Detection of NTRK gene fusions in sarcomas: a comparative study of Pan-TRK immunohistochemistry, FISH, and RNA-Based NGS

Background. Fusion involving NTRK1, NTRK2 and NTRK3 are oncogenic driver occurring in several adult and pediatric tumor types. In sarcomas they are mostly found in infantile fibrosarcoma, inflammatory (IFS), inflammatory myofibroblastic tumor (IMT) and in the so-called “NTRK-rearranged spindle cell neoplasm” entity described in the current WHO (2020) classification, including lipofibromatosis-like neural tumor, fibrosarcoma-like and malignant peripheral nerve sheath tumor-like spindle cell neoplasms.

Methods. We retrospectively reviewed 92 soft tissue and bone sarcomas diagnosed at the Rizzoli Institute between 2019 and 2023, in which pan-TRK IHC was performed. 17 tumours showed positive staining and were further assessed using FISH for NTRK1, NTRK2, and NTRK3 rearrangements. A subset of 12 cases underwent RNA-based NGS for fusion detection.

Results. In total, we collected data from 17 patients who tested positive for pan-TRK antibody and compared pan-TRK IHC and molecular testing for the detection of NTRK rearrangement in sarcomas. FISH analysis detected NTRK rearrangements in 4/17 cases (23.5%), while NGS confirmed NTRK fusions in 3/12 cases (25%). All NTRK fusion-positive cases confirmed by both FISH and NGS showed diffuse pan-TRK staining. Two additional cases exhibited pan-TRK diffuse positivity but were NTRK wild-type by NGS and harboured BCOR::MAML1 and EWSR1::NACC2 fusions, respectively. 1 case with focal positivity by immunohistochemistry and NTRK rearrangement by FISH was not confirmed by NGS.

Conclusions. Pan-TRK IHC can be considered an initial screening tool to identify sarcomas potentially harbouring NTRK fusions. However, the presence of diffuse pan-TRK immunoreactivity in NTRK-wild-type tumours highlights the need for cautious interpretation of IHC results. FISH may represent a useful intermediate diagnostic tool when NGS is unavailable but requires cautious interpretation particularly in cases with atypical or isolated signals. NGS-based molecular confirmation remains essential for the definitive identification of NTRK fusions.