Identification of 4 oncogenic NTRK point mutations in patient samples. (A) Among the 185 patient samples sequenced using a custom capture library consisting of 1862 kinase and kinase-associated genes, 10 patients harbored a mutation in 1 of the NTRK genes. The NTRK2^R458G mutation was found in 2 patients. Mutations are organized by gene and diagnosis. Patient specimen identifier (ID) is also provided. (B) NTRK2^A203T, NTRK2^R458G, NTRK3^E176D, and NTRK3^L449F mutations transform the murine Ba/F3 pro-B cell line and enable interleukin 3 (IL-3)–independent growth. No growth was observed in Ba/F3 cells harboring an empty vector (pMX-puro) or wild-type (WT) NTRK2 or NTRK3. Total viable cells are plotted over time and cell growth was measured after the withdrawal of IL-3. This experiment was repeated at least twice with consistent results. Additional validation studies are found in supplemental Figure 2. (C-F) Electropherograms from Sanger sequencing of patient genomic DNA confirm the presence of NTRK2^A203T, NTRK2^R458G, NTRK3^E176D, and NTRK3^L449F mutations. Peaks correspond to the following nucleotides: A (green), T (red), C (blue), and G (black). Arrows indicate direction of sequencing. (G) Immunoblot analysis of total and phosphorylated TrkB (p-TrkB) and TrkC (p-TrkC) on patient samples with known NTRK^A203T (12-00171), NTRK^R458G (12-00337 and 13-00187), and NTRK3^L449F (10-00828) mutations. (H) Gene schematics depict the location of prioritized NTRK2 and NTRK3 point mutations. The location of the following domains is included: leucine-rich repeat (LRR) N-terminal domain (LRRNT; NTRK2-specific), LRR 1, LRR 2, LRR C-terminal domain (LRRCT), immunoglobulin (Ig)-like C2-type 1, Ig-like C2-type 2, transmembrane (TM), juxtamembrane, and tyrosine kinase. aCML, atypical CML; CML, chronic myeloid leukemia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MPN, myeloproliferative neoplasm; NOS, not otherwise specified; VAF, variant allele frequency.