Ocular Immunology & Inflammation, 2014; 22(1): 34–41 ! Informa Healthcare USA, Inc. ISSN: 0927-3948 print / 1744-5078 online DOI: 10.3109/09273948.2012.752507
ORIGINAL ARTICLE
Vitreoretinal Lymphoma versus Uveitis: Cytokine Profile and Correlations Juline N. Caraballo, MD1, Melissa R. Snyder, PhD2, Patrick B. Johnston, MD, PhD3, Brian P. O Neill, MD4, Harish Raja, MD1, Joseph G. Balsanek, MS2, Brian E. Peters, BS2, and Jose S. Pulido, MD, MBA, MPH, MS1 1
Department of Ophthalmology, 2Department of Laboratory Medicine and Pathology, Immunology Laboratory, 3 Hematology Division, and 4Department of Neurology,Mayo Clinic, Mount Sinai School of Medicine, Rochester, MN
ABSTRACT Purpose: To compare the cytokine spectrum of vitreoretinal lymphoma to uveitis and correlate cytokine concentrations with disease activity. Methods: Retrospective case series of 10 patients with vitreoretinal lymphoma and 7 patients with uveitis. Aqueous humor concentration of IFN-g, TNF-a, TNF-b, IL-1ra, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, MCP-1, G-CSF, GM-CSF, and VEGF-A was determined using a bead-based assay (Luminex). Variance between groups, correlation coefficients, and longitudinal behavior of cytokines were analyzed. Results: No statistically significant difference in cytokines was found when comparing groups. IL-10 was positively correlated with IL-6 and MCP-1. IL-6 was positively correlated with G-CSF, IL-1ra, IL-8, and IL-10. A relationship between concentration of any cytokine, aside from IL-10, and disease activity was not found. Conclusion: IL-10 and IL-6 are good immunologic markers to be used as complementary diagnostic tools. IL-10 is the only IL that could be used for monitoring purposes. Keywords: Cytokines, interleukin-6, interleukin-10, uveitis, vitreoretinal lymphoma
Primary vitreoretinal lymphoma (PVRL) is the most common intraocular lymphoma; in most cases, it represents a high-grade B-cell non-Hodgkin lymphoma, usually a diffuse large B-cell lymphoma (DLBCL).1 PVRL is considered a subset of primary central nervous system lymphoma (PCNSL).2 PVRL may present in isolation or in association with central nervous system (CNS) involvement. Approximately 15–25% of patients with PCNSL will develop vitreoretinal lymphoma,3,4 while 60–80% of patients with PVRL will have progression to CNS lymphoma.4,5 Vitreoretinal lymphoma with CNS involvement represents about 1% of intracranial tumors and less than 1% of intraocular tumors.6 Because it is so uncommon, the true incidence is difficult
20 14
to estimate, but an increased number of cases in immunocompetent, immunosuppressed, and immunodeficient individuals have been reported in the past two decades.7,8 PVRL diagnosis represents a challenge. It is frequently misdiagnosed as posterior uveitis, and these patients show some response to topical corticosteroids, which confounds the diagnosis even more.9 The reported time interval from the onset of ocular symptoms to the final diagnosis ranges from 6 to 24 months,2,10 sometimes diagnosis can be achieved only when the progression to brain involvement has occurred. This highlights the importance of an accurate and early diagnosis of this aggressive malignancy. The techniques used for the diagnostic study of PVRL
Received 5 June 2012; revised 19 November 2012; accepted 20 November 2012; published online 31 January 2014 Correspondence: Jose S. Pulido MD, MBA, MPH, MS, Mayo Clinic, Department of Ophthalmology 200 First Street, SW Rochester, MN 55905. Phone: 507-284-3721 / Fax: 507-284-4612. E-mail: [email protected]
34
Vitreoretinal Lymphoma versus Uveitis include morphological analysis of vitreous and chorioretinal biopsies with additional immunohistochemistry or flow cytometry, molecular analysis using PCR for immunoglobulin rearrangements, as well as biochemical studies such as cytokines assays, in particular, IL-10:IL-6 ratio.11–13 Although cytology is the cornerstone of the diagnostic workup of PVRL, it has a high false-negative rate due to the fragility of the malignant cells, which suffer rapid degradation if the sample is not stored, transported, and handled correctly.10 Alternatively, corticosteroid therapy before the vitrectomy, seen in those patients, may cause paucity of lymphoma cells in the sample, increasing the difficulty of diagnosis.14 This is why significant effort has gone toward developing more specific and sensitive diagnostic tools. The cytokine analysis for diagnostic workup of PVRL is based primarily on the study of intraocular concentrations of IL-6 and IL-10. The IL-6 levels are elevated predominantly in inflammatory conditions like uveitis, while the IL-10 levels are increased in B-cell lymphoma, acting as a cell-mediated immunity suppressor. An IL-10:IL-6 ratio 41.0 is highly suggestive of PVRL15–17 and this can be measured in the vitreous fluid as well as in the aqueous humor due to diffusion of interleukins from the posterior to the anterior chamber.18 Even though the determination of IL-10 and IL-6 concentration in the anterior chamber appears to have insufficient sensitivity and specificity to be used as the sole diagnostic tool,19 we have recently demonstrated that IL-10 concentration in aqueous humor correlates with disease activity and therefore may be useful to evaluate the effect of treatment in PVRL20. As no studies have examined aqueous concentration of cytokines other than IL-10 and IL-6 in PVRL, the purpose of this study was to analyze, in samples from patients with PVRL and uveitis, other cytokines that have been linked to inflammatory processes in the eye,21 comparing interleukin concentration with disease activity and treatment history, as well as their correlations with IL-10 and IL-6, to determine if they can be used as a complementary diagnostic tool or follow-up method when PVRL is suspected.
