Comprehensive Genomic Analysis Identifies Frequent Met Juxtamembrane Domain Deletions As An Actionable Genomic Alteration In Pulmonary Sacromatoid Carcinoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PABackground:Pulmonary sarcomatoid carcinoma (SC) is a rare and highly aggressive lung tumor, and it is associated with poor prognosis and high rate of resistance to conventional chemotherapy. We performed whole-exome next generation sequencing followed by a careful ranking and validation strategy to understand its unique differentiation pattern and to identify actionable targets.Methods:Twenty-nine patients with pulmonary SC diagnosed at our institution between 1998 and 2013 were enrolled and divided into a discovery set (n = 10) and a validation set (n = 19). Whole-exome sequencing was performed on genomic DNA samples extracted from fresh frozen tumor tissue and paired normal tissue in the discovery set. TruSeq Amplicon-Cancer 48 gene panel and bidirectional Sanger sequencing was performed to validate mutations. Validated genes were then screened for mutations using Sanger sequencing on FFPE-DNA of the patients in the validation set. Total RNA and protein were extracted from fresh frozen tumor tissues and paired normal tissues when available. RT-PCR and Western blotting were performed to validate MET juxtamembrane domain deletion. Ablation of MET signaling by siRNA or pharmacological inhibitors was conducted in cell line models harboring MET exon 14 skipping, and cell line models harboring wild-type MET. MTS assay and western blotting were then performed to assess cell proliferation and MET downstream signaling changes.Results:Previously reported mutations in TP53 (7/10) KRAS (2/10) and PIK3CA (2/10), as well as novel mutations in RASA1(2/10), CDH4 (2/10), CDH7 (2/10), LAMB4 (3/10), MET (2/10), SCAF1 (2/10), and LMTK2 (2/10), were detected and validated in the discovery set. CDH4, CDH7, SCAF1, LMTK2, and MET were screened in the validation set, and two SCAF1 mutations and five MET mutations were identified. In total, 24% (7/29) of pulmonary SC harbor somatic mutations in the splice sites of MET exon 14, including five D1010H point mutations in the 3p splice acceptor site. RT-PCR and Western blotting confirmed the presence of exon 14 skipped MET variant. Inactivation of MET using the MET inhibitor Crizotinib decreased cell proliferation and inhibited downstream AKT and mitogen-activated protein kinase activation in cell lines harboring MET exon14 skipping (H596 lung adenocarcinoma and Hs746T gastric cancer), whereas effects of MET inhibition on cell proliferation and downstream protein activation were negligible in MET wild-type cell lines. Further functional studies utilizing specific siRNA and MET exon 14 expression constructs are ongoing.Conclusions:Our study finds an unprecedently high frequency of exon 14 skipping MET mutations in pulmonary SC and suggests that MET activation might contribute to the mesenchymal differentiation and aggressive biology and defines MET inhibition as a promising novel therapeutic strategy for MET-mutated pulmonary SC.Citation Format: Xuewen Liu, Yuxia Jia, Yufeng Shen, Haiying Cheng, Sanjay Koul, Alain C. Borczuk, Balazs Halmos. Comprehensive genomic analysis identifies frequent MET juxtamembrane domain deletions as an actionable genomic alteration in pulmonary sacromatoid carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 696. doi:10.1158/1538-7445.AM2015-696