Immunogenomic Profiles of EBV-Associated Classic Hodgkin Lymphoma in an HIV-Inclusive Cohort

Introduction: Classic Hodgkin lymphoma (cHL) is a common hematolymphoid neoplasm globally with high relapse-related mortality. Furthermore, cHL is highly associated with HIV which is increasing in incidence after the introduction of antiretroviral therapy, population growth, and aging. Tumor-associated Epstein-Barr virus (EBV) is identified in nearly all cHL in people with HIV and 30% of cHL in HIV-uninfected patients. Our group recently reported that patients with HIV+ cHL have better progression free survival compared to patients without HIV and ABVD treatment is safe and effective in this population. Recent studies in high-income countries have shown that checkpoint inhibitors (ICIs) can improve outcomes while decreasing cytotoxicity for cHL, which has the potential to be beneficial in low- and middle-income countries with limited supportive care. To our knowledge, the immunogenomic features related to the success of ICIs have not been evaluated in the African context. In this study, we aim to evaluate tumor mutational burden (TMB) and T-cell receptor (TCR) repertoire diversity in a well-annotated and previously understudied cohort of cHL patients from Malawi.

Methods: The Kamuzu Central Hospital Lymphoma Study has prospectively enrolled patients, both adult and pediatric, with newly diagnosed lymphomas in Malawi since 2013. EBV tumor status was determined by Epstein-Barr encoding region in situ hybridization (EBER-ISH). DNA was extracted from n=24 bulk pre-treatment formalin-fixed, paraffin-embedded (FFPE) cHLs (QIAmp DNA FFPE Advanced). TCR sequencing was performed (immunoSEQ, Adaptive Biotechnologies) and epitope sequences were annotated using the McPAS human database. After passing quality control, repertoires were randomly downsampled to 100 productive templates for analysis due to count variation. Whole exome sequencing was conducted on tumor samples paired with blood to determine somatic mutations (Aligent SuperSelect Human Exon v6, Novogene). We considered three groups of interest based on systemic HIV status and tumor EBV status: EBV-/HIV- (n=8), EBV+/HIV- (n=11), EBV+/HIV+ (n=5).

Results: In our cohort, the median age was 22 years, ranging from 13 to 59 years, and 62% were male. EBV+ tumors were associated with improved overall patient survival (HR=0.10, p=0.04) compared to EBV- tumors. There was no difference in TMB by EBV status alone, however, EBV+/HIV+ cHL had increased median TMB compared to the EBV+/HIV- cases (p=0.03). The most commonly mutated genes within the entire cohort include histone methyltransferases, chromosome stability proteins, mRNA processing and transport proteins. KMT2C/D, genes previously associated with cHL, are mutated more often in the EBV+ (KMT2C: Χ²= 3.8 p=0.05) and HIV+ groups (KMT2D: X²=7.4 p=0.01). TCR diversity is increased in the EBV- compared to the EBV+ group (p=0.06). 71% of the EBV+/HIV+ TCR epitopes targeted known pathogens, compared to 52% of the EBV-/HIV- cases (p=0.07). TMB and TCR metrics were not associated with stage, age and other clinical factors other than EBV and HIV status.

Conclusions: In this Malawian cohort treated with ABVD chemotherapy, patient HIV and tumor EBV status are prognostic, where EBV-/HIV- cHL is associated with inferior overall survival compared to HIV+ and EBV+ tumors. TCR diversity is associated with tumor EBV expression, while TMB was associated with patient HIV status. Increased TMB and TCR diversity are known biomarkers for increased response to ICIs in cHL. In our cohort, EBV-/HIV- patients are enriched for these predictive immunogenomic characteristics and have the worst outcomes. These patients could potentially benefit from earlier ICI treatment. Other immunotherapies should be considered in this context and responses evaluated in light of the immunogenomic features described above.