Trans-national patterns of pancreas cancer ras mutations and discovery of a new ras mutation with oncogenic synergy when found with ras codon 12 mutations
Authors: Zhimin Guo, Yingnian Lu, Sarah Oakes, Amber Britton, Thomas King, Kai-wen Huang, Vesela Tzenova, John Ferraro, David Apelian, Alex Franzusoff
Journal: Clinical Cancer Research (2008)

B35    Background : K-Ras oncoproteins with mutations at amino acids 12, 13, or 61 drive uncontrolled cell proliferation and tumorigenesis and a high frequency of K- ras  codon 12 mutations in pancreas cancer (>80%) is reported. We have developed yeast-based immunotherapy targeting Ras point mutations (GI-4000 tarmogens) and we analyzed the incidence of specific  ras  point mutations among patients with pancreas cancer in Bulgaria, India, Taiwan and the US.   Experimental procedures : Tumor biopsies were obtained from 172 patients in Bulgaria, India, Taiwan and the US. DNA sequences were characterized for tumor-associated mutations in K- ras  exons 2 and 3 by nested PCR amplification including peptide-nucleic acid oligomer clamping, followed by DNA sequencing. Single G12V, Q61R, E76G or E76K mutations or double G12V+E76G, Q61R+E76G mutations were encoded into the mouse K- ras  gene then transfected into BALB/3T3 fibroblast cells. The transfected cells were seeded for growth in soft agar or implanted into BALBc athymic nude mice for tests of tumorigenesis. The intracellular content of GTP-bound Ras protein, a marker of Ras activation, was assayed with cells transfected to express extra copies of wild type Ras, or Ras with single or double mutations.   Results : Notable differences from previously reported patterns included i) a different profile of  ras  point mutation frequencies found in pancreas cancer in different countries; ii) regional differences in frequency and type of  ras  mutations within a single country; iii) the unanticipated frequency of amino acid 61 Ras mutations (Ras Q61H); and iv) the discovery of a new Ras mutation at amino acid 76 (Ras E76G or E76K) that was detected in patient tumors as a single mutation or simultaneously with amino acid 12 Ras mutations (G12).  The role of Ras oncoprotein harboring E76 mutations alone or in combination with G12 mutations in tumorigenesis was investigated in preclinical studies. Cells transfected with K- ras  encoding single G12V, Q61R or E76K mutations or double G12V+E76G, Q61R+E76G or Q61R+E76K mutations all exhibited the transformed phenotype by forming colonies in soft agar. Cells expressing singly mutated Ras had increased intracellular levels of GTP-bound Ras protein compared to overexpression of wild type Ras protein and GTP-bound Ras levels was further amplified in cells expressing Ras with double G12+E76 mutations. To confirm tumorigenesis  in vivo , tumors stemming from double G12V+E76G mutations implanted into BALBc mice showed more aggressive growth rates compared to those expressing any single mutant Ras.   Conclusions:  Tumor genotyping reveals differences in trans-national and regional incidence of pancreas cancer-associated  ras  mutations and led to discovery of Ras-E76 mutations. Tumors expressing double Ras G12+E76 mutations exhibit more aggressive growth in preclinical studies and identifying  ras  double mutations may be prognostic of clinically more aggressive malignancies. These results highlight the importance of  ras  genotyping in all countries and predict that Ras E76 mutations represent a new cancer biomarker and target for cancer immunotherapy.