HUMAN MUTATION Mutation in Brief #923 (2006) Online MUTATION IN BRIEF Somatic Mutations of GUCY2F, EPHA3, and NTRK3 in Human Cancers Laura D. Wood1, Eric S. Calhoun1, Natalie Silliman1, Janine Ptak1, Steve Szabo1, Steve M. Powell2, Gregory J. Riggins1,3, Tian-Li Wang1, Hai Yan4, Adi Gazdar5, Scott E. Kern1, Len Pennacchio6, Kenneth W. Kinzler1, Bert Vogelstein1, and Victor E. Velculescu1* 1The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute at the Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland; 2Divison of Gastroenterology/Hepatology, University of Virginia Health System, Charlottesville, Virginia; 3Department of Neurosurgery, Johns Hopkins Medical Institutions, Baltimore, Maryland; 4Department of Pathology, Duke University Medical Center, Durham, North Carolina; 5Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; 6U.S. Department of Energy Joint Genome Institute, Walnut Creek, California *Correspondence to: Victor E. Velculescu, M.D., Ph.D., The Ludwig Center for Cancer Genetics and Therapeutics at the Johns Hopkins Kimmel Cancer Center, 1650 Orleans Street, Room 5M05, Baltimore, Maryland 21231; E- mail: [email protected] Communicated by Richard Wooster Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high- throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients. Published 2006 Wiley- Liss, Inc. KEY WORDS: tyrosine kinases; human cancer; genetic mutation; GUCY2F; EPHA3; NTKR3 INTRODUCTION Tyrosine kinases are important regulators of signal transduction pathways, including those important for cell growth and apoptosis. Several studies have systematically evaluated the sequences of protein kinase genes in distinct types of human tumors (Bardelli, et al., 2003; Davies, et al., 2005; Parsons, et al., 2005; Stephens, et al., 2005). In our previous study of tyrosine kinase genes in colorectal tumors, we identified mutations in a small subset of genes in this family, including the EPHA3, FES, GUCY2F, KDR, MLK4, NTRK2, and NTRK3 genes (Bardelli, et al., 2003). In the current study, we further analyzed this subset of genes in other tumor types and identified somatic mutations in three different genes in breast, lung, and pancreatic cancers. Received 12 April 2005; accepted revised manuscript 23 May 2006. PUBLISHED 2006 WILEY-LISS, INC. DOI: 10.1002/humu.9452 2 Wood et al. METHODS Primers for polymerase chain reaction (PCR) and sequencing reactions were synthesized by MWG (High Point, NC, USA; http://www.mwg-biotech.com) and were identical to those previously described (Bardelli, et al., 2003). All exons encoding the kinase domains of EPHA3 (MIM# 179611; GenBank Accession NM_005233.3), FES (MIM# 190030; GenBank Accession NM_002005.2), KDR (MIM# 191306; NM_002253.1), MLK4 (GenBank Accession NM_032435.1), NTRK2 (MIM# 600456; GenBank Accession NM_006180.3), and NTRK3 (MIM# 191316; GenBank Accession NM_001012338.1) were PCR amplified and directly sequenced in a panel of 94 tumors comprising 23 lung carcinomas, 11 breast carcinomas, 12 pancreatic carcinomas, 12 gastric carcinomas, 12 ovarian carcinomas, 12 glioblastomas, and 12 medulloblastomas (Supplementary Tables 1 and 2; see Appendix). For GUCY2F (MIM# 300041; GenBank Accession NM_001522.1), all exons (including those not in the kinase domain) were sequenced because the kinase domain of this gene is thought to be inactive (Lucas, et al., 2000) and previous analyses identified mutations throughout the gene sequence (Bardelli, et al., 2003). All tumor samples were obtained in accordance with the Health Insurance Portability and Accountability Act regulations. PCR amplification and sequencing reactions were performed using 384 capillary automated sequencing apparatuses (Spectrumedix, State College, PA, USA; http://www.spectrumedix.com) as previously described (Wang, et al., 2002). In all cases, sequence traces were assembled and analyzed to identify potential somatic mutations using the Mutation Surveyor software package (SoftGenetics, State College, PA, USA; http://www.softgenetics.com). Of the 71 exons sequenced, 94% were successfully analyzed. Any exon with a potential somatic mutation was independently amplified and sequenced in the corresponding tumor and normal DNA to confirm the observed alteration. Statistical evaluation of mutation frequencies was performed using TRAB (Wang, et al., 2002) (http://astor.som.jhmi.edu/~gp/software/trab/). To assess evolutionary