B-Cell Clonality Determination Using an Immunoglobulin κ Light Chain Polymerase Chain Reaction Method

Abstract

To augment the detection of clonality in B-cell malignancies, we designed a consensus primer kappa light chain gene (Igkappa) polymerase chain reaction (PCR) assay in combination with a consensus primer immunoglobulin heavy chain gene (IgH) PCR assay. Its efficacy was then evaluated in a series of 86 paraffin tissue samples comprising neoplastic and reactive lymphoproliferations. Analysis after PCR was accomplished by 10% native polyacrylamide gel electrophoresis after heteroduplex pretreatment of PCR products and by a post-PCR chip-based capillary electrophoresis analytic method. Overall, 49 of 68 (72%) of mature B-cell neoplasms yielded discrete Igkappa gel bands within the predicted size range with no clonotypic Igkappa products observed among reactive lymphoid or T-cell proliferations. The application of Igkappa PCR improved overall sensitivity from 81% with IgH PCR alone to 90% with combined Igkappa/IgH PCR, with this effect being most notable in germinal center-related lymphomas. Sequencing of positive Igkappa rearrangements revealed that most rearrangements involved members of the Vkappa1 (40%) and Vkappa2 (34%) gene families along with Jkappa1 (26%), Jkappa2 (23%), and Jkappa4 (51%) gene segments. Involvement of Vkappa pseudogenes was identified in 24% of cases with Vkappa-KDE rearrangements. Our results demonstrate the efficacy of Igkappa PCR in improving the detection rate of clonality in B-cell neoplasms and further introduce a novel post-PCR chip-based capillary electrophoresis analytic method for rapid PCR fragment size evaluation.