35
second consisted of 10 patients with diagnosis of vitreoretinal lymphoma (VRL). The diagnosis of VRL or uveitis was made on the basis of their clinical history, ocular exam, and vitreoretinal cytopathology. A diagnosis of VRL was made only if there was histologic confirmation either from an ocular or other tissue source. For better interpretation of the study’s results, the samples from the VRL group were divided into 2 groups: active and inactive vitreoretinal lymphoma, depending on the eye exam findings from within a week prior to the day the anterior chamber paracentesis was performed. Active disease was defined by vitreous cell count 1+ or qualitative description of findings using the words ‘‘some,’’ ‘‘significant,’’ and ‘‘dense.’’ Conversely, inactive disease was considered when the findings were 51+ vitreous cells or the description included words like ‘‘rare’’ or ‘‘trace.’’ Disease progression and treatment history information was collected by reviewing the patient’s medical record. Aqueous humor samples were obtained by anterior chamber paracentesis at multiple times during the evolution of disease, with removal of 0.1 mL of aqueous humor each time for analysis. The concentration of cytokines in the undiluted samples of aqueous humor was determined using a bead-based assay run on a Luminex-based platform (manufacturer: Affymetrix). Linearity, limit of detection was used to establish the reportable range, and inter- and intraassay precisions were verified. Cytokines measured were interferon-g (IFN-g), tumoral necrosis factor a and b (TNF-a and TNF-b), interleukin (IL)-1 receptor antagonist (IL-1ra), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, monocyte chemotactic protein (MCP-1), granulocyte colony stimulating factor (G-CSF), granulocyte/monocyte colony stimulating factor (GM-CSF), and vascular endothelial growth factor-A (VEGF-A). Detection limits were 50.56 pg/mL for IL-2, IL-4, IL-5, IL-8, IL-10 and IL-12(p40); 51.67 pg/mL for IL-6, IL-7, IL-12(p70), IL-13, IL-17, IL-1b, IL-1ra, and VEGF-A; 51.75 pg/mL for G-CSF; 55 pg/mL for GM-CSF, IFN-g, TNF-a, and TNF-b; and 41215 mg/mL for MCP-1.
Statistical Analysis PATIENTS AND METHODS This retrospective study was performed with the approval of the institutional review board of Mayo Clinic. Patients were evaluated and diagnosed in the Ocular Oncology Service of the Department of Ophthalmology at Mayo Clinic (Rochester, Minnesota, USA). Seventeen patients participated in the study. The patients were sorted into 2 groups: the first comprised 7 patients diagnosed with uveitis, and the !
2014 Informa Healthcare USA, Inc.
Univariate analysis was applied to the data using statistical software from Wassa P.22 The active vitreoretinal lymphoma (AVRL) group, inactive vitreoretinal lymphoma (IVRL) group, and uveitis group were compared using one-way ANOVA test. To assess the correlations between cytokines in each group, Pearson’s correlation coefficient (r) and determination coefficient (r2) were calculated. Twotailed test results were statistically significant at p50.05. Significant correlations were defined by r40.7 (r240.5).
36 J. N. Caraballo et al.
RESULTS A total of 34 aqueous humor samples were obtained and analyzed and distributed as follows: 25 samples from 10 patients with diagnosis of VRL and 9 samples from 7 patients with diagnosis of uveitis. Considering the criteria described above, aqueous humor samples obtained from the patients with VRL were later classified into 2 groups: AVRL group (17 samples) and IVRL group (8 samples). The median, range, mean, and standard deviation for each cytokine in the three groups are summarized in Table 1.
Aqueous Humor Cytokine Levels We previously studied the IL-10 and IL-6 levels in this case series, and our results showed that the median of IL-10 level in aqueous samples in the AVRL group was markedly higher when compared to the uveitis group. Conversely, the uveitis group showed higher aqueous IL-6 concentrations than those reported in the AVRL group and IVRL group20. The rest of the cytokines and their concentrations in the aqueous humor samples of the AVRL group, IVRL group, and uveitis group were compared, and no significant difference was noted in any of the cytokine levels between the disease groups by ANOVA (p40.05). Subgroup analysis was then performed. We observed that IL-1ra showed a broader range of concentrations in the aqueous humor of AVRL group (51.67– 883.08 pg/mL) when compared with the IVRL (51.67–106.53 pg/mL) and the uveitis group (51.67– 62.25 pg/mL) (Figure 1A). Concentrations of IL-8 were higher in the uveitis group (median 2.94 pg/ mL, mean 6.0 ± 6.7 pg/mL) than levels in the AVRL group (median 1.29 pg/mL, mean 2.88 ± 5.26 pg/mL) and IVRL group (median 1.5 pg/mL, mean 1.7 ± 1.59 pg/mL) (Figure 1B). VEGF-A levels had a median of 8.84 pg/mL (mean 14.9 ± 11.4 pg/mL) in AVRL group, 13.07 pg/mL (mean 15.57 ± 7.61 pg/mL) in IVRL group, and 18.28 pg/mL (mean 20.83 ± 17.48 pg/mL) in uveitis group, with no statistical difference noted between them (Figure 1C). G-CSF showed a higher concentration in the uveitis group (median 14.72 pg/mL, mean 51.48 ± 107.99 pg/mL) when compared with the AVRL group (median 51.75 pg/mL, mean 20.27 ± 74.6 pg/mL) and the IVRL group (median 51.75 pg/mL, mean 0.67 ± 1.17 pg/mL). Likewise, a broader range of concentrations was detected in the uveitis group (51.75–353.67 pg/mL) than in the other two groups (Figure 1D). The aqueous humor concentration of GM-CSF, IL-12 (p70), and IL-13 were below detection limits in all samples from the three groups. The concentrations of TNF-a, TNF-b, IFN-g, IL-1b, IL-2, IL-4, IL-5, and IL-17 were rarely positive and showed borderline
concentrations when compared with the lower detectable limit.
