Automated kappa and lambda light chain mRNA expression for the assessment of B-cell clonality in cutaneous B-cell infiltrates: Its utility and diagnostic application

Introduction: Primary cutaneous B-cell lymphoma (1°CBCL) accounts for 25% of all lymphomas. The difficulty in distinction of reactive from neoplastic B-cell infiltrates prompts the use of molecular diagnostic adjuncts. While T-cell clonality can be seen in various reactive states, clonal B-cell infiltrates are often neoplastic; standard assays employed include polymerase chain reaction (PCR) or Southern blot analysis to assess heavy chain rearrangement. We sought to assess the utility of kappa (κ) and lambda (λ) mRNA expression using the Ventana automated assay (Ventana Medical Systems, Tucson, AZ, USA) in the analysis of atypical cutaneous B-cell lymphoid infiltrates. Materials and methods: Multiple 4 μm sections of paraffin-embedded, formalin-fixed skin biopsies from 31 patients with CBCL were placed on silane-coated slides, deparaffinized, then digested in pepsin (5 mg/ml) for 30 min at 37°C. Fluorescein-tagged oligoprobes and tissue mRNA were denatured at 80°C for 5 min, hybridized for 2 h at 37°C, and incubated with antifluorescein alkaline phosphatase conjugates. Detection of the probe target complex employed nitroblue tetrazolium and bromochloroindolyl phosphate conjugates with a nuclear fast red counterstain. A κ:λ ratio > 3:1 was held to represent κ light chain restriction and a κ:λ ratio ≤ 1:1 to indicate λ light chain restriction. Results: The diagnosis in each case was determined by careful integration of clinical, histologic, and phenotypic data. The diagnoses included: pseudolymphoma (PL), marginal zone lymphoma (MZL), 1°CBCL of the trunk, scalp or leg, 2° lymphoma, and plasma cell dyscrasia. All but one case of lymphoma were light chain restricted. All cases of PL were proven to be polyclonal by this methodology. In non-plasmacytic small cell lymphomas, only 5-10% of the infiltrate expressed κ or λ, with clonality established through the abnormal κ:λ ratio. Interpretations were most difficult in the 2° small cell-dominant follicular center cell lymphomas and easiest in cases with significant plasmacytic differentiation (i.e. MZL, immunocytomas, or plasma cell dyscrasias). Conclusion: The Ventana κ/λ assay is a reliable, quick, and inexpensive way to determine B-cell clonality in cutaneous lymphoid infiltrates in paraffin-embedded formalin-fixed tissue.