Background: Epstein-Barr virus (EBV)-positive lymphoproliferative disorders can exhibit overlapping Hodgkin-like morphological features but show a wide spectrum of clinical behaviours, and include indoent diseases such as EBV-positive mucocutaneous ulcer (EBVMCU), and malignant systemic diseases such as EBV+ classical Hodgkin lymphoma (EBVCHL) and EBV+ diffuse large B-cell lymphoma (EBVDLBCL). Diagnostic distinction of these lesions is imperative to ensure appropriate treatment; however, there is a dearth of diagnostic biomarkers to differentiate these lesions. Furthermore, the underlying biological basis of this divergent behaviour is unclear. This study aimed to identify the immunobiological and genetic basis of this divergent clinical behaviour using multimodal spatial profiling of the immune microenvironment and tumour cells, and single-cell genomic sequencing.

Methods: Multiplex immunofluorescence (mIF) and Nanostring GeoMX spatial profiling (GeoMX-DSP) were performed on formalin-fixed paraffin-embedded (FFPE) sections, comparing indolent EBVMCU to malignant EBVCHL and EBVDLBCL. Single HRS and HRS-like cells were isolated from EBVMCU, EBVCHL and EBVDLBCL using the DepArray platform, and low-pass whole genome sequencing was performed. Chromosome 9 ploidy and PDL1/PDL2 amplification status were assessed by DNA fluorescent in situ hybridisation (FISH) combined with CD30 immunofluorescence. PDL1 protein expression was assessed by PAX5/PDL1 dual chromogenic immunohistochemistry.

Results: MIF showed an increase in CD4+ T-regulatory and PDL1+IDO1+M1 macrophages in EBVMCU (p<0.05), whilst EBVDLBCL showed an increase in immunosuppressive IDO1+M2 macrophages (p<0.05). Gene set enrichment analysis performed on GeoMX-DSP data showed that EBVMCU was characterised by marked antiviral responses, with upregulation of pathways involved in T-regulatory chemotaxis in HRS cells (odds ratio (OR) 120; p <0.0001), interferon-alpha; (OR 50; p <0.0005) and interferon-beta (OR 40; 0 < 0.0004) responses in macrophages. Furthermore, EBVMCU HRS-like cells, in comparison to EBVDLBCL HRS-like cells, showed upregulation of type 1 interferon responses (OR 20; p< 0.00025). Differential gene expression analysis shows increased expression of tumour suppressor CRIP1 (FDR>0.001), chemotactic CXCL10 and pro-inflammasome IL1RN (FDR>0.001) in EBVMCU, and upregulation of anti-inflammasome IL1R2 (FDR>0.001) in EBVCHL and EBVDLBCL. Low-pass whole-genome sequencing of single HRS/HRS-like cells showed few copy number variations in EBVMCU, whilst EBVCHL and EBVDLBCL showed greater genomic complexity. Moreover, EBVCHL showed recurrent chromosome 9 amplifications. On chromosome9/PDL1/PDL2 FISH, EBVMCU showed no chromosome 9 aneuploidy above 3n compared to 50% in EBVDLBCL and 33% EBVCHL. Amplification of PDL1/PDL2 (>2n+1) was not present in EBVMCU or EBVDLBCL, but was present in 66% of cHL. Assessment of proteomic expression of PDL1 by immunohistochemistry showed ubiquitous, strong expression of PDL1 in HRS cells in EBVCHL, whilst HRS-like cells in EBVMCU and EBVDLBCL showed patchy weak expression.

Conclusions: EBVMCU showed evidence of active inflammation, T-regulatory cell chemotaxis and anti-viral response, whereas EBV+cHL and EBVDLBCL showed evidence of immune permissive, and immune suppressive microenvironments, with loss of inflammasome related signalling and decreased tumour suppressor mechanisms. EBVMCU showed little genomic complexity whilst EBVDLBCL and EBVCHL showed greater genomic complexity. Chromosome 9 amplification was a recurrent aberration in EBVMCU. FISH for PDL1/L2 and PDL1 immunohistochemistry are potential diagnostic discriminatory biomarkers.

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2025
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