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

Jos Rijntjes,Patricia J.T.A. Groenen,Diede A.G. van Bladel,Demi L.C.M. Haacke,Michiel van den Brand,Blanca Scheijen,J. Han J.M. van Krieken,Samhita Pamidimarri Naga,Konnie M. Hebeda,Jeroen A.C.W. Luijks

doi:10.1038/s41379-021-00983-8

Abstract

AbstractClonality 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.

Highlights

Classic Hodgkin lymphoma is a B-cell neoplasm, characterized by the presence of a small number of malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, residing in a background of inflammatory cells, including reactive B cells and plasma cells[1,2]
Most classic Hodgkin lymphoma (cHL) cases are diagnosed based on clinical presentation and histopathology, the molecular detection of clonal immunoglobulin (IG) gene rearrangements is helpful for diagnosing cases with a more atypical presentation and a histology similar to T cell lymphoma, or for clonal comparison in patients with recurrent disease[11,12]
IG-next-generation sequencing (NGS) was analyzed on all 16 cases, both formalinfixed paraffin-embedded (FFPE) and fresh frozen (FF), while BIOMED-2/EuroClonality analysis could only be performed on FFPE cases, due to limited DNA yield for case 13, but included all FF cases

Summary

Introduction

Classic Hodgkin lymphoma (cHL) is a B-cell neoplasm, characterized by the presence of a small number of malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, residing in a background of inflammatory cells, including reactive B cells and plasma cells[1,2]. In cHL, the HRS cells represent functionally impaired B lymphocytes that have lost the capacity to express immunoglobulins and many of the B-cell markers, but show CD15 and CD30 expression[3]. Evidence for their B-cell origin has been obtained by the detection of clonal rearrangements of the immunoglobulin genes, i.e., the immunoglobulin heavy (IGH) and light chain gene loci[4,5,6,7]. The presence of somatic hypermutation (SHM) within the rearranged variable (V) genes demonstrates that HRS cells originate from (post-) germinal center B lymphocytes[8,9,10]. The combination of both IGH and IGK rearrangement analysis, including incomplete rearrangements not undergoing SHM, has certainly enhanced the detection rate of clonal rearrangements in cHL, more sensitive techniques can improve clonality assessment in cHL

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