conservation of mutated residues, human protein sequences were compared to those of other available species. For GUCY2F these included sequences from cow, mouse, and rat; for EPHA3 these included sequences from chicken, mouse, and rat; and for NTRK3 these included sequences from chicken, mouse, rat, pig, macaque, and chimpanzee. Sequences were aligned using the Clustal Method (DNASTAR, Inc., Madison,WI, USA; http://www.dnastar.com) and identical residues were considered conserved. RESULTS Analysis of the seven tyrosine kinase genes in 94 human tumors identified a total of 6 nonsynonymous somatic mutations: two missense changes in EPHA3, both in lung tumors, and 4 missense changes in GUCY2F, affecting two pancreatic tumors, one lung tumor, and one breast tumor (Table 1, examples in Fig. 1). One synonymous somatic mutation in EPHA3 was also detected in a lung tumor. Because of the high frequency of GUCY2F mutations in the 12 pancreatic tumors examined and because of the previously identified importance of NTRK3 in pancreatic cancer (Miknyoczki, et al., 1999a; Miknyoczki, et al., 1999b), we further analyzed the sequence of these two genes in 48 additional pancreatic tumors (Supplementary Table 3). These analyses identified one nonsynonymous somatic mutation in NTRK3 and no additional changes in GUCY2F (Table 1, Fig. 1). Table 1. Somatic mutations detected in GUCY2F, EPHA3, and NTRK3 Tumor type and Tumor type and NCBI Tumor Mutation Gene name Other names fraction affected fraction affected accession sample (nucleotide and amino acid) (this study) (other studies) pancreas (2/60) Px147 c.2015A>C (p.Lys672Thr) GUC2DL, GC-F, Px117 c.3187A>C (p.Lys1063Gln) colon (10/182), lung GUCY2F RetGC-2, ROS-GC2, NM_001522.1 lung (1/23) NCI-H2009 c.3155A>G (p.Lys1052Arg)‡ (3/89), breast (1/81) CYGF breast (1/11) HCC2157 c.29G>C (p.Arg10Pro)*‡ lung (3/23) HCC15 c.1979C>A (p.Thr660Lys) ETK, ETK1, TYRO4, colon (2/182), EPHA3 NM_005233.3 HCC515 c.2798C>T (p.Thr933Met)* HEK, HEK4 lung (4/89) HCC515 c.2832C>A (p.Ala944Ala)* colon (6/182), lung NTRK3 TRKC NM_002530.2 pancreas (1/60) Px21 c.1795C>T (p.His599Tyr)* (2/89), breast (1/81) For each tumor type, the fraction of samples with somatic mutations is indicated. The nucleotide position of each mutation corresponds to the position of that change in the coding sequence of each gene, where position 1 is the A of the ATG. Mutations indicated by a star are homozygous, presumably due to loss of heterozygosity (LOH) of the wild-type allele in the tumor. All other mutations are heterozygous. All mutations affected evolutionarily conserved residues except the G to C change in GUCY2F at nucleotide position 29. Mutations described in previous studies include the following reports: colon cancer (Bardelli et al., 2003), breast cancer (Stephens et al., 2005), and lung cancer (Davies et al., 2005). The mutations indicated by a "‡" in GUCY2F in breast and lung tumors were identified in the same samples previously described in the Stephens et al. and Davies et al. studies, respectively. NTRK3, EPHA3, and GUCY2F Mutations in Cancer 3 GUCY2F EPHA3 NTRK3 Normal C Tumor c.2015A>C c.2798C>T c.1795C>T (p.Lys672Thr) (p.Thr933Met) (p.His599Tyr) Figure 1. Examples of somatic mutations in GUCY2F, EPHA3, and NTRK3. Representative examples of mutations in the three genes. In each case the bottom sequence chromatogram was obtained from tumor DNA, and the top chromatogram was obtained from normal DNA from the same patient. Arrows indicate the location of missense somatic mutations, and the nucleotide and amino acid alterations are indicated below the traces. DISCUSSION One of the questions that can be addressed by studies such as the one described above is whether certain genes play a role in specific tumor types or are more generally important in tumorigenesis. The results presented here suggest that some tyrosine kinases may play unique roles in specific tumors. For example, while MLK4 is mutated in >5% of colorectal tumors (Bardelli, et al., 2003), no mutations were detected in MLK4 in any of seven different tumor types examined in this study (p<0.05, chi-square test). Additionally, no mutations in MLK4 were identified in independent studies analyzing protein kinases in lung, breast, and testicular tumors (Davies, et al., 2005; Stephens, et al., 2005; Bignell, et al., 2006). Thus, while MLK4 may play a role in colorectal tumorigenesis,