Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior

Abstract

Burkitt lymphoma (BL) is characterized by a tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of Epstein-Barr virus-positive (EBV+) BL with granulomatous reaction compared with 8 cases of EBV+ BL and 8 EBV-negative (EBV-) BL, both with typical starry sky pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated using multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (cluster 2), EBV- BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky," both EBV+ and EBV-. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, whereas BLs with "starry sky" were characterized by upregulation of M2 polarization and protumor response. Moreover, the analysis of additional signatures revealed an upregulation of the dark zone signature and epigenetic signature in BL with a typical starry sky. Tumor-associated macrophages and epigenetic regulators may be promising targets for additional therapies for BL lymphoma, opening novel immunotherapeutic strategies.

Graphical abstract

Figure 1.

PCA and heat map showed 3 clusters of BL. PCA (A) and heat map (B) revealed the presence of 3 distinct clusters. Cluster 1 consisted of the 5 BL with a diffuse granulomatous reaction (blue), cluster 2 had 5 EBV⁺ BL with typical starry sky pattern (orange), and cluster 3 includes 8 EBV^– BL with typical starry sky (violet), 2 cases of BL with a partial granulomatous reaction, and 3 cases of EBV⁺ BL with typical starry sky. Heat map visualizing the expression levels of immune-related genes (rows) in each BL sample (column). Upregulated or downregulated genes were selected for subsequent analysis if their expression values were found to exceed an adjusted P value cutoff of <.05, after multiple testing correction using a moderated t statistic.

Figure 2.

Immune differences between BL with granulomatous reaction and BL with typical starry sky. GEP showed that BL with typical starry sky pattern both EBV⁺ (orange) and EBV^– (violet) were enriched in and M2-related chemokines (A), M2 macrophages (B), and immune exhaustion signature (C).

Figure 2.

Immune differences between BL with granulomatous reaction and BL with typical starry sky. GEP showed that BL with typical starry sky pattern both EBV⁺ (orange) and EBV^– (violet) were enriched in and M2-related chemokines (A), M2 macrophages (B), and immune exhaustion signature (C).

Figure 3.

BL with starry sky was characterized by DZ signatures and epigenetic modification signatures. Analysis of additional signatures showed that BL with starry sky was enriched in DZ signature (A) genes and epigenetic modulation signature (B).

Figure 4.

Multiplex immunostaining to validate M1 and M2 polarization. In BL granulomatous cases, the fraction of macrophages was significantly high, with a lower density of M2/TAM polarized macrophages (A). Conversely, BL with starry sky pattern, both EBV⁺ (B) and EBV^– (C) showed an higher density of M2 macrophages. Original magnification, ×40.

Figure 5.

mRNA in situ hybridization for IFNG and immunohistochemistry (IHC) of CD4 and CD8. Representative microphotographs and quantitative analyses of mRNA in situ hybridization for IFNG and IHC for CD4 and CD8 showing the increase in the prevalence of CD8⁺ cytotoxic lymphocytes expressing IFNG in BL case. Original magnification, ×200 and ×400.

Figure 6.

Quantitative analysis of immunostaining. HALO image analysis software and statistical analysis using PRISM supported the results of immunostaining (A) and combined RNAscope and IHC.

Figure 7.

BL can show 2 different biological and clinical settings. M1 polarized macrophages prevail, and the immune response may be able to control neoplastic growth. In contrast, in cases characterized by the starry sky pattern, M2 macrophages dominate and may be responsible for a protumor immune response, resulting in disease rapid progression and dissemination.