Molecular Modeling and Functional Analysis of Exome Sequencing–Derived Variants of Unknown Significance Identify a Novel, Constitutively Active FGFR2 Mutant in Cholangiocarcinoma
Authors: Egan Jan B. , Marks David L. , Hogenson Tara L. , Vrabel Anne M. , Sigafoos Ashley N. , Tolosa Ezequiel J. , Carr Ryan M. , Safgren Stephanie L. , Enriquez Hesles Elisa , Almada Luciana L. , Romecin-Duran Paola A. , Iguchi Eriko , Ala’Aldeen Aryan , Kocher Jean-Pierre A. , Oliver Gavin R. , Prodduturi Naresh , Mead David W. , Hossain Asif , Huneke Norine E. , Tagtow Colleen M. , Ailawadhi Sikander , Ansell Stephen M. , Banck Michaela S. , Bryce Alan H. , Carballido Estrella M. , Chanan-Khan Asher A. , Curtis Kelly K. , Resnik Ernesto , Gawryletz Chelsea D. , Go Ronald S. , Halfdanarson Thorvardur R. , Ho Thai H. , Joseph Richard W. , Kapoor Prashant , Mansfield Aaron S. , Meurice Nathalie , Nageswara Rao Amulya A. , Nowakowski Grzegorz S. , Pardanani Animesh , Parikh Sameer A. , Cheville John C. , Feldman Andrew L. , Ramanathan Ramesh K. , Robinson Steven I. , Tibes Raoul , Finnes Heidi D. , McCormick Jennifer B. , McWilliams Robert R. , Jatoi Aminah , Patnaik Mrinal M. , Silva Alvin C. , Wieben Eric D. , McAllister Tammy M. , Rumilla Kandelaria M. , Kerr Sarah E. , Lazaridis Konstantinos N. , Farrugia Gianrico , Stewart A. Keith , Clark Karl J. , Kennedy Eileen J. , Klee Eric W. , Borad Mitesh J. , Fernandez-Zapico Martin E. 
Published: August 2017

Purpose Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician’s ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. Materials and Methods Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. Results The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522 VUSs of interest, including a large number of kinases. Ten receptor tyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. Conclusion The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians’ ability to make informed treatment decisions.