EML4-ALK-targeted and other antitumor drugs, such as the EGFR inhibitor erlotinib, cyclin-dependent kinase inhibitor, and riboxy and heat shock protein 90 inhibitors (91)(92)(93). Combined use of these drugs can synergistically enhance antitumor activity and inhibit ALK kinase activity (94).

Summary and outlook
The EML4-ALK fusion gene is one of the important tumor driver genes discovered in NSCLC in recent years, and it is an important molecular target affecting the diagnosis and treatment of NSCLC. In particular, the detection rate of the EML4-ALK fusion gene is higher in patients with NSCLC who are young, non-smoking, females without EGFR and other gene mutations. Although the current detection technologies have their own shortcomings, they can meet the needs of current clinical diagnosis and treatment. Most patients with NSCLC with EML4-ALK fusion gene positivity can benefit significantly from molecular targeted therapy, but drug resistance is an important factor that plagues current targeted therapy. It is believed that with the successful development of a new generation of EML4-ALK-targeted drugs and the elucidation of the drug resistance mechanism, the survival of patients with NSCLC with EML4-ALK fusion gene mutation will be further improved.

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Authors' contributions
YuL, YaL and JJW designed the theme of the review. YuL, YaL, XS and JJW retrieved the relevant literature. XS wrote and reviewed the article. Data authentication is not applicable. All authors read and approved the final manuscript.

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