Cure rates of classical Hodgkin Lymphoma (cHL) in children and young adult patients currently exceed 90%. Nonetheless, survivors are confronted with chronic therapy-related health conditions such as infertility, cardiovascular disease, and high rates of novel second cancers. This calls for development of new targeted and less toxic treatments. cHL is characterized by a low frequency (~0.1-5%) of malignant Hodgkin Reed-Sternberg (HRS) cells, while most of the tissue is composed of nonmalignant immune cells. It is thought that the HRS cells depend on interactions with the tumor microenvironment (TME) for their survival. Indeed, a vast number of interactions between different immune and HRS cells have been reported; however, most of these reports are based on immunohistochemistry or in vitro studies. Here, we systematically characterized the in vivo interactions by applying single-cell RNA sequencing (scRNAseq) to nine primary pediatric and adolescent cHL biopsies and three noncancerous control biopsies of reactive lymph nodes. With scRNAseq, we first sorted live cells to get an unbiased overview of the TME. Then we used a previously published flow cytometry antibody panel to enrich for HRS cells, allowing us to directly assess interactions on a per-tumor basis. Tumor cell identity was confirmed by marker expression as identified by pathology, single-cell copy-number status and the ratio of immunoglobulin kappa/lambda expression. Immune cell identity was determined by canonical marker expression. Using the scRNAseq data, we found that the TME expression profiles in cHL and control biopsies mostly overlap but harbor some differences. First, we identified genes that are consistently overexpressed in HRS cells. These included transcription factors, neural markers, multiple interleukins and other signaling molecules. Second, the extensively described immunosuppressive interactions between HRS, T and NK cells (expressing CTLA-4, TIM-3, and LAG-3) were the strongest and most common interactions that we could identify in HL but not in noncancerous reactive lymph nodes. Third, while the inflammation in the reactive lymph nodes was driven by IFN-g signaling, this pathway was inactive in HL tumors. Other interactions like recruitment of CXCR3+ and CCR4+ T cells, CD47 signaling and interleukin signaling were less pronounced in cHL compared to the controls or were less consistent between tumors. These findings were validated in bulk RNA sequencing of 45 HL tumors. A model arises in which the presence of HRS cells induces inflammation that in most ways resembles lymph node infections. This inflammation and HRS survival are controled by patient-specific interactions between HRS cells and the TME, and by T cell exhaustion, which is universal and the most essential interaction in cHL.

Citation Format: Jurrian K. de Kanter, Thanasis Margaritis, Auke Beishuizen, Marijn Scheijde-Vermeulen, Liset Westera, Arianne M. Brandsma, Ruben van Boxtel, Friederike Meyer-Wentrup. Single-cell RNA sequencing reveals that childhood classical Hodgkin Lymphoma resembles normal inflammation except for T cell exhaustion [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A37.