2.1. Study Cohort and Study Samples

This study included patients from the biobank program U-CAN (Uppsala Umeå Comprehensive Cancer Consortium). The U-CAN program has, since 2010, collected data and created a biobank with blood and tissue samples with various cancer diagnoses including lymphoma. The current cohort consisted of 88 patients included in U-CAN and was confined to cHL patients with available plasma samples and frozen diagnostic biopsies diagnosed between 2010 and 2019 (n = 27). One patient was included at relapse with a first diagnosis in 1989 (Figure 1). As controls, 30 healthy study subjects with lymph nodes classified histopathologically as reactive lymphadenopathy were used. The controls were matched for age and gender.

Flowchart of patients included in the current study: UCAN = Uppsala Umeå Comprehensive Cancer Consortium biobank program, cHL = classical Hodgkin lymphoma, NLPHL = nodular lymphocyte-predominant Hodgkin lymphoma, QC = quality control, PEA = proximity extension assay, and Immuno-ONC = Immuno-Oncology.

The diagnostic biopsies for patients with cHL (n = 27) were obtained from lymph nodes in the axilla (n = 2), neck (n = 21), and mediastinum (n = 2). Two patients had missing information on lymph node location. Biopsy location from non-malignant lymph nodes (n = 30) were neck (n = 18), groin (n = 5), axilla (n = 5), and mesenterium (n = 1). One patient had missing data on lymph node location. Of the cHL plasma samples (n = 26), 25 were from the same patients as the tissue samples from cHL patients. Plasma control samples (n = 27) were obtained after consent and matched for age and gender as the tissue controls. Clinical data were obtained from patient hospital records. Stage of disease was defined according to the Ann Arbor classification, and advanced stage was defined as IIB-IVB [21,22]. Analysis of EBV infection in HRS cells was performed using immunohistochemistry (IHC) for latent membrane protein 1 (LMP1) and in situ hybridization (ISH) for Epstein–Barr virus (EBV)-encoded small RNAs (EBERs).