Detection of NTRK1/2/3 rearrangements in pigmented spindle cell naevus of Reed: a comparative study of optimized pan‐ TRK immunohistochemistry, FISH and RNA ‐ NGS

AIMS: Pigmented spindle cell naevus of Reed (PSCN) is mainly characterized by MYO5A::NTRK3 fusion. However, the prevalence of NTRK fusions/rearrangements in PSCNs ranges from 15.8% to 57% in the literature, and the agreement among techniques for detecting these alterations has not been analysed. Here, we analysed NTRK fusions/rearrangements in a series of PSCNs using an optimized pan-TRK protocol, RNA-NGS fusion panel and FISH (NTRK1/2/3). METHODS AND RESULTS: Seventy-nine PSCNs were collected, reclassified (2025 WHO criteria and negative for BRAF V600E/RAS Q61R stains), and stained with a modified version of a recently proposed pan-TRK protocol (additional amplification steps and Red chromogen). In 64 and 57 cases with available material, we performed RNA-NGS and FISH (NTRK1/2/3) analyses, respectively. Pan-TRK was positive in 16 (20.3%) cases, all showing diffuse, granular, cytoplasmic staining of moderate intensity. In 59 cases with evaluable RNA-NGS data, 33 (55.9%) harboured MYO5A::NTRK3 fusion, 3 (5.1%) had NTRK3 imbalance, and 1 (1.7%) case each showed MYO5A::ROS1 and ESR1::CCDC170 fusion. In 51 cases with evaluable FISH (NTRK3) data, 33 (64.7%) were positive for NTRK3 rearrangement. Both the comparison of pan-TRK versus RNA-NGS and versus FISH (NTRK3) revealed high values of specificity (SP) and positive predictive value (PPV) (92.3%-96.2%) for the detection of NTRK3 fusions/rearrangements, despite a limited sensitivity (SE) of 36.4%. In the comparison of pan-TRK versus RNA-NGS and FISH (NTRK3), both SP and PPV were 100%, with a SE of 33.3%. CONCLUSIONS: Our 'optimized' pan-TRK protocol achieved high SP and PPV for the detection of NTRK3 fusions/rearrangements in PSCNs, despite a limited sensitivity compared to molecular assay. These results suggest that this protocol may serve as a diagnostic tool in routine practice, effectively guiding molecular testing and refining the diagnostic workflow for PSCNs.