Clonality assessment and detection of clonal diversity in classic Hodgkin lymphoma by next-generation sequencing of immunoglobulin gene rearrangements

• Published in: Modern pathology Vol. 35; no. 6; pp. 757 - 766
• Main Authors: van Bladel, Diede A.G., van den Brand, Michiel, Rijntjes, Jos, Pamidimarri Naga, Samhita, Haacke, Demi L.C.M., Luijks, Jeroen A.C.W., Hebeda, Konnie M., van Krieken, J. Han J.M., Groenen, Patricia J.T.A., Scheijen, Blanca
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01.06.2022; Nature Publishing Group US; Elsevier Limited
• Subjects: 45/77; 631/1647/1513/2216; 631/250/248; Bioinformatics; Copy number; DNA Copy Number Variations; Gene Rearrangement; Genes, Immunoglobulin; Heavy chains; High-Throughput Nucleotide Sequencing; Hodgkin Disease - diagnosis; Hodgkin Disease - genetics; Hodgkin's lymphoma; Humans; Immunoglobulin Heavy Chains - genetics; Immunoglobulin kappa-Chains - genetics; Immunoglobulins; Laboratory Medicine; Lymphocytes B; Lymphocytes T; Lymphoma; Medicine; Medicine & Public Health; Next-generation sequencing; Paraffin; Pathology; T-cell lymphoma
• ISSN: 0893-3952; 1530-0285
• DOI: 10.1038/s41379-021-00983-8

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma.