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Background:Current diagnostic criteria for mature B-cell tumors require a combination of histopathological, immunophenotypic, cytogenetic, and genetic evaluations on tissue biopsy specimens (Campo, Blood2022; Alaggio, Leuk2022). Despite this battery of techniques, substantial heterogeneity remains in specific subgroups that can be further resolved using additional molecular diagnostic methods, including gene expression profiling (GEP). For example, among aggressive B-cell tumors, a subset recognized as high-grade B-cell lymphomas can be identified as harboring MYC+BCL2 lesions by FISH (HGBCL-DH-BCL2). However, a significant remaining high-risk subset also have a characteristic tumor gene expression “double-hit signature” or “dark zone signature” (DZsig) (Ennishi, JCO2018; Alduaij, Blood2023).

Introduction:The scarcity of malignant Hodgkin and Reed-Sternberg (HRS) cells has hampered comprehensive genomic profiling of classic Hodgkin lymphoma (cHL) from tumor tissue. Multiple recent studies have demonstrated that plasma cfDNA profiling facilitates molecular characterization of cHL. Leveraging noninvasive genotypes and Latent Dirichlet Allocation, we recently defined 2 genetic cHL subtypes in a large international cohort comprising 366 patients including pediatric and adult patients of all ages (Alig et al., ASH2022 and ICML2023). Cluster H1 comprised ~2/3 of cases and was dominated by a high somatic mutational burden, and non-silent mutations in genes canonically involved in NF-κB, JAK/STAT and PI3K signaling as well as in B2M. Conversely, cluster H2 (~1/3 of cases) was primarily characterized by recurrent somatic copy number aberrations as well as mutations in TP53and KMT2D. Herein, we validate these previously identified genetic subtypes in external datasets as well as through orthogonal methods including tissue-based and noninvasive transcriptional and immune profiling.

Introduction: Classic Hodgkin lymphoma (cHL) is distinguished from other cancers by the minute fraction of Hodgkin/Reed-Sternberg (HRS) cells (1-5%) in the tumor mass. Therefore, the immune-cell rich microenvironment plays a pivotal role in cHL's development, progression, and response to therapy. However, comprehensive characterization of the malignant and immune cell states in cHL has posed significant challenges due to the rarity of HRS cells and the intrinsic heterogeneity of the disease. To address this challenge, we aimed to characterize the immune cell states of cHL using a combination of experimental and bioinformatics approaches.