Correlations AVRL Group Statistically significant positive correlations were observed between G-CSF and IL-6 (r = 0.7, p50.001, r2 = 0.5), and IL-1ra and IL-6 (r = 0.7, p50.001, r2 = 0.5) (Figure 2). IVRL Group Analysis of the data showed positive correlations between MCP-1 and IL-12p40 (r = 0.9, p50.001, r2 = 0.8), IL-8 and IL-12p40 (r = 0.7, p50.05, r2 = 0.5), IL-8 and IL-6 (r = 0.7, p50.05, r2 = 0.5). Uveitis Group This group showed positive correlations between MCP-1 and IL-12p40 (r = 0.8, p50.05, r2 = 0.6), MCP-1 and IL-1ra (r = 0.8, p50.05, r2 = 0.6), MCP-1 and IL-10 (r = 0.8, p50.05, r2 = 0.6), IL-8 and IL-12p40 (r = 0.7, p50.05, r2 = 0.5), IL-10 and IL-6 (r = 0.7, p50.05, r2 = 0.5) (Figure 3). No other significant correlation could be detected for analyzed cytokines in aqueous humor of patients with AVRL, IVRL, or uveitis.
Effect of Treatment in Cytokine Profile We previously reported the IL-10 concentration in aqueous humor in 3 cases with diagnosis of vitreoretinal lymphoma to serial treatment with methotrexate (MTX) and rituximab (RTX) administered by intraocular injections. Derived from those results, we concluded that IL-10 levels in aqueous humor correlated with disease activity and could possibly be used as monitoring instrument for therapy response20. In this study we used those 3 patients and analyzed the longitudinal behavior of the cytokines in relation to treatment administration and disease activity. In one of the patients we observed that aqueous humor IL-6 and IL-8 were correlated with treatment (Figure 4), but this finding wasn’t consistent in the other patients. Even though some changes were observed in the aqueous concentration of cytokines in relation to treatment administration, those changes were variable in each patient and, as opposed to IL-10 levels, there was no definite pattern of response with treatment.
DISCUSSION Due to the challenge that diagnosis of vitreoretinal lymphoma represents and its high rate of relapse, Ocular Immunology & Inflammation
!
2014 Informa Healthcare USA, Inc.
51.75 55 55 51.67 41215 55 55 51.67 51.67 50.56 50.56 50.56 51.67 51.67 50.56 50.56 50.56 51.67 51.67 51.67
0.42 1.00 0.47 0.54 0.55 0.50 0.62 0.62 0.62 0.21 0.76 0.80 0.18 0.07 0.23 0.23 0.56 1.00 1.00 0.21
p Value 51.75 55 55 8.84 165.7 55 55 51.67 4.43 50.56 50.56 50.56 88.19 2.37 1.29 9.21 1.09 51.67 51.67 51.67
Median 51.75–318.49 – 55–32.53 2.69–39.37 9.2–41215 55–11.48 55–5.04 51.67–2.08 51.67– 883.08 50.56–4.28 50.56–1.56 50.56–1.05 51.67–41215 51.67–7.2 50.56–17.88 50.56–381.44 50.56–14.89 – – 51.67–2.98
Range
Cytokine concentrations are expressed in picogram/milliliter (pg/ml).
