In Vitro and In Vivo Model of EBV-Positive Non-Hodgkin Plasmablastic Lymphoma with Focal Plasmacytic Differentiation
Authors: Nichele Ilaria , Zamò Alberto , Bertolaso Anna , Bifari Francesco , Tinelli Martina , Franchini Marta , Stradoni Roberta , Aprili Fiorenza , Pizzolo Giovanni , Krampera Mauro 
Published: October 2019

Abstract
Abstract 2668

Introduction.
Diffuse large B cell lymphoma (DLBCL) represents a group of aggressive lymphoid neoplasms with morphological variants. Plasmablastic lymphoma has been recently classified as a clinical-pathological variant with plasmablastic features and terminal B-cell differentiation. Diffuse large B-cell lymphoma with both plasmablastic and plasmacytic features has been previously reported, but cell lines and animal models have been never described. However, the use of human lymphoma cell lines and animal models contributes significantly to a better understanding of the pathophysiology of hematopoietic tumors.
We established and characterized a new plasmablastic lymphoma cell line with plasmacytic differentiation and we tested its tumorigenicity in vivo.

Methods.
Mononuclear cells from human DLBCL bone marrow, were seeded at concentration of 1×106/mL in RPMI 10% FBS medium added with 1% of antibiotics. Cells showed spontaneous proliferative capacity and a cell line (VR09) was established after a few months of continuous culture. VR09 cells were evaluated by May-Grünwald-Giemsa staining, flow cytometry, immunohistochemistry, molecular biology, standard karyotyping and fluorescence in situ hybridization (FISH). Epstein-Barr Virus (EBV) positivity was also tested. Moreover, cells were inoculated subcutaneously into 6 immunodeficient Rag2−/−γ-chain−/− mice. Subcutaneously-growing tumors were evaluated by immunohistochemistry and FISH. Disaggregated cells from masses were cultured again to confirm their biological features and proliferative capacity.

Results.
Cells in suspension formed large clumps with round shape by ten weeks of continuous culture. Cells appeared morphologically as medium/large-size cells with plasmablastic/plasmacytic appearance and, sometimes, bizarre shape. They had a round-ovoid or irregular nucleus with compact chromatin, no nucleoli and an abundant basophilic cytoplasm.
When subcutaneously injected in mice, VR09 cells grew as an isolated spherical tumor (100% incidence). Moreover, cells maintained their proliferative capacity once disaggregated from mass and re-cultured.
Cells displayed the phenotype of plasmablastic lymphoma with secretory differentiation (CD19+ CD3- CD5- CD20+ CD79a+ CD79b+/− CD138+ CD38+ cycline D1- Ki6780% IgM+ IgD+ MUM1+ MDNA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-). The same markers were detected on tissues derived from growing tumors and cell suspension.
FISH analysis revealed the trisomy of chromosome 12 and the negativity for the MYC rearrangement both in VR09 cell line and in tumors. Cell suspension from mass showed an additional structural chromosome aberration involving chromosomes 7 and 9.
EBER probe detected episomal EBV genome in both VR09 cell line and tumors.
Genetic analysis in VR09 cell line showed the somatic hypermutation in the VH region, wild type p53 and a novel synonymous mutation of CD79B-variant 2, involving the insertion of alanine in the exon 2.

Conclusions.
We have established and characterized a new plasmablastic EBV-positive cell line with focal plasmacytic differentiation and with tumorigenic potential in immunodeficient mice. VR09 cell line represents a model for in vitro and in vivo studies regarding pathogenesis and drug sensitivity.

Disclosures:
No relevant conflicts of interest to declare.