Epstein–Barr virus reactivation influences clonal evolution in human herpesvirus-8-related lymphoproliferative disorders

Background: Human herpesvirus-8 (HHV8) is a lymphotropic virus associated with different lymphoproliferative disorders, including primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD), diffuse large B-cell lymphomas, not otherwise specified, and the rare entity known as germinotropic lymphoproliferative disorder (GLPD). In PELs and GLPD the neoplastic cells also contain Epstein–Barr virus (EBV). In addition, occasional cases with atypical and overlapping features among these entities have been recognised, suggesting that the spectrum of the HHV8-related lesions may not be fully characterised. Aims: Here, we report two cases of lymphoproliferative disorder associated with HHV8 and EBV that further expand the spectrum of HHV8/EBV-positive lymphoproliferative disease. Methods and results: Case 1 represented HHV8/EBV positive extracavitary nodal PEL followed by pleural PEL. The striking characteristic of this case was the almost focal and intrasinusoidal localisation of the neoplastic cells and the association with Castleman’s disease features. In the second case, we found the entire spectrum of HHV8-related disorders, i.e. MCD, GLPD, and PEL, coexisting in the same lymph node, underlining the variability, possible overlap and evo lution among these entities. Both cases were well analysed with immunohistochemistry, determination of the EBV latency programme, and molecular analysis for clonality of immnoglobulin genes. In both patients, the disease followed an unexpected indolent course, both being still alive after 8 and 12 months, respectively. Conclusion: Our findings represent further evidence of the overlap among HHV8/EBV-positive lymphoproliferative disorders, and underline a grey zone that requires further study; they further confirm the experimental evidence that lytic EBV replication influences HHV8-related tumorigenesis

Additional file 1: Table S1. of Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation

List of inter and intra chromosomal gene fusion detected by fusion-detection pipeline. (XLS 89 kb

Additional file 3: Table S3. of Burkitt lymphoma beyond MYC translocation: N-MYC and DNA methyltransferases dysregulation

Gene set enrichment analysis for gene ontology categories of 64 genes predicted as targets of microRNAs differentially expressed in MYC translocation-positive and -negative BLs. (DOC 67 kb

Immunophenotype of rosetting T cells in different variants of NLPHL and THRLBCL^*.

*<p>Cases were scored positive if positive rosetting T cells were observed around at least 5% of the tumor cells.</p

Top 5 upregulated genes in pairwise comparisons between tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL.

<p>p-values <0.05. FDR (false discovery rate)<0.3. vs = versus.</p

Validation on protein level of genes expressed in both THRLBCL-like NLPHL and THRLBCL identified by gene expression profiling.

<p><b>a., b. and c.:</b> Strong expression of BAT3/BAG6 in the tumor cells of typical NLPHL (pattern A), THRLBCL-like NLPHL and THRLBCL, 200x. Inset: Positive tumor cells in 400x. <b>d., e. and f.:</b> Expression of HIGD1A in the tumor cells of typical NLPHL (pattern A), THRLBCL-like NLPHL and THRLBCL, 200x. Inset: Positive tumor cells in 400x. <b>g., h. and i.:</b> Expression of UBD/FAT10 in the tumor cells of typical NLPHL (pattern A), THRLBCL-like NLPHL and THRLBCL, 200x. Inset: Positive tumor cells in 400x. <b>j., k. and l.:</b> Expression of CXCL13 in rosetting T cells of typical NLPHL (pattern A) and in the tumor cells of THRLBCL-like NLPHL and THRLBCL, 200x. Insets in 400x. <b>m., n. and o.:</b> Expression of ICOS in the tumor cells of typical NLPHL (pattern A), THRLBCL-like NLPHL and THRLBCL, 200x. Inset: Positive tumor cells in 400x.</p

Unsupervised hierarchical clustering and principal component analysis of gene expression profiles of microdissected tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL, as well as sorted tonsillar GC B cells.

<p><b>a.</b> Unsupervised hierarchical clustering of the gene expression profiles of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL as well as CD77⁺ GC B cells. All probesets with a standard deviation ≥0.9 (479 probesets) were considered. GC B cell samples are indicted by a grey bar. <b>b.</b> Principal component analysis of the gene expression profiles of the tumor cells of NLPHL, THRLBCL-like NLPHL and THRLBCL. All probesets with a standard deviation ≥1.2 (79 probesets, 40.18% diversity) were considered.</p

Immunohistochemical validation of genes expressed in the tumor cells of NLPHL and THRLBCL in an independent set of cases.

<p>Immunohistochemical validation of genes expressed in the tumor cells of NLPHL and THRLBCL in an independent set of cases.</p

Activin A induces Langerhans cells differentiation in epidermis-depleted skin explants.

<p>Langerin expression was evaluated in the dermal layer, separated from skin explants by dispase digestion and subsequently treated for 72 hrs after i.d. injection with 100 ng Activin A (magnification 100X, inset 400X.).</p

Quantification of the microenvironment in typical NLPHL (patterns A and C), THRLBCL-like NLPHL (pattern E) as well as THRLBCL.

<p><b>a.</b> Numbers of CD4⁺ T cells/mm² in typical NLPHL (pattern A: n = 14 and pattern C: n = 13), THRLBCL-like NLPHL (n = 14) and THRLBCL (n = 25). (*p<0.05, **p<0.01, unpaired t-test). <b>b.</b> Numbers of CD8⁺ T cells/mm² in typical NLPHL (pattern A: n = 14, pattern C: n = 15) and THRLBCL-like NLPHL (n = 12) as well as THRLBCL (n = 22). <b>c.</b> Numbers of CD163⁺ macrophages/mm² in NLPHL (pattern A and C as well as THRLBCL-like NLPHL: n = 14, each) and THRLBCL (n = 25), (***p<0.001, Mann-Whitney-test).</p