G-CSF GM-CSF IFN-g VEGF MCP-1 TNF-a TNF-b IL-1b IL-1ra IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-12(p40) IL-12(p70) IL-13 IL-17
Detection limit
AVRL
20.27 ± 74.6 – 2.68 ± 7.75 14.9 ± 11.4 259.38 ± 280.41 1.17 ± 3.25 0.29 ± 1.18 0.12 ± 0.48 69.06 ± 208.23 0.39 ± 1.04 0.09 ± 0.36 0.5 ± 1.73 206.77 ± 337.08 2.1 ± 1.95 2.88 ± 5.26 42.29 ± 92.59 2.89 ± 4.34 – – 0.65 ± 1.05
Mean ± SD 51.75 55 55 13.07 153.1 55 55 51.67 3.77 50.56 50.56 50.56 8.77 3.95 1.5 50.56 1.4 51.67 51.67 51.67
Median 51.75–2.99 – – 5.39–27.95 22.36–257.16 – – – 51.67–106.53 – – 50.56–1.06 1.94–356.58 51.67–5.98 50.56–5.25 50.56–9.59 50.56–3.62 – – 51.67–2.11
Range
IVRL
0.67 ± 1.17 – – 15.57 ± 7.61 158.82 ± 76.89 – – – 18.2 ± 33.96 – – 0.13 ± 0.35 84.61 ± 135.62 3.67 ± 1.85 1.7 ± 1.59 1.19 ± 3.17 1.87 ± 1.15 – – 0.51 ± 0.89
Mean ± SD 14.72 55 55 18.28 236.1 55 55 51.67 9.25 50.56 50.56 50.56 232.16 1.92 2.94 50.56 3.4 51.67 51.67 51.67
Median
51.75–353.67 – 55–32.49 51.67–63.6 19.86–650.34 55–7.57 – – 51.67–62.25 50.56–2.78 50.56–0.58 50.56–1.05 28.33–936.24 51.67–3.99 0.69–22.98 50.56–4.84 50.56–9.37 – – –
Range
Uveitis
51.48 ± 107.99 – 4.8 ± 10.34 20.83 ± 17.48 273.31 ± 208.81 1.59 ± 2.99 – – 15.95 ± 18.29 0.88 ± 1.25 0.06 ± 0.18 0.4 ± 0.46 373.67 ± 351.14 1.54 ± 1.53 6.0 ± 6.7 1.03 ± 1.61 3.78 ± 2.85 – – –
Mean ± SD
TABLE 1. Detection limit, p value, median, range, mean and standard deviation (SD) of cytokines concentration in aqueous humor samples of active vitreoretinal lymphoma (AVRL) group, inactive vitreoretinal lymophoma (IVRL) group and uveitis group.
Vitreoretinal Lymphoma versus Uveitis 37
38 J. N. Caraballo et al.
FIGURE 1. Boxplot graphs showing aqueous humor concentrations of (A) IL-1ra, (B) IL-8, (C) VEGF, and (D) G-CSF in patients with uveitis, active vitroretinal lymphoma (AVRL), and inactive vitreoretinal lymphoma (IVRL). X’s are outliers.
FIGURE 2. Dispersion graphs showing aqueous humor concentration of (A) G-CSF versus IL-6 and (B) IL-1ra versus IL-6 in patients with active vitreoretinal lymphoma.
significant effort has gone to develop more specific and sensitive diagnostic tools, as well as disease monitoring methods. To our knowledge, only one study analyzing aqueous concentration of cytokines other than IL-10 and IL-6 in vitreoretinal lymphoma has been published, and that was during the revision phase of this article.23 As opposed to our study, these authors evaluated the cytokines in the vitreous at the time of diagnosis. They had 21 Asian patients and 27 controls. Their controls were patients that had
structural disease in the retina and did not have uveitis, and thus no cellular infiltration of the vitreous. Interestingly, they found elevation of cytokine levels similar to ours, but since they did not have a uveitis control group, they could only state that there was a difference compared to their nonuveitis group. While their data are important, our study compares the levels to those in patients with uveitis, since it is this group that is most often confused with VRL. Ocular Immunology & Inflammation
Vitreoretinal Lymphoma versus Uveitis
39
FIGURE 3. Dispersion graphs showing AqH concentration of (A) MCP-1 versus IL-12p40, (B) MCP-1 versus IL-1ra, (C) MCP-1 versus IL-10, (D) IL-8 versus IL-12p40, and (E) IL-10 versus IL-6 in patients with uveitis.
FIGURE 4. Longitudinal measurements of IL-10, IL-8, and IL-6 in one case with vitreoretinal lymphoma. Time of administration of intraocular methotrexate and rituximab in indicated (arrows).
We measured and compared the concentrations of other known cytokines in the aqueous humor of patients with vitreoretinal lymphoma and uveitis where we had also obtained IL-10 and IL-6 levels. No statistically significant differences in the aqueous concentration of these additional cytokines were found when comparing the 3 groups (active and inactive vitreoretinal lymphoma and uveitis). Our data showed that measurement of these cytokines was not sufficient to differentiate lymphomatous from uveitic inflammation in the eye. However, we previously observed there was a difference in median aqueous concentration of IL-6 and IL-10 when comparing the active vitreoretinal lymphoma group with the uveitis group (manuscript submitted). We found a positive correlation of IL-10 with IL-6 and MCP-1 in the uveitis group. IL-6 showed a positive correlation with G-CSF and IL-1ra in the active !
