VR09 cell line: an EBV-positive lymphoblastoid cell line with in vivo characteristics of diffuse large B cell lymphoma of activated B-cell type

Abstract

Background: small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro.

Design and method: we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features.

Results: VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2(-/-) γ-chain(-/-) mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/- CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, λ- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53.

Conclusion: This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features.

Figure 1. Morphology of primary malignant cells and VR09 cell line.

Morphological features of patient’s primary cells and VR09 cells in suspension, as assessed by May-Grünwald-Giemsa staining. (A) Patient’s peripheral blood cells. (B) Patient’s bone marrow cells. (C) VR09 cells: small and round clumps in suspension. (D) VR09 cells: plasmacytoid appearance, with irregular nucleus, compact chromatin and abundant basophilic cytoplasm.

Figure 2. Immunophenotype.

Immunophenotyping of VR09 cell line (grey and filled curves) and cell suspensions from in vivo tumor mass (black and filled curves), as compared with isotype control (white curves).

Figure 3. Cell line growth in vivo. (

A) Spherical subcutaneous mass (arrow) in Rag-2^−/− γ-chain^−/− mice 36 days after VR09 cell line injection (representative case). (B) Timing of tumor development in vivo (6 mice s.c. injected with 5×10⁶ cells/mouse).

Figure 4. Representative histological markers detected on tumors.

High magnification (20X) of histological sections of tumors developing after in vivo injection of VR09 cell line. Lymphoid infiltrates display large size, plasmablastic-plasmacytic features and high Ki-67 index. Cells express CD19, CD20, CD138, CD79a, IgM, IgG and EBV protein (EBER).

Figure 5. EBV positivity in the original tumor and in the VR09 cell line.

PCR products analysis by Agilent 2100 Bioanalyzer showed the presence of the same 151 bp specific amplicon for EBV RPMS1 gene, thus demonstrating that EBV infection was present in the original cells from patient. A normal DNA from pancreas was used as negative control.

Figure 6. Karyotyping and FISH of secondary culture.

(A) Trisomy of chromosome 12 and structural chromosome aberration involving chromosomes 7 and 9 on secondary culture detected by karyotype; (B) Chromosome 9 staining by FISH of secondary culture.