Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma

Abstract

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma’s that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

Clonal Relationship and Mutation Analysis in Lymphoplasmacytic Lymphoma/Waldenström Macroglobulinemia Associated With Diffuse Large B-cell Lymphoma

“…However, it is still unclear whether in all cases this progression to DLBCL results from transformation of the malignant LPL B‐cell clone(s), or represents a de novo DLBCL that is clonally unrelated to LPL/WM. With the current availability of NGS‐based clonality assessment, providing detailed information of (sub)clonal IG gene rearrangements, we investigated the clonal B‐cell dynamics and clonal relationship of LPL and DLBCL presentations in a cohort of 13 LPL/WM patients with the EuroClonality IG‐NGS assay 26–28 . In addition, the MYD88 and CXCR4 mutational status of the LPL and DLBCL biopsies was determined by employing a targeted smMIP panel, combined with the analysis of several candidate genes associated with extranodal DLBCL development and transformation.…”

“…DNA concentrations were determined using Qubit (dsDNA BR Assay Kit; Life Technologies, Carlsbad, CA). NGS‐based clonality analysis was performed as previously described by the EuroClonality‐NGS Working Group to detect productive and unproductive IGH and IGK gene rearrangements 26–28 . The 4 standard targets of the EuroClonality IG‐NGS assay (framework‐3 [FR3] IGHV‐IGHD‐IGHJ, IGHD‐IGHJ, IGKV‐IGKJ, and IGKV/Intron RSS‐KDE) were analyzed for all LPL and DLBCL samples using 40 ng DNA input (or a minimum of 10 ng for samples with limited material available) for each of the 3 multiplex PCR reactions with M13‐tailed primers.…”

“…NGS-based clonality analysis was performed as previously described by the EuroClonality-NGS Working Group to detect productive and unproductive IGH and IGK gene rearrangements. [26][27][28] The 4 standard targets of the EuroClonality IG-NGS assay (framework-3 [FR3] IGHV-IGHD-IGHJ, IGHD-IGHJ, IGKV-IGKJ, and IGKV/ Intron RSS-KDE) were analyzed for all LPL and DLBCL samples using 40 ng DNA input (or a minimum of 10 ng for samples with limited material available) for each of the 3 multiplex PCR reactions with M13-tailed primers. Library preparations were generated in a second step with adaptor-tailed M13 primers compatible with sequencing on an Illumina platform.…”

“…The clonal B-cell composition in LPL tissue biopsies was assessed by NGS-based clonality assays as developed by the Euroclonality-NGS Working Group. [26][27][28] IG-NGS clonality analysis detected clonal B-cells in all but 2 LPL tissue samples (due to inferior genomic DNA quality), based on the presence of clonal IG gene rearrangements at either the IGH loci (n = 3/26; 12%) or a combination of IGH and IGK loci (n = 23/26; 88%) (Suppl. Table S3).…”

Large B-cell Lymphomas of Immune-Privileged Sites Relapse via Parallel Clonal Evolution from a Common Progenitor B Cell

“…All reads with exactly the same CDR3 were assigned to the V-gene with the longest fragment that map to the V-genes, because this fragment has the highest mapping reliability. The data was visualized with VDJtools (v1.2.1) 35 . Samples with the same CDR3-length and similar CDR3 sequence were considered clonal.…”