Correlation between measurement of IL‐10 and IL‐6 in paired aqueous humour and vitreous fluid in primary vitreoretinal lymphoma

Editor, P rimary vitreoretinal lymphoma (PVRL) is a rare and aggressive malignancy almost always of large diffuse B-cell origin that predominantly arises in the retina and adjacent structures (Chan et al. 2011). PVRL often masquerades as uveitis and is easily misdiagnosed. As PVRL is potentially lethal, early and accurate diagnosis is crucial for adequate treatment and prognosis. The gold standard for diagnosing PVRL requires detection of malignant lymphoid cells in vitreous or retinochoroidal biopsies that can be supported by flowcytometric evidence of monoclonality of lymphocytes in vitreous fluid (VF) (Gonzales & Chan 2007). However, outcome of cytology is frequently false negative due to fragility of cells in obtained specimens. An enzyme-linked immunosorbent assay (ELISA) measured vitreal IL-10 to IL-6 ratio greater than 1.0 is currently considered to be typical for PVRL over non-infectious uveitis (Wolf et al. 2003). Here, we present a less invasive alternative using modern multicytokine analysis on aqueous humour (AqH) instead of VF. Paracentesis to obtain AqH of the anterior chamber is safe and less invasive compared to collection of VF. However, the AqH samples consist of smaller volumes compared to undiluted VF and potentially limit the evaluation of both IL-10 and IL-6 levels by ELISA. The emerging use of multiplex bead-based array platform (Luminex), however, allows the detection and evaluation of multiple cytokines in small sample volume (<25 ll) (de Jager et al. 2003). Exploiting this exciting technique, we measured IL-10 and IL-6 levels in paired AqH and VF samples of patients (n = 11) with proven PVRL between 2005 and 2014, to investigate whether the use of AqH could replace VF. The samples were collected during diagnostic vitrectomy to confirm the diagnosis PVRL, and the remainders of the samples were used to measure IL10 to IL-6 ratios with approval from the medical–ethical institutional review board. The diagnosis of PVRL was based upon cytology/immunocytology of vitreous cells, histopathology of retinal biopsy or proven CNS lymphoma. None of the patients was treated for PVRL with intra-ocular methotrexate, rituximab, systemic chemotherapy or irradiation of the eye during/before the diagnostic vitrectomy. The samples were preserved at minus 80°C degrees immediately after harvesting. IL-10 and IL-6 levels were measured in 25 ll of undiluted sample. Correlation of the biomarkers with AqH and VF was analysed by computing the nonparametric Spearman’s rank correlation coefficient. The female/male ratio was 4/7 and the mean age of developing of lymphoma was 68.8 years (range 63– 82 years). All patients suffered from PVRL and two patients had additional CNS involvement. The geometric mean of the levels of IL-10 in AqH was 863.6 (95% CI: 169.3–4405) and in VF was 2188 (95% CI: 607.6–7880). The mean of the levels of IL-6 in AqH was 81.9 (95% CI: 45.7–147.1) and in VF was 79.1 (95% CI: 40.7–153.6). The levels of IL-10 (r = 0.92; p = 0.0002) and the levels of IL-6 (r = 0.83, p = 0.003) in paired AqH and VF strongly correlated. Accordingly, the IL-10 to IL-6 ratio in the paired AqH and VF correlated (r = 0.73, p = 0.01) (Fig. 1). The individual levels and the ratio of IL-10 and IL-6, in paired AqH and VF of PVRL patients, strongly correlate and demonstrate that the relative proportion of IL-10 to IL-6 is similar for both AqH and VF. We emphasize that the general opinion is that definite diagnosis must be made by cytologic or histopathologic evaluation of vitreous or retinochoroidal specimens that can be supported by IL-10 to IL-6 ratio. The present results suggest that the AqH IL-10 to IL-6 ratio may be as useful as the previously suggested vitreal IL-10 to IL-6 ratio and, thus, provides a less invasive alternative for the evaluation of these cytokines to support histopathology and immunocytologic evidence, or follow-up after treatment, for PVRL.