Wang, Shi

Abstract Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a rare and aggressive lymphoma with molecular heterogeneity not well characterize. In this study, we performed next-generation sequencing analysis for a large number of DNA and RNA samples from patients with PT-DLBCL. DNA sequencing analysis identified ≥ 3 chromosomes with copy number variations (CNVs) and microsatellite instability as prognostic biomarkers, rather than MYD88 mutations and genetic subtypes. Remarkably, targeted RNA-seq analysis in 195 patients revealed that TP53 mutations with a ≥ 40% variant allele frequency had significantly adverse prognostic impact, and that a 150-gene expression signature subdivided PT-DLBCL into two distinct clusters, termed as testicular lymphoma tumor (TLT) and microenvironment (ME) subtypes. The TLT subtype featured upregulation of genes involved in B-cell receptor signaling, cell cycle, DNA damage and repair, higher frequencies of CNVs and MYD88 mutations, elder ages, larger tumor sizes, and significantly poorer survival. Genomic microRNA profiling analysis identified significantly differentially expressed microRNAs between 113 PT-DLBCL and 180 systemic DLBCL patients, and further subdivided the PT-DLBCL cohort by microRNA signatures. The subcohort with upregulation of 16 microRNAs associated with PT-DLBCL and testicular tissue expression had significantly better survival. This study revealed characteristic genetic, gene expression, and microRNA profiles and heterogeneity in PT-DLBCL.

Introduction: Primary diffuse large B-cell lymphoma (DLBCL) of the central nervous system (CNS), traditionally referred to as primary CNS lymphoma (PCNSL), is a rare type of primary large B-cell lymphoma of immune-privileged sites with significantly poorer prognosis than systemic DLBCL. Genetic profiles of PCNSL were widely studied, whereas the tumor microenvironment has not been well characterized. Patients and Methods: A series of molecular analyses were performed using DLBCL FFPE tissues collected by the International Lymphoma Consortium. First, multiplex fluorescent immunohistochemistry (mfIHC) using antibodies for CD20, PAX5, CD3, CD4, CD8, FOXP3, CD45RO, CD68, CD56, PD-1, PD-L1, PD-L2, and CTLA-4 was performed on PCNSL tissues from 121 immunocompetent patients and de novo systemic DLBCL tissues from 405 patients. Digital quantification results for 405 patients with EBV-negative systemic DLBCL or primary testicular (PT) DLBCL have been reported previously. The preliminary mfIHC results of PCNSL will be presented. Second, mfIHC for multiple B cell biomarkers, CD3, CD4, CD8, CD68, CD163, CD11c, and CD31 were performed for 10 PCNSL validation cases, 24 PT-DLBCL and 451 systemic DLBCL tissues (unpublished). Third, RNA samples were extracted and sequenced for the targeted 1408 cancer-associated genes, successfully in 142 PCNSL cases, including 102 cases evaluated by either of these two mfIHC panels and additional 40 PCNSL cases, Results: First, in the first large mfIHC cohort, PCNSLs compared with de novo systemic nodal or extranodal DLBCL had significantly higher mean/median CD56+ cell densities whereas lower T cell densities (consistent with the immune privilege), as well as lower CD45RO+, PD-1+, and FOXP3+ percentages in T cells whereas higher CD4¯CD8¯ double-negative percentages in T cells. Prognostic analysis in the PCNSL cohort showed that high CD56+ cell densities and high CD45RO+CD8+ T cell densities were associated with significantly poorer and better patient survival, respectively, which are opposite to their prognostic effects in systemic DLBCL as previously reported. Twelve patients had very high PD-L1 expression in B cells and significantly poorer survival in the PCNSL cohort. Second, as validation, the 10 PCNSL cases in the second mfIHC study showed significantly less T cells than systemic DLBCL and PT-DLBCL cases, and low CD8 T cell densities and having low percentage of CD8 T cells adjacent to tumor cells were associated with significantly poorer survival. Immunofluorescence landscapeand spatial analysisrevealed significant differences in immune privilege between PCNSL and PT-DLBCL. Third, gene expression profiling analysis by targeted RNA-seq identified 282 significantly differentially expressed genes between PCNSL and systemic DLBCL, referred to as the PCNSL signature. Unsupervised clustering analysis divided the PCNSL cohort into two clusters, which had no significant difference in MYD88 mutation frequencies analyzed at the mRNA level. Importantly, the cluster with higher expression of many neural glial genes in the PCNSL signature had significantly poorer survival, which remained significant in multivariate analysis adjusting for clinical factors and/or treatments, two PCNSL subcohorts (as training and validation cohorts), and methotrexate-treated patients. NCAM1 (Neural Cell Adhesion Molecule 1, encoding CD56) was among the upregulated PCNSL signature genes, explaining the poorer prognosis associated with higher CD56+ cell densities in PCNSL by the mfIHC analysis. Summary: Our studies represent the largest immunophenotypic and gene expression profiling studies currently in PCNSL, a rare type of primary DLBCL of immune-privileged sites. Multiplex immunofluorescence revealed the immune landscape in PCNSL in comparison with systemic and testicular DLBCL, and identified prognostic immune biomarkers. Targeted RNA-seq identified a PCNSL gene signature enriched with neuronal-glial genes and showing significant prognostic effect in PCNSL. These novel findings in the PCNSL microenvironment warrant further studies.