GSK2292767 ALK fusion positive NSCLC is clinically

ALK fusion-positive NSCLC is clinically actionable because it can be targeted by several FDA-approved drugs, including the first generation TKI crizotinib, which is a dual inhibitor to MET and ALK [15], and the second generation inhibitors, alectinib and ceritinib, both of which are highly-selective ALK inhibitors. The latter two were not only approved as the first line treatment for ALK-positive NSCLC [16], [17], but also in treating crizotinib-resistant or GSK2292767 metastatic NSCLC [18], [19]. Nowadays high-throughput sequencing technology increases the chance of identifying new fusion genes that have activities similar to classical ALK fusions [20], [21], potentially matching more patients to existing drugs. Here, we identified a rare ALK fusion partner, GCC2 (GRIP and coiled-coil domain-containing protein 2), in a patient with NSCLC by targeted next generation sequencing (NGS). Our in vitro functional study and the clinical observations of the patient proved that GCC2-ALK has similar kinase activities as EML4-ALK, and that the patient with this variant can be treated with existing ALK inhibitors.
Materials and methods
Results
Discussion The ALK receptor tyrosine kinase undergoes genetic rearrangements in a variety of human cancers, including anaplastic large-cell lymphoma [32], diffuse large B-cell lymphoma [33], colon, breast and lung cancers [34]. ALK translocations drive tumorigenesis mainly by enabling constitutive, ligand-independent activation of the ALK kinase. The fusion of EML4-ALK is the most common ALK fusion in NSCLCs [35]. Given the well-established transforming activities of EML4-ALK and its sensitivity to crizotinib [36], attempts to identify more partner genes to ALK that have the same effects in tumorigenesis and clinical responses to ALK inhibitors have recently been made [37], [38]. In this study, we identified a rare GCC2-ALK fusion (G13:A20) in NSCLC by targeted NGS. A similar GCC2-ALK fusion (G12:A20) has been reported recently in one of the 158 ALK-positive patients in a study of 3000 lung cancer patients from Korea [6] without any functional and clinical follow-ups. Herein, we found that introduction of GCC2-ALK (G13:A20) into Ba/F3 cells enabled their IL-3-independent cell growth that can be suppressed by crizotinib. Molecular pathway studies in both HEK-293 and 293T cells uncovered the enhanced ALK kinase activity of GCC2-ALK and the excess downstream RAS-MAPK, JAK-STAT and PI3K-AKT signals. In the absence of direct ligand binding, GCC2 is proposed to facilitate the oligomerization of the ALK kinase domain through its tandem coiled-coil domains similar to other fusion partners [39], and therefore leads to the constitutive activation of the ALK kinase and its downstream signaling pathways. As reported, resistant mechanisms to crizotinib include secondary ALK mutations L1196M and G1269A, ALK amplification, cKIT amplification, EGFR or KRAS activation [40], [41], [42], [43]. In this patient, we have not identified any newly acquired mutations after drug resistance except ALK E1407K at a MAF of 1%. However, it is unlikely that this mutation will influence the ALK activity or crizotinib binding since the mutation site is located outside of the kinase domain. We also observed a dramatic increase of GCC2-ALK MAF from 5% in the primary tumor to 20% in the recurrent lesion, and the new lesion responded well to the second generation of ALK inhibitor ceritinib, suggesting that the relapsed tumor is still ALK-driven. However, the mechanism for crizotinib-resistance in this patient is still unknown. In summary, our study identified a targetable ALK fusion, GCC2-ALK, in an advance NSCLC patient. In vitro functional studies and clinical observations of the patient consistently show that GCC2-ALK is constitutively activated and can be inhibited by crizotinib, suggesting the benefit of including it into clinical practice for diagnosis and targeted treatment.