Understanding the pathogenesis of classic Hodgkin lymphoma in persons living with HIV through conventional and spatial transcriptomics

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 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.