Characteristics of Lung Cancers Harboring NRAS Mutations
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Published OnlineFirst March 20, 2013; DOI: 10.1158/1078-0432.CCR-12-3173 Clinical Cancer Predictive Biomarkers and Personalized Medicine Research Characteristics of Lung Cancers Harboring NRAS Mutations Kadoaki Ohashi1, Lecia V. Sequist6,7, Maria E. Arcila8, Christine M. Lovly1, Xi Chen2, Charles M. Rudin10, Teresa Moran11, David Ross Camidge12, Cindy L. Vnencak-Jones4, Lynne Berry3, Yumei Pan1, Hidefumi Sasaki13, Jeffrey A. Engelman6,7, Edward B. Garon14, Steven M. Dubinett14, Wilbur A. Franklin12, Gregory J. Riely9, Martin L. Sos15,16,17, Mark G. Kris9, Dora Dias-Santagata5, Marc Ladanyi8, Paul A. Bunn Jr12, and William Pao1 Abstract Purpose: We sought to determine the frequency and clinical characteristics of patients with lung cancer harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS-mutant lung cancer cells. Experimental Design: We reviewed clinical data from patients whose lung cancers were identified at six institutions or reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) to harbor NRAS mutations. Six NRAS-mutant cell lines were screened for sensitivity against inhibitors of multiple kinases (i.e., EGFR, ALK, MET, IGF-1R, BRAF, PI3K, and MEK). Results: Among 4,562 patients with lung cancers tested, NRAS mutations were present in 30 (0.7%; 95% confidence interval, 0.45%–0.94%); 28 of these had no other driver mutations. 83% had adenocarcinoma histology with no significant differences in gender. While 95% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS-mutated lung tumors than KRAS-mutant non–small cell lung cancer [NSCLC; NRAS: 13% (4/30), KRAS: 66% (1772/2733), P < 0.00000001]. Five of 6 NRAS-mutant cell lines were sensitive to the MEK inhibitors, selumetinib and trametinib, but not to other inhibitors tested. Conclusion: NRAS mutations define a distinct subset of lung cancers (1%) with potential sensitivity to MEK inhibitors. Although NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking. Clin Cancer Res; 19(9); 1–8. Ó2013 AACR. Introduction Authors' Affiliations: 1Vanderbilt-Ingram Cancer Center, the Department Recent advances have been made in targeting molecularly of Medicine/Division of Hematology-Oncology, 2Department of Biostatis- tics/Division of Cancer Biostatistics, 3Center for Quantitative Science, defined subsets of non-small cell lung cancers (NSCLC) that Vanderbilt University School of Medicine; 4Departments of Pathology, depend on specific molecular alterations for cell survival. Microbiology and Immunology, Vanderbilt University Medical Center, Prime examples include tumors which harbor mutations in Nashville, Tennessee; 5Department of Pathology, 6Massachusetts General Hospital Cancer Center; 7Harvard Medical School, Boston, Massachu- the gene encoding the EGF receptor (EGFR) or transloca- setts; 8Department of Pathology, 9Thoracic Oncology Service, Division of tions in the gene encoding the anaplastic lymphoma kinase Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering ALK Cancer Center, New York, New York; 10Johns Hopkins University, Balti- ( ). Patients with these tumors can derive substantial more, Maryland; 11Catalan Institute of Oncology, Universitat Autonoma clinical benefit from EGFR (gefitinib, erlotinib) or ALK de Barcelona and Hospital Universitari Germans Trias i Pujol, Barcelona, (crizotinib) tyrosine kinase inhibitors (TKI), respectively Spain; 12University of Colorado Cancer Center, Aurora, Colorado; 13Department of Surgery II, Nagoya City University Medical School, (1–8). Nagoya, Japan; 14Translational Oncology Research Laboratory, Hematol- To date, many other potential "driver mutations" occur- ogy/Oncology Division, David Geffen School of Medicine at UCLA, Santa ring in genes encoding cellular signaling proteins have also Monica; 15Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, Califor- been identified in NSCLCs. Genomic alterations include nia; 16Max Planck Institute for Neurological Research with Klaus Joachim mutations in the GTPase KRAS (25%; refs. 9, 10), the Zulch€ Laboratories of the Max Planck Society and the Medical Faculty; and ERBB2 17Department I of Internal Medicine and Center of Integrated Oncology receptor tyrosine kinase (2%–3%; refs. 11, 12), the Koln€ – Bonn, University of Cologne, Cologne, Germany lipid kinase PIK3CA (2%–4%; refs. 10, 13, 14), the serine– BRAF Note: Supplementary data for this article are available at Clinical Cancer threonine kinase (2%–4%; refs. 9, 10, 15), and the Research Online (http://clincancerres.aacrjournals.org/). serine–threonine kinase MEK1 (1%; ref. 16), as well as ROS1 Corresponding Author: William Pao, Vanderbilt-Ingram Cancer Center, translocations in the tyrosine kinases (1%–2%; 2220 Pierce Ave 777 PRB, Nashville, TN 37232. Phone: 615-343-9454; Fax: refs. 17–19) and RET (1%; refs. 19–21). A tumor with a 615-343-7602; E-mail: [email protected] mutation in one of these genes rarely harbors a mutation in doi: 10.1158/1078-0432.CCR-12-3173 another (22). Although targeted therapies have not yet been Ó2013 American Association for Cancer Research. approved for all of these molecular subsets of lung cancer, www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst March 20, 2013; DOI: 10.1158/1078-0432.CCR-12-3173 Ohashi et al. and clinical stage were recorded. All chart review/tissue Translational Relevance collection was carried out under institutional review Recent advances in lung cancer biology and molecular board/privacy board–approved protocols or waivers. tumor profiling have allowed for rational prioritization of targeted therapies in patients. NRAS mutations have Genotype analysis been reported to occur in lung cancers, but as yet, no gDNA was extracted from patient samples (>70% comprehensive report has focused on the characteristics tumor cells) and cell lines using standard procedures. of patients whose tumors harbor NRAS mutations. Here, Tumor specimens were obtained as standard of care for we describe clinical characteristics associated with 30 clinical management or with patients’ consent under unique patients with NRAS-mutated lung cancers Institutional Review Board–approved protocols. A mass among 4,562 patients tested (0.7%). While 95% of spectrometry–based (Sequenom; ref. 22) or SNapShot patients were former or current smokers, smoking-relat- assay (32, 33) was conducted for genotyping as described. ed G:C>T:A transversions were significantly less frequent Cell lines were genotyped using SNapShot and/or direct in NRAS-mutated lung tumors than in KRAS-mutant sequencing. non–small cell lung cancer (NSCLC). NRAS mutations were, for the most part, mutually exclusive with other Statistical analysis known driver mutations, suggesting that NRAS muta- Fisher’s exact tests (for small sample size) were applied to tions define a distinct molecular subset. In preclinical test associations among NRAS mutations, smoking history, models, 5 of 6 NRAS-mutant NSCLC cell lines were and race. The c2 tests were applied to compare the frequency sensitive to MEK inhibitors. Our data suggest the possi- of transversions in KRAS- versus NRAS-mutant cancers. bility of personalized treatment in this subset of lung cancers. Cell culture H1299, H2347, H2087, and SW1271 were purchased from American Type Culture Collection. HCC15 were obtained as described before (34). HCC1195 was kindly preclinical and emerging clinical data suggest that molec- provided by Dr. Roman Thomas. H1299, H2347, HCC15, ular subtyping will allow for the rational prioritization of and HCC1195 cells were cultured in RPMI-1640 media treatment options for patients with lung cancer (23). (Mediatech) supplemented with 10% heat-inactivated FBS NRAS KRAS is a GTPase related to , originally identified in (Atlanta Bio) and pen–strep solution (Mediatech; final neuroblastoma cell lines as a third RAS family member concentration 100 U/mL penicillin, 100 mg/mL streptomy- KRAS HRAS following and (24). RAS GTPases regulate cell cin). H2087 and SW1271 cells were cultured in Dulbeccos’ growth, proliferation, and differentiation. Although the 3 Modified Eagle’s Media (DMEM; Mediatech) with the same family members share conserved sequences, their protein supplements. Cells were grown in a humidified incubator products generate distinct signal outputs (25, 26) and have with 5% CO2 at 37C. distinct roles in development (27, 28) and tumorigenesis in mice (29, 30). Growth inhibition assay NRAS mutations have been reported to occur in lung Cells were seeded in 96-well plates at a density of 500 to cancers (31), but as yet, no comprehensive report has 5,000 cells per well and exposed to drugs alone or in focused on the characteristics of patients whose tumors combination the following day. At 120 hours after drug NRAS harbor mutations. Here, we used retrospective clin- addition, Cell Titer Blue Reagent (Promega) was added, and ical data as well as preclinical models to define the clinical fluorescence was measured on a Spectramax spectropho- NRAS relevance of mutations in lung cancer. tometer (Molecular Devices), according to the manufac- turer’s instructions. All experimental points were set up in Materials and Methods hextuplicate replicates and were conducted at least