Combined cellular and soluble mediator analysis for improved diagnosis of vitreoretinal lymphoma

Abstract

Purpose: Primary vitreoretinal lymphoma [(P)VRL]) is a rare malignancy of the eye localized in the retina, vitreous or choroid. Here, we aim to determine the value of the combination of innovative diagnostic methods for accurate differentiation between (P)VRL and non-(P)VRL in patients with suspect uveitis or vitritis.

Methods: Multicolour flow cytometric immunophenotyping of cells in the vitreous samples was performed using the EuroFlow small sample tube. Additionally, cytokines/chemokines and growth factors were measured in the vitreous specimens using a multiplex immunoassay. Data were evaluated in predefined clinical subgroups using omniviz unsupervised Pearson's correlation visualization and unsupervised heatmap analysis.

Results: A total of 53 patients were prospectively included in the period 2012-2015. In the (P)VRL subgroup (n = 10), nine cases showed aberrant surface membrane immunoglobulin (SmIg) light chain expression. In the non-(P)VRL group (n = 43) clearly skewed SmIg light chain expression was observed in two multiple sclerosis-related uveitis cases, but not in other uveitis types. Soluble mediator measurement revealed high interleukin (IL)-10/IL-6 ratios, and high IL-1RA levels in 9/10 (P)VRL cases, but not in any non-(P)VRL case. Further correlation and heatmap analysis revealed a minimal signature of cellular parameters (CD19+ B cells, aberrant SmIg light chain expression) and cytokine parameters (IL-10/IL-6 ratio >1, high IL-10, high IL-1 RA, high monocyte chemotactic protein-1, high macrophage inflammatory protein-1β) to reliably distinguish (P)VRL from non-(P)VRL.

Conclusion: Here, we show the power of a combined cellular and proteomics strategy for detecting (P)VRL in vitreous specimens, especially in cases with minor cellular (P)VRL infiltrates.

Keywords: diagnostics; immunology; multiparameter flow cytometry; soluble mediators; uveitis; vitreoretinal lymphoma.

Figure 1

Flow cytometric analysis of B cells in vitreous specimens. (A, B) primary vitreoretinal lymphoma; all CD19+ B cells (marked in red) show monotypic surface membrane immunoglobulin lambda (SmIgλ) expression; (C, D) Multiple sclerosis‐related uveitis; all CD19+ B cells (marked in red) show monotypic SmIgλ expression; (E, F) Uveitis in the context of sarcoidosis; CD19+ B cells either show SmIgκ (blue) or SmIgλ (purple) expression.

Figure 2

Omniviz‐based visualization of soluble mediators in vitreous specimens. (A) HeatMapper analysis of cytokine, chemokine, growth factor levels, including interleukin (IL)‐10/IL‐6 ratio, shows clustering of 9/10 primary vitreoretinal lymphoma [(P)VRL], while 1 (P)VRL (sample 25) does not cluster (red arrow). Colours represent different diagnosis: green: (P)VRL; blue: multiple sclerosis (MS)‐related uveitis; yellow: other uveitis. (B) Same analysis with top seven of cellular and soluble mediator parameters shows clustering of 9/10 (P)VRL, except for the same (P)VRL as in (A) (sample 25; red arrow). Notably, the (P)VRL with a small B‐cell infiltrate (samples 22, 23, 24) form a subcluster, with coclustering of one non‐(P)VRL case (sample 45). Colours represent different diagnosis: green: (P)VRL; blue: MS‐related uveitis; yellow: other uveitis.