Branched-chain in situ hybridization for κ and λ light chains: A powerful ancillary technique for determining B-cell clonality in cytology samples

Background: Current immunohistochemical and in situ hybridization (ISH) assays are generally inconclusive for clonality unless plasmacytic differentiation is present. This study examined a series of cytology specimens and explored the ability of a branched-chain RNA (bRNA) ISH assay for immunoglobulin κ constant (IGKC) and immunoglobulin λ constant (IGLC) to detect a clonal population of B lymphocytes.

Methods: Pathology databases were used to identify fine-needle aspiration biopsies (n = 28) and exfoliative cytology samples (n = 20). Demographic, flow cytometry, and excision biopsy results were recorded. bRNA ISH was performed on the Leica Bond platform with the following probes: IGKC, IGLC, immunoglobulin λ-like polypeptide 5 (IGLL5), and a housekeeping gene (HKG).

Results: The bRNA ISH assay was validated with 30 surgical biopsies. On bRNA ISH, a clonal B-cell population (light-chain ratio > 10:1) was detected in 22 of 28 cases with a final diagnosis of lymphoma. In 2 cases, a κ predominance was present, although the ratio was <10:1. Eleven of the 17 κ-clonal lymphomas also expressed IGLL5, the latter recognized by the presence of an intranuclear signal. Two B-cell lymphomas lacked IGKC and IGLC, whereas 2 cases were negative for the HKG. In 12 of the 20 cases with reactive lymphoid tissue, bRNA ISH identified a polyclonal lymphoid population. No light-chain messenger RNA was detected in 6 cases (typically those associated with very few B cells).

Conclusions: The automated bRNA ISH platform is a robust technique for detecting a clonal B-cell population in cytology material.