Immunoglobulin Light Chain mRNA In Situ Hybridization Refines Classification Across the NLPHL-cHL Spectrum

Nodular lymphocyte-predominant Hodgkin/B-cell lymphoma (NLPHL/BL) and classic Hodgkin lymphoma (cHL) are biologically distinct entities, exhibiting preserved versus lost B-cell differentiation and immunoglobulin (IG) transcription. However, cases with overlapping morphologic and immunophenotypic features between NLPHL and lymphocyte-rich cHL (cHL-LR), here designated as NLPHL-OL, remain biologically ambiguous. We applied a novel, highly sensitive dual κ/λ mRNA in situ hybridization assay to assess IG light-chain expression across this spectrum. We analyzed 53 lymph node biopsies, including NLPHL-OL cases (n =15), cHL-LR (n = 11), along with typical NLPHL (n = 13) and cHL of other subtypes (n = 14) as controls. Distribution, intensity, and patterns of κ/λ mRNA expression in neoplastic cells was systematically evaluated and correlated with histology, architecture, immunophenotype, and Epstein-Barr virus (EBV) status. Diffuse, strong cytoplasmic light chain mRNA expression, representing the canonical pattern, was detected in the tumor cells of all NLPHL (13/13) and the majority of NLPHL-OL cases (12/15, 80%). In contrast, faint perinuclear or granular cytoplasmic signals (non-canonical patterns) were detected in 6/11 (55%) cHL-LR and 4/14 (28%) of other cHL subtypes. Canonical κ/λ expression was thus restricted to NLPHL and NLPHL-OL, confirming their classification as NLPHL with aberrant features, whereas cHL was negative or displayed exclusively non-canonical patterns. Non-canonical staining was also seen in 3/15 (20%) NLPHL-OL, including two EBV-positive cases. Rare aberrant light chain profiles in NLPHL and NLPHL-OL cases included single-cell κ/λ coexpression, and distinct LP-cell populations with mutually exclusive κ and λ expression. In summary, κ/λ mRNA ISH refines diagnostic classification across the NLPHL-cHL spectrum, is particularly useful in cases with overlapping features, and reveals canonical, non-canonical, and negative staining patterns that may reflect B-cell program integrity, placing these cases along a biological continuum.