2014 Informa Healthcare USA, Inc.
vitreoretinal lymphoma group, with IL-8 in the inactive vitreoretinal lymphoma group, and with IL-10 in the uveitis group. Alternatively, none of the cytokines analyzed showed changes related to treatment response, being inadequate for monitoring disease activity, opposed to what we have previously observed with IL-10 concentrations in aqueous humor. Measurements of IL-10 and IL-6 in the aqueous humor are good complementary tools in the diagnostic workup of PVRL.18,24 IL-10 shows higher concentrations in active vitreoretinal lymphoma.25 Conversely, IL-6 shows higher concentrations in uveitis.13,26 Even though in our previous study we did not find any statistically significant difference between the groups, median concentrations of IL-10 and IL-6 were remarkably different when we compared the AVRL group and the uveitis group. Similar
40 J. N. Caraballo et al. behavior to IL-6 was observed in the concentrations of G-CSF, which was higher in the uveitis group. G-CSF is a growth factor that stimulates bone marrow and proliferation of granulocytes and it is supposed to be increased in inflammatory responses like uveitis. Alternatively, IL-8 is a major proinflammatory cytokine and constitutes a general marker of active uveitis; we detected IL-8 in higher concentrations in the uveitis group. These results correlate with those described by Valentinincic et al.,26 who determined several interleukin levels in serum and aqueous humor of patients with different uveitis entities and found increased IL-8 and IL-6 levels in uveitis group when compared to control groups. The concentration of VEGF-A was higher in the uveitis group, with no statistical difference noted between the three groups. VEGF, through its promotion of angiogenesis and increased vascular permeability, plays an important role in the pathogenesis of uveitis and uveitic complications, such as cystoid macular edema (CMO), choroidal neovascularization (CNV), and retinal neovascularization (RNV).27 In uveitis, there is a major inflammatory process with expression of multiple cytokines, including IL-6, which can upregulate the expression of VEGF through various molecular pathways.28 This would explain why the concentration of VEGF is higher in the uveitis group, in comparison to the AVRL group, where the IL-6 concentration is lower due to prevalence of an anti-inflammatory status. The concentrations of VEGF-A found in our study were lower than those reported in age-related macular degeneration29,30 and diabetic retinopathy (nonproliferative and proliferative).31,32 In the AVRL group, IL-10 did not appear to be directly or indirectly associated with other cytokines, but it showed positive association with IL-6 and MCP1 in the uveitis group. MCP-1 is a chemoattractant cytokine that has been found to be elevated in aqueous humor in active stages of anterior uveitis.33 Some studies have hypothesized that the increase of IL-10 in uveitis is a response to control the inflammation.34 The levels of IL-10 in vitreous and aqueous humor samples have been shown to be correlated with the active vitreoretinal lymphoma.35,36,25 Sou et al.35 reported the vitreous concentration of IL-10 in 6 patients (10 eyes) who underwent intravitreal injections with MTX and noted that once chemotherapy was completed with clinical remission of PVRL, the concentrations of IL-10 in the vitreous fluid reached barely detectable levels. We previously analyzed the IL-10 levels in aqueous humor in 3 patients with PVRL receiving intraocular chemotherapy with MTX and RTX and observed that IL-10 aqueous levels correlate quite well with disease activity (reflected by number of cells in vitreous found in ophthalmologic examination). This could possibly be used as a monitoring
instrument for treatment response (manuscript submitted). In this study we analyzed the longitudinal measurements of multiple cytokines in those 3 patients. The changes observed in the different cytokine concentrations in the aqueous humor in response to treatment administration were not consistent in every patient. No other cytokine was found to have similar response to treatment as does IL-10. The analysis of cytokines in aqueous humor of patients with suspected PVRL is a relatively simple process that can provide us with useful information. The study of immunologic markers like cytokines allows us to better understand the pathogenic features of PVRL, and this study shows that IL-10 and IL-6 are the best immunologic markers to be used as complementary diagnostic and monitoring tools for vitreoretinal lymphoma. PVRL is an uncommon malignancy, which makes its study very difficult. This is why the majority of the studies, including ours, have small sample sizes. As shown recently by Usui et al. in an Asian cohort, though, it appears that some of the cytokines aside from IL-10 might be increased in VRL as compared to controls without cellular infiltration of the vitreous. Only IL-10 levels appear to be important as a way to follow treatment and to help with the diagnosis of VRL.23 Larger collaborative series with longitudinal measurement of aqueous humor interleukin concentrations and their response to treatment are necessary to verify these findings.
DECLARATION OF INTEREST This study was performed at the Mayo Clinic, Rochester, MN and was supported in part by an unrestricted grant from Research to Prevent Blindness, Inc, NY, and from the Mayo Cancer Center. The authors have no proprietary interests in this work.
