Gene Expression Profiling of Primary Vitreoretinal Lymphoma

Background: Primary vitreoretinal lymphoma (PVRL) is a rare form of non-Hodgkin lymphoma, whose original lesions are limited in the intraocular tissues: the vitreous and the retina. Although its morphological and genetic findings show that most cases are diffuse large B-cell lymphoma (DLBCL), the characteristics of the tumor cells of PVRL, including their origin, have not been defined to date. DLBCL is subdivided into 3 groups: activated B-cell (ABC) type, germinal center B-cell (GCB) type, and primary mediastinal B-cell lymphoma on the basis of genes expressed in differentiation or biologic response of B-cell. In order to determine the origin of the tumor cells of PVRL, we performed gene expression profiling analysis.

Methods: PVRL was diagnosed using the following criteria: (1) typical eye involvement: a cloudy vitreous body and/or subretinal proliferative lesions, (2) presence of lymphoma cells in the vitreous fluid, and (3) clonality of the infiltrating lymphoma cells in the vitreous fluid using either PCR analysis of IgH gene rearrangements, or flow cytometry analysis. Patients who had (1) accompanied by either (2) or (3) were diagnosed with VRL. VRL confined to the eyes at diagnosis was defined as PVRL. We used the samples from nodal DLBCL whose types were pathologically determined according to Hans classifier (Blood, 103, p 275, 2004) as control. RNA was extracted from the vitreous fluid and the lymph nodes from PVRL and nodal DLBCL patients, respectively. Since the extracted RNA from PVRL was very small in amount, all RNA samples were amplified with WTA reaction. Then, one-color microarray-based gene expression analysis was performed using SurePrint G3 Human GE Microarray 8x60K v2 (Agilent Technology). Data were extracted and the agglomerative hierarchical clustering was performed on the basis of the genes discriminating GCB/ABC signatures that were published initially by Alizadeh et al. (Nature, 403, p503, 2000). Gene ontology (GO) analysis was performed using DAVID data base. We calculated Z-scores and ratios (non-log scaled fold-change) from the normalized signal intensities of each probe for comparison between 2 groups.

Results: We performed gene expression profiling analysis for 7 samples from PVRL patients. We also examined 6 samples from nodal DLBCL patients as control; 4 samples were ABC type, and 2 samples were GCB type. Six of 7 PRVL showed different gene expression profiling from ABC type nodal DLBCL. They were close to that of GCB type. Genes regulating cell adhesion were enriched by GO analysis for genes whose expression was different between PRVL and GCB. Next we examined the relation between the gene expression and the outcomes of PVRL. Four GCB-like PRVL samples were obtained from the patients treated with the same strategy: intravitreal administration of methotrexate (MTX) followed by systemic high dose MTX. Among them, 2 patients revealed early relapse in the CNS a year after the treatment whereas the others did not. We compared the gene expression between them. Three genes, SLC35F1, ADAM19, and SMC1A related to neurological disorders, cell migration, and cell division, respectively, were significantly upegulated in the PRVL with early relapse.

Conclusion: The majority of PVRL in the present study were different from ABC type and close to GCB type DLBCL. Further studies are required to confirm that and to determine the genes predicting the outcome.