PROJECT SUMMARY/ABSTRACT The incidence of hematological malignancies is higher in persons living with PLWH (Persons living with HIV (Human Immunodeficiency Virus)). The risk of developing classic Hodgkin lymphoma (CHL) in PLWH is 5-26 times the general population. The US (United States) HIV/AIDS Cancer Match study documented a 1.63-fold increase in the incidence of CHL in PLWH after the introduction of ART compared to the pre-ART era. CHL is an unusual B-cell malignancy with <5% tumor cells amidst >95% benign cells composed of immune and stromal cells. Most are of the mixed cellularity histological subtype of CHL, and 80-100% of CHL in PLWH are EBV-associated. Our previous study, using a limited panel of antibodies, showed that the immune microenvironment of CHL in PLWH differs from those seen in immunocompetent patients. Furthermore, the immune microenvironment plays a crucial role in the pathogenesis of CHL. DNA from leukocytes in untreated HIV-infected individuals show DNA methylation changes indicative of aging. Combination ART can partly reverse epigenetic aging but does not entirely correct it. We hypothesize that the persistent epigenetic alterations within the immune cells in PLWH on long- term combination ART result in altered and aberrant gene expression in the immune cells. In the context of the presence of sufficient numbers of CD4+ T cells, the abnormal immune cells contribute to the pathogenesis of classic Hodgkin lymphoma; they are responsible for the increased relative risk of developing CHL in PLWH on combination ART. We propose to identify persistent transcriptomic changes in immune cells in benign lymph nodes of PLWH on combination ART and address whether these or related changes can be documented in CHL samples of PLWH. We will select benign/reactive lymph nodes from 21 PLWH on ART and 21 age-matched non-PLWH groups, and CHL samples from 26 PLWH and 26 age-matched and EBV-association matched non-PLWH individuals. We will include paired benign lymph node and CHL samples from the same patients in the study. RNA extracted from the samples will be evaluated by Bulk RNA Barcoding and Sequencing (BRB-seq) and by Nanostring nCounter analysis. For nCounter analysis, we will use a panel ~100 genes representative of immune response to hematolymphoid tumors, to identify differentially expressed RNAs and to validate the results of BRB-Seq. Tissue microarrays will be prepared from the paraffin blocks, and spatial transcriptomics and protein expression will be undertaken using the CosMXTM Spatial Molecular Image analysis system to evaluate the tissue microenvironment in a single-cell and spatial context. The differential RNA expression not addressed by CosMXTM will be addressed by multiplex-immunohistochemistry and in-situ hybridization (RNAscope). The innovative BRB-seq that we propose to validate will significantly reduce the costs of RNA sequencing and expression analysis.