Clinical Utilization of Next-Generation Sequencing-Based Clonality Assays for Immunoglobulin Heavy Chain in Evaluating Suspected B-Cell Proliferation

Abstract

Abstract Objectives B-cell clonality assays of the immunoglobulin heavy chain (IGH) and light chain (IGK) genes have been used widely in the workup of B-cell neoplasms. PCR is the current gold standard test for clonality. Recently, NGS has been suggested to both improve sensitivity and identify the specific V-(D-)J sequence required to track clones. Here we report our experience with using NGS for complementing our current procedures. Methods Our validated NGS test is performed by amplifying patients’ DNA using IGH-FR1 assay panel. Pooled amplicons get sequenced. The sequence data then get analyzed using in-house bioinformatics software, which sorts sequences and generates frequency distributions. Each sequence is ranked by its frequency. A clonal population is designated if the top sequence read is at least 1% of the total reads. For the standard PCR gene rearrangement test, we use a BIOMED-2 PCR-based B-cell assay. Results We retrospectively searched our database for all cases in which IGH and IGK clonality was tested by either standard PCR or NGS from 8/2016 to 7/2017. A total of 253 cases were tested by PCR while 21 cases were tested by NGS, which showed higher success rates. Of the 253 cases tested by conventional PCR, 39% (n = 98) were clonal (either IGH, IGK, or both), 34% (n = 86) were polyclonal, and the rest 28% (n = 69) showed a weak inconclusive signal or failed. Of the 21 cases analyzed by NGS, 71% (n = 15) were clonal, 24% (n = 5) were polyclonal, and just 5% (n = 1) failed. Conclusion Our experience indicates that NGS for IGH clonality, even more expensive, is an effective and more sensitive tool that produces higher informational content and yields higher success rates when compared to conventional PCR assay. NGS captures multidimensional data and provides a more precise determination of the clonal relationship between different lymphoid proliferations, which also is essential for the detection and measurement of minimal residual disease.