REFERENCES 1. Coupland SE, Damato B. Understanding intraocular lymphomas. Clin Exp Ophthalmol. 2008;36:564–578. 2. Grimm SA, Pulido JS, Jahnke K, et al. Primary intraocular lymphoma: an International Primary Central Nervous System Lymphoma Collaborative Group report. Ann Oncol. 2007;18:1851–1855. 3. Choi JY, Kafkala C, Foster CS. Primary intraocular lymphoma: a review. Semin Ophthalmol. 2006;21:125–133. 4. Chan CC, Rubenstein JL, Coupland SE, et al. Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium. Oncologist. 2011;16: 1589–1599. 5. Akpek EK, Ahmed I, Hochberg FH, et al. Intraocularcentral nervous system lymphoma: clinical features, diagnosis, and outcomes. Ophthalmology. 1999;106:1805–1810. Ocular Immunology & Inflammation
Vitreoretinal Lymphoma versus Uveitis 6. Bardenstein DS. Intraocular lymphoma. Cancer Control. 1998;5:317–325. 7. Schabet M. Epidemiology of primary CNS lymphoma. J Neurooncol. 1999;43:199–201. 8. Corn BW, Marcus SM, Topham A, et al. Will primary central nervous system lymphoma be the most frequent brain tumor diagnosed in the year 2000? Cancer. 1997;79: 2409–2413. 9. Rothova A, Ooijman F, Kerkhoff F, et al. Uveitis masquerade syndromes. Ophthalmology. 2001;108:386–399. 10. Merle-Beral H, Davi F, Cassoux N, et al. Biological diagnosis of primary intraocular lymphoma. Br J Haematol. 2004;124:469–473. 11. Davis JL. Diagnosis of intraocular lymphoma. Ocul Immunol Inflamm. 2004;12:7–16. 12. Coupland SE, Bechrakis NE, Anastassiou G, et al. Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with masquerade syndrome. Graefes Arch Clin Exp Ophthalmol. 2003;241:860–870. Epub 2003 Sep 30. 13. Chan CC, Wallace DJ. Intraocular lymphoma: update on diagnosis and management. Cancer Control. 2004;11: 285–295. 14. Cassoux N, Merle-Beral H, Leblond V, et al. Ocular and central nervous system lymphoma: clinical features and diagnosis. Ocul Immunol Inflamm. 2000;8:243–250. 15. Buggage RR, Whitcup SM, Nussenblatt RB, et al. Using interleukin 10 to interleukin 6 ratio to distinguish primary intraocular lymphoma and uveitis. Invest Ophthalmol Vis Sci. 1999;40:2462–2463. 16. Chan CC, Gonzalez JA. Primary Intraocular Lymphoma. Singapore: World Scientific; 2007:1–267. 17. Chan CC. Molecular pathology of primary intraocular lymphoma. Trans Am Ophthalmol Soc. 2003;101:275–292. 18. Cassoux N, Giron A, Bodaghi B, et al. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007;48:3253–3259. 19. Akpek EK, Maca SM, Christen WG, et al. Elevated vitreous interleukin-10 level is not diagnostic of intraocular-central nervous system lymphoma. Ophthalmology. 1999;106: 2291–2295. 20. Raja H, Snyder MR, Johnston PB, et al. Effect of intravitreal methotrexate and rituximab on interleukin-10 levels in aqueous humor of treated eyes with vitreoretinal lymphoma. PLoS One. 2013 Jun 4;8(6):e65627. 21. Ooi KG, Galatowicz G, Calder VL, et al. Cytokines and chemokines in uveitis: is there a correlation with clinical phenotype? Clin Med Res. 2006;4:294–309.
!
2014 Informa Healthcare USA, Inc.
41
22. Wessa P. Free Statistic Software. Office for Research Development and Education, version 1.1.23-r7, 2012. 23. Usui Y, Wakabayashi Y, Okunuki Y, et al. Immune mediators in vitreous fluids from patients with vitreoretinal B-cell lymphoma. Invest Ophthalmol Vis Sci. 2012;53: 5395–5402. 24. Hirakata A, Inami T, Saito M, et al. Diagnostic value of interleukin-10 and interleukin-6 in the vitreous of intraocular malignant lymphoma patients. Nihon Ganka Gakkai Zasshi. 2004;108:359–367. 25. Chan CC, Whitcup SM, Solomon D, et al. Interleukin-10 in the vitreous of patients with primary intraocular lymphoma. Am J Ophthalmol. 1995;120:671–673. 26. Valentincic NV, de Groot-Mijnes JD, Kraut A, et al. Intraocular and serum cytokine profiles in patients with intermediate uveitis. Mol Vis. 2011;17:2003–2010. 27. Gulati N, Forooghian F, Lieberman R, et al. Vascular endothelial growth factor inhibition in uveitis: a systematic review. Br J Ophthalmol. 2011;95:162–165. 28. Cohen T, Nahari D, Cerem LW, et al. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996 Jan 12;271(2):736–41. 29. Funk M, Karl D, Georgopoulos M, et al. Neovascular agerelated macular degeneration: intraocular cytokines and growth factors and the influence of therapy with ranibizumab. Ophthalmology. 2009;116:2393–2399. 30. Roh MI, Kim HS, Song JH, et al. Concentration of cytokines in the aqueous humor of patients with naive, recurrent and regressed CNV associated with amd after bevacizumab treatment. Retina. 2009;29:523–529. 31. Zhou L, Sun H, Xu J, et al. Level of vascular endothelial growth factor and interleukin-6 in aqueous humor in diabetic retinopathy patients. Yan Ke Xue Bao. 2010;25: 26–30. 32. Selim KM, Sahan D, Muhittin T, et al. Increased levels of vascular endothelial growth factor in the aqueous humor of patients with diabetic retinopathy. Indian J Ophthalmol. 2010;58:375–379. 33. Verma MJ, Lloyd A, Rager H, et al. Chemokines in acute anterior uveitis. Curr Eye Res. 1997;16:1202–1208. 34. Jabs DA, Nussenblatt RB, Rosenbaum JT. Standardization of uveitis nomenclature for reporting clinical data: results of the First International Workshop. Am J Ophthalmol. 2005; 140:509–516. 35. Sou R, Ohguro N, Maeda T, et al. Treatment of primary intraocular lymphoma with intravitreal methotrexate. Jpn J Ophthalmol. 2008;52:167–174. 36. Kawamura H, Yasuda N, Kakinoki M, et al. Interleukin-10 and interleukin-6 in aqueous humor during treatment of vitreoretinal lymphoma with intravitreally injected methotrexate. Ophthalmic Res. 2009;42:172–174.
