Proof‐of‐Concept Evaluation of T‐Cell Receptor Beta Constant Region 1 and 2 Transcript in Situ Hybridization for Clonality Assessment in Mycosis Fungoides

Background Mycosis fungoides (MF) may be difficult to distinguish from inflammatory dermatoses. T‐cell receptor (TCR) gene rearrangement assays provide ancillary evidence of clonality but lack spatial resolution. We evaluated the feasibility of RNA in situ hybridization (RNA‐ISH) targeting TCR beta constant isoforms, TRBC1 and TRBC2, in routine skin biopsies. Methods We retrospectively analyzed 15 MF biopsies, predominantly Stage IA–IB, and 11 inflammatory controls using TRBC1 and TRBC2 RNA‐ISH on separate sections. Expression was assessed semiquantitatively in matched intraepidermal regions using exploratory thresholds. Available TCR gamma PCR results were reviewed. Results RNA‐ISH was successful in all 26 samples. Nine MF cases (60%) showed TRBC skewing, three (20%) were indeterminate, and three (20%) were double negative. All controls showed balanced polytypic expression. Exploratory sensitivity was 60% (95% CI, 32.3%–83.7%) and specificity 100% (95% CI, 71.5%–100%). Substantial discordance with available PCR results was observed. Conclusions TRBC1/TRBC2 RNA‐ISH is technically feasible in archival skin and can demonstrate spatial isoform skewing in a subset of MF biopsies. Technical and interpretive limitations preclude diagnostic validation; larger multiplexed studies are warranted.