Abstract SY14-04: Discovery and clinical application of EML4-ALK oncogene in lung cancer
Authors: Mano Hiroyuki 
Published: May 2019

Abstract
Lung cancer remains the leading cause of cancer deaths worldwide. Conventional chemotherapeutic regimens improve only marginally the outcome of individuals with non-small-cell lung cancer (NSCLC), resulting in a median survival time of &lt;1 year. In order to discover novel transforming genes in lung cancer, we have developed a sensitive retroviral expression library system for full-length cDNAs, and applied this technology to an NSCLC specimen without EGFR mutations. A 3T3 fibroblast-focus formation assay with the library led to the discovery of a novel fusion gene between the echinoderm microtubule-associated protein-like 4 (EML4) and the anaplastic lymphoma kinase (ALK) genes. Oncogenic activity of EML4-ALK requires the N-terminal coiled-coil domain within EML4 that leads to the constitutive dimerization and, thereby, activation of the fusion protein (Soda et al. Nature 448:561).
Since both of the EML4 and ALK genes are closely mapped in an opposite direction to the same short arm of human chromosome 2, a small chromosome inversion involving the two genes is likely to be the underlining mechanism for the generation of the gene fusion, which was indeed evidenced by both of FISH and genomic PCR analyses. As in the case for BCR-ABL, PCR-based detection of the EML4-ALK fusion points should be a highly accurate and sensitive diagnostic tool for NSCLC harboring the fusion gene. As expected, RT-PCR readily detected the fusion cDNA even from sputum containing 10 cells/mL of EML4-ALK-positive cells. It should be noted, however, that dozens of variants may be present for EML4-ALK in NSCLC, in which various introns of EML4 is used for the connection to ALK (Mano. Cancer Sci 99:2349, Horn et al. J Clin Oncol 27:4232). Hence, to detect reliably NSCLC with all these variants, multiplex RT-PCR systems should be used in clinical settings, which can capture all in-frame fusions of the two genes (Choi et al. Cancer Res 68:4971, Takeuchi et al. Clin Cancer Res 14:6618, Takeuchi et al. Clin Cancer Res 15:3143).
In order to prove the essential role of EML4-ALK in the carcinogenesis of NSCLC, we generated transgenic mice expressing the fusion gene in lung epithelial cells. Interestingly, such mice developed hundreds of adenocarcinoma nodules in both lungs only at a few weeks after birth (Soda et al. PNAS 105:19893). Further, oral administration of a specific inhibitor to ALK successfully cleared such nodules from the mice, proving in vivo that EML4-ALK is the primary genetic event to generate NSCLC with the fusion kinase, and, therefore, specific inhibitors against EML4-ALK could be an effective molecular targeted therapy for the fusion-positive NSCLC.
A specific inhibitor to the ALK enzymatic activity (PF-02341066) is already under clinical trials as reported in the 2009 annual meeting of America Society of Clinical Oncology (abstract #3509) (Kwak et al. J Clin Oncol 27:15s suppl; abstr 3509) and of European Cancer Organization and Congress of the European Society for Medical Oncology (abstract #G6). Knowing the marked therapeutic efficacy of the compound, we tried to develop a reliable strategy to diagnose NSCLC with EML4-ALK.
Immunohistochemistry (IHC)-based detection of EML4-ALK is highly difficult, probably, owing to the weak activity of the EML4 promoter that drives the expression of EML4-ALK messages. We had thus examined a suitability of commercially available antibodies to ALK for IHC, and successfully developed the intercalated antibody-enhanced polymer (iAEP) method that enables a reliable detection of EML4-ALK among formalin-fixed and paraffin-embedded specimens (Takeuchi et al. Clin Cancer Res 15:3143). The screening results with iAEP and our multiplex RT-PCR were almost 100% concordant between each other, with only a few exceptional cases. From such disconcordant specimens (IHC-positive but RT-PCR-negative), we further discovered a novel fusion of ALK in NSCLC, that is KIF5B-ALK.
Using a combination of multiplex RT-PCR and iAEP, we constructed in March 2009 a Japanese nation-wide diagnostic network for NSCLC with ALK-fusions. This “ALK-Lung Cancer Study Group (ALCAS)” initiative has already collected hundreds of NSCLC specimens, and successfully served as a screening system to select individuals to be treated with ALK inhibitors.
These data demonstrate that a subset of lung cancer express previously unidentified fusion kinases that are promising candidates for a therapeutic target as well as for a diagnostic molecular marker for this intractable disorder.
Citation Format: Hiroyuki Mano. Discovery and clinical application of EML4-ALK oncogene in lung cancer [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY14-04