EBV Dictates Lower Tumor Neoepitopes Scores and Intra-Tumoral Repertoire Diversity in NHL from Immuno-Compromized Patients

Non-hodgkin lymphomas (NHL) are among the most common and fatal cancers in HIV-infected patients and transplant recipients. They are frequently associated with the Epstein Barr Virus (EBV) and a central nervous system (CNS) localization. Cancers neoantigens derived from the somatic tumor variants are a key factor of anti-tumor activity and response to immunotherapies, but the immunogenomics and tumor microenvironment (TME) characteristics of NHL from immunodeficient (ID) patients are lacking. We hypothesized they might be influenced by the low immunological pressure, the viral stimulation and/or the immune-privileged site. We report the comprehensive and prospective study of the tumor mutational burden (TMB), numbers of tumor neoepitopes, neoepitopes specific T cell responses and TME in ID patients, according to the EBV status, the immune status and the disease localization.

Consecutive HIV-infected patients or transplant recipients with treatment-naïve NHL were included and compared with immunocompetent patients with diffuse large B-cell lymphomas (DLBCL). A whole exome sequencing (WES) was carried out in parallel on tumor and on PBMC for TMB assessment and a whole genome RNA sequencing was performed on frozen tumor biopsies. The neoepitopes derived from the somatic tumor variants (called in WES and RNAseq data) were predicted in silico using a bioinformatic pipeline including pVACseq and NetMHC and filtered according to: VAF≥10%, gene expression≥1 TPM and affinity binding score ≤500 nM. Neoepitopes specific T cell responses were assessed by ELISPOT assays after a 10 days PBMC co-culture with peptides from the Top 50 best predicted autologous neoepitopes (positivity defined as spot forming cells >50/10⁶ cells). For the TME study, the profiling of cell type abundance and the T-cell receptor (TCR) sequencing were performed with deconvolution tools (EPIC) and MiXCR respectively. All statistical tests used R.

Sixty-seven patients were included so far: 33 post-transplant lymphoproliferative disorders (PTLD) and 16 HIV-positive NHL, 70% of whom having DLBCL, compared to 18 immunocompetent (IC)-DLBCL. Diseases were systemic in 73% and localized in CNS in 27% cases. The median age was 58 years (range 21-85) and 70% were male. Immuno-molecular data are available for 57 patients so far. The TMB was 3.7/Mb for all patients but was lower in EBV-positive NHL (2.7/Mb) compared to the negative ones (4/Mb) (p=0.012, t-test) (Fig 1) without difference according to the ID status. The overall median number of neoepitopes per tumor predicted from the non-immunoglobulin (Ig) variants was 113 (range 11-720) but was lower in EBV-positive NHL (49 vs 243, p= 0.04, t-test), correlating with the TMB (r=0.8, p<0.0001). Most neoepitopes were MHC-class II restricted (ratio MHC-class II/ MHC- class I= 4.1) independently of the EBV status. The median number of neoepitopes predicted from the Ig heavy chain (IgH) genes was 17.5 (range, 6-36) with a MHC-class II/MHC-class I ratio of 1.4. Neoepitopes specific T cell responses were detectable among 71% cases out of 14 patients tested so far, independently of ID status or CNS localization (Fig 2). All 3 positive responses against Ig-derived neoepitopes out of 5 tested cases were directed against the IgH, MHC-class II restricted and immunodominant compared to those against the non-Ig neoepitopes. There was no shared immunogenic neoepitopes between patients. The TME study showed a higher frequency of CD4+ and CD8+ T cells mean frequency in EBV-positive compared to EBV-negative NHL (11% vs 4% and 10% vs 2% respectively, p<0.03).The intra-tumoral TCR repertoire distribution was mostly polyclonal with a median frequency of dominant clones of 8% (range 1-61) and a lower repertoire diversity, assessed on the ratio of clonotypes number/ total TCR-β reads number, in EBV-positive NHL (29) than in EBV-negative ones (37) (p=0.02), without difference according to the immune status.

Our data demonstrate that both immunogenomics and TME of NHL from immuno-compromized patients are preferentially influenced by the EBV status, with lower numbers of tumor neoepitopes correlating with lower TMB, and a lower intra-tumoral TCR diversity in EBV-positive versus EBV-negative NHL. We further demonstrated that anti-tumor immune responses can be mounted despite ID status or CNS localization, especially against MHC-class II presented Ig-variants, and might be targeted for therapeutic strategies.