Copyright of Ocular Immunology & Inflammation is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
ORIGINAL ARTICLE
Vitreoretinal Lymphoma versus Uveitis: Cytokine Profile and Correlations Juline N. Caraballo, MD1, Melissa R. Snyder, PhD2, Patrick B. Johnston, MD, PhD3, Brian P. O Neill, MD4, Harish Raja, MD1, Joseph G. Balsanek, MS2, Brian E. Peters, BS2, and Jose S. Pulido, MD, MBA, MPH, MS1 1
Department of Ophthalmology, 2Department of Laboratory Medicine and Pathology, Immunology Laboratory, 3 Hematology Division, and 4Department of Neurology,Mayo Clinic, Mount Sinai School of Medicine, Rochester, MN
ABSTRACT Purpose: To compare the cytokine spectrum of vitreoretinal lymphoma to uveitis and correlate cytokine concentrations with disease activity. Methods: Retrospective case series of 10 patients with vitreoretinal lymphoma and 7 patients with uveitis. Aqueous humor concentration of IFN-g, TNF-a, TNF-b, IL-1ra, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, MCP-1, G-CSF, GM-CSF, and VEGF-A was determined using a bead-based assay (Luminex). Variance between groups, correlation coefficients, and longitudinal behavior of cytokines were analyzed. Results: No statistically significant difference in cytokines was found when comparing groups. IL-10 was positively correlated with IL-6 and MCP-1. IL-6 was positively correlated with G-CSF, IL-1ra, IL-8, and IL-10. A relationship between concentration of any cytokine, aside from IL-10, and disease activity was not found. Conclusion: IL-10 and IL-6 are good immunologic markers to be used as complementary diagnostic tools. IL-10 is the only IL that could be used for monitoring purposes. Keywords: Cytokines, interleukin-6, interleukin-10, uveitis, vitreoretinal lymphoma
Primary vitreoretinal lymphoma (PVRL) is the most common intraocular lymphoma; in most cases, it represents a high-grade B-cell non-Hodgkin lymphoma, usually a diffuse large B-cell lymphoma (DLBCL).1 PVRL is considered a subset of primary central nervous system lymphoma (PCNSL).2 PVRL may present in isolation or in association with central nervous system (CNS) involvement. Approximately 15–25% of patients with PCNSL will develop vitreoretinal lymphoma,3,4 while 60–80% of patients with PVRL will have progression to CNS lymphoma.4,5 Vitreoretinal lymphoma with CNS involvement represents about 1% of intracranial tumors and less than 1% of intraocular tumors.6 Because it is so uncommon, the true incidence is difficult
20 14
to estimate, but an increased number of cases in immunocompetent, immunosuppressed, and immunodeficient individuals have been reported in the past two decades.7,8 PVRL diagnosis represents a challenge. It is frequently misdiagnosed as posterior uveitis, and these patients show some response to topical corticosteroids, which confounds the diagnosis even more.9 The reported time interval from the onset of ocular symptoms to the final diagnosis ranges from 6 to 24 months,2,10 sometimes diagnosis can be achieved only when the progression to brain involvement has occurred. This highlights the importance of an accurate and early diagnosis of this aggressive malignancy. The techniques used for the diagnostic study of PVRL
Received 5 June 2012; revised 19 November 2012; accepted 20 November 2012; published online 31 January 2014 Correspondence: Jose S. Pulido MD, MBA, MPH, MS, Mayo Clinic, Department of Ophthalmology 200 First Street, SW Rochester, MN 55905. Phone: 507-284-3721 / Fax: 507-284-4612. E-mail: [email protected]
34
Vitreoretinal Lymphoma versus Uveitis include morphological analysis of vitreous and chorioretinal biopsies with additional immunohistochemistry or flow cytometry, molecular analysis using PCR for immunoglobulin rearrangements, as well as biochemical studies such as cytokines assays, in particular, IL-10:IL-6 ratio.11–13 Although cytology is the cornerstone of the diagnostic workup of PVRL, it has a high false-negative rate due to the fragility of the malignant cells, which suffer rapid degradation if the sample is not stored, transported, and handled correctly.10 Alternatively, corticosteroid therapy before the vitrectomy, seen in those patients, may cause paucity of lymphoma cells in the sample, increasing the difficulty of diagnosis.14 This is why significant effort has gone toward developing more specific and sensitive diagnostic tools. The cytokine analysis for diagnostic workup of PVRL is based primarily on the study of intraocular concentrations of IL-6 and IL-10. The IL-6 levels are elevated predominantly in inflammatory conditions like uveitis, while the IL-10 levels are increased in B-cell lymphoma, acting as a cell-mediated immunity suppressor. An IL-10:IL-6 ratio 41.0 is highly suggestive of PVRL15–17 and this can be measured in the vitreous fluid as well as in the aqueous humor due to diffusion of interleukins from the posterior to the anterior chamber.18 Even though the determination of IL-10 and IL-6 concentration in the anterior chamber appears to have insufficient sensitivity and specificity to be used as the sole diagnostic tool,19 we have recently demonstrated that IL-10 concentration in aqueous humor correlates with disease activity and therefore may be useful to evaluate the effect of treatment in PVRL20. As no studies have examined aqueous concentration of cytokines other than IL-10 and IL-6 in PVRL, the purpose of this study was to analyze, in samples from patients with PVRL and uveitis, other cytokines that have been linked to inflammatory processes in the eye,21 comparing interleukin concentration with disease activity and treatment history, as well as their correlations with IL-10 and IL-6, to determine if they can be used as a complementary diagnostic tool or follow-up method when PVRL is suspected.
