Next-Generation Sequencing-Based Clonality Assessment of Ig Gene Rearrangements: A Multicenter Validation Study by EuroClonality-NGS

J Mol Diagn. 2021 Sep;23(9):1105-1115. doi: 10.1016/j.jmoldx.2021.06.005. Epub 2021 Jun 26.


Ig gene (IG) clonality analysis has an important role in the distinction of benign and malignant B-cell lymphoid proliferations and is mostly performed with the conventional EuroClonality/BIOMED-2 multiplex PCR protocol and GeneScan fragment size analysis. Recently, the EuroClonality-NGS Working Group developed a method for next-generation sequencing (NGS)-based IG clonality analysis. Herein, we report the results of an international multicenter biological validation of this novel method compared with the gold standard EuroClonality/BIOMED-2 protocol, based on 209 specimens of reactive and neoplastic lymphoproliferations. NGS-based IG clonality analysis showed a high interlaboratory concordance (99%) and high concordance with conventional clonality analysis (98%) for the molecular conclusion. Detailed analysis of the individual IG heavy chain and kappa light chain targets showed that NGS-based clonality analysis was more often able to detect a clonal rearrangement or yield an interpretable result. NGS-based and conventional clonality analysis detected a clone in 96% and 95% of B-cell neoplasms, respectively, and all but one of the reactive cases were scored polyclonal. We conclude that NGS-based IG clonality analysis performs comparable to conventional clonality analysis. We provide critical parameters for interpretation and discuss a first step toward a quantitative scoring approach for NGS clonality results. Considering the advantages of NGS-based clonality analysis, including its high sensitivity and possibilities for accurate clonal comparison, this supports implementation in diagnostic practice.

Publication types

  • Multicenter Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • B-Lymphocytes / immunology*
  • Clone Cells / immunology*
  • Data Accuracy
  • Gene Rearrangement*
  • Genes, Immunoglobulin*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Immunoglobulin Heavy Chains / genetics*
  • Immunoglobulin kappa-Chains / genetics*
  • Lymphoma, B-Cell / genetics*
  • Lymphoma, Follicular / genetics*
  • Multiplex Polymerase Chain Reaction / methods
  • Phenotype
  • Sensitivity and Specificity


  • Immunoglobulin Heavy Chains
  • Immunoglobulin kappa-Chains