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.