Molecular and cytogenetic features of NTRK fusions enriched in BRAF and RET double-negative papillary thyroid cancer

Rare NTRK-driven malignant neoplasms can be effectively inhibited by anti-TRK agents. The discovery of NTRK1/2/3-rich tumours in papillary thyroid cancer (PTC) patients is a precondition for the rapid identification of NTRK fusion tumours. Knowledge of NTRK gene activation is critical to accurately detect NTRK status. A total of 229 BRAF V600E-negative samples from PTC patients were analysed in this study. Break-apart fluorescence in situ hybridisation (FISH) was performed to detect RET fusion. NTRK status was analysed using FISH, DNA- and RNA-based next-generation sequencing (NGS), and quantitative reverse transcription-polymerase chain reaction (RT-qPCR). In 128 BRAF and RET double-negative cases, 56 (43.8%, 56/128) NTRK rearrangement tumours were found, including 1 NTRK2, 16 NTRK1, and 39 NTRK3 fusions. Two novel NTRK fusions, EZR::NTRK1 and EML4::NTRK2, was found in the NTRK rearrangement tumors.Dominant break-apart and extra 3' signal patterns accounted for 89.3% (50/56) and 5.4% (3/56) of all NTRK-positive cases, respectively, as determined by FISH. In our cohort, there were 2.3% (3/128) FISH false-negative and 3.1% (4/128) FISH false-positive cases identified. NTRK fusions are highly recurrent in BRAF and RET double-negative PTCs. FISH or RNA-based NGS is a reliable detection approach. NTRK rearrangement can be precisely, rapidly, and economically detected based on the developed optimal algorithm.