Single-step next-generation sequencing of immunoglobulin heavy chain genes to detect clonality in classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) diagnosis occasionally requires clonality assessment. However, the paucity of neoplastic Hodgkin and Reed-Sternberg cells among abundant reactive elements dramatically reduces the sensitivity of standard polymerase chain reaction (PCR)-based BIOMED-2/EuroClonality protocols, which fail to detect clonality in approximately one-third of cases. Next-generation sequencing (NGS)-based approaches have been proposed to overcome this limitation, but their performance in routine diagnostics for cHL remains insufficiently evaluated. We analyzed seven cHL cases and five follicular lymphoid hyperplasia (FLH) controls, all showing polyclonal patterns by standard PCR/capillary electrophoresis. DNA extracted from formalin-fixed paraffin-embedded lymph node biopsies was subjected to immunoglobulin heavy chain (IGH) gene rearrangement analysis using the commercially available LymphoTrack® IGH assay on the MiSeq platform. Next-generation sequencing identified clonal IGH rearrangements in 3 of 7 (43%) cHL cases previously undetectable by conventional methods. Clonal cases exhibited a dominant rearrangement representing > 2.5% of total reads, with the second most frequent rearrangement being less than half of the first. The remaining cHL cases and all FLH controls displayed polyclonal patterns. The LymphoTrack® IGH NGS assay demonstrated superior sensitivity over standard PCR-based protocols for detecting clonality in cHL, supporting its implementation in routine diagnostic workflows for challenging cases.