NTRK gene aberrations in triple-negative breast cancer: detection challenges using IHC, FISH, RT-PCR, and NGS

Triple-negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type-agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan-TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real-time polymerase chain reaction (RT-PCR), and RNA-based next-generation sequencing (NGS). Of 83 cases, 16 showed pan-TRK positivity although no cases had NTRK-fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT-PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan-TRK IHC. Our data demonstrate that IHC has a high false-positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.