35
second consisted of 10 patients with diagnosis of vitreoretinal lymphoma (VRL). The diagnosis of VRL or uveitis was made on the basis of their clinical history, ocular exam, and vitreoretinal cytopathology. A diagnosis of VRL was made only if there was histologic confirmation either from an ocular or other tissue source. For better interpretation of the study’s results, the samples from the VRL group were divided into 2 groups: active and inactive vitreoretinal lymphoma, depending on the eye exam findings from within a week prior to the day the anterior chamber paracentesis was performed. Active disease was defined by vitreous cell count 1+ or qualitative description of findings using the words ‘‘some,’’ ‘‘significant,’’ and ‘‘dense.’’ Conversely, inactive disease was considered when the findings were 51+ vitreous cells or the description included words like ‘‘rare’’ or ‘‘trace.’’ Disease progression and treatment history information was collected by reviewing the patient’s medical record. Aqueous humor samples were obtained by anterior chamber paracentesis at multiple times during the evolution of disease, with removal of 0.1 mL of aqueous humor each time for analysis. The concentration of cytokines in the undiluted samples of aqueous humor was determined using a bead-based assay run on a Luminex-based platform (manufacturer: Affymetrix). Linearity, limit of detection was used to establish the reportable range, and inter- and intraassay precisions were verified. Cytokines measured were interferon-g (IFN-g), tumoral necrosis factor a and b (TNF-a and TNF-b), interleukin (IL)-1 receptor antagonist (IL-1ra), IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, monocyte chemotactic protein (MCP-1), granulocyte colony stimulating factor (G-CSF), granulocyte/monocyte colony stimulating factor (GM-CSF), and vascular endothelial growth factor-A (VEGF-A). Detection limits were 50.56 pg/mL for IL-2, IL-4, IL-5, IL-8, IL-10 and IL-12(p40); 51.67 pg/mL for IL-6, IL-7, IL-12(p70), IL-13, IL-17, IL-1b, IL-1ra, and VEGF-A; 51.75 pg/mL for G-CSF; 55 pg/mL for GM-CSF, IFN-g, TNF-a, and TNF-b; and 41215 mg/mL for MCP-1.
Statistical Analysis PATIENTS AND METHODS This retrospective study was performed with the approval of the institutional review board of Mayo Clinic. Patients were evaluated and diagnosed in the Ocular Oncology Service of the Department of Ophthalmology at Mayo Clinic (Rochester, Minnesota, USA). Seventeen patients participated in the study. The patients were sorted into 2 groups: the first comprised 7 patients diagnosed with uveitis, and the !
2014 Informa Healthcare USA, Inc.
Univariate analysis was applied to the data using statistical software from Wassa P.22 The active vitreoretinal lymphoma (AVRL) group, inactive vitreoretinal lymphoma (IVRL) group, and uveitis group were compared using one-way ANOVA test. To assess the correlations between cytokines in each group, Pearson’s correlation coefficient (r) and determination coefficient (r2) were calculated. Twotailed test results were statistically significant at p50.05. Significant correlations were defined by r40.7 (r240.5).
36 J. N. Caraballo et al.
RESULTS A total of 34 aqueous humor samples were obtained and analyzed and distributed as follows: 25 samples from 10 patients with diagnosis of VRL and 9 samples from 7 patients with diagnosis of uveitis. Considering the criteria described above, aqueous humor samples obtained from the patients with VRL were later classified into 2 groups: AVRL group (17 samples) and IVRL group (8 samples). The median, range, mean, and standard deviation for each cytokine in the three groups are summarized in Table 1.
Aqueous Humor Cytokine Levels We previously studied the IL-10 and IL-6 levels in this case series, and our results showed that the median of IL-10 level in aqueous samples in the AVRL group was markedly higher when compared to the uveitis group. Conversely, the uveitis group showed higher aqueous IL-6 concentrations than those reported in the AVRL group and IVRL group20. The rest of the cytokines and their concentrations in the aqueous humor samples of the AVRL group, IVRL group, and uveitis group were compared, and no significant difference was noted in any of the cytokine levels between the disease groups by ANOVA (p40.05). Subgroup analysis was then performed. We observed that IL-1ra showed a broader range of concentrations in the aqueous humor of AVRL group (51.67– 883.08 pg/mL) when compared with the IVRL (51.67–106.53 pg/mL) and the uveitis group (51.67– 62.25 pg/mL) (Figure 1A). Concentrations of IL-8 were higher in the uveitis group (median 2.94 pg/ mL, mean 6.0 ± 6.7 pg/mL) than levels in the AVRL group (median 1.29 pg/mL, mean 2.88 ± 5.26 pg/mL) and IVRL group (median 1.5 pg/mL, mean 1.7 ± 1.59 pg/mL) (Figure 1B). VEGF-A levels had a median of 8.84 pg/mL (mean 14.9 ± 11.4 pg/mL) in AVRL group, 13.07 pg/mL (mean 15.57 ± 7.61 pg/mL) in IVRL group, and 18.28 pg/mL (mean 20.83 ± 17.48 pg/mL) in uveitis group, with no statistical difference noted between them (Figure 1C). G-CSF showed a higher concentration in the uveitis group (median 14.72 pg/mL, mean 51.48 ± 107.99 pg/mL) when compared with the AVRL group (median 51.75 pg/mL, mean 20.27 ± 74.6 pg/mL) and the IVRL group (median 51.75 pg/mL, mean 0.67 ± 1.17 pg/mL). Likewise, a broader range of concentrations was detected in the uveitis group (51.75–353.67 pg/mL) than in the other two groups (Figure 1D). The aqueous humor concentration of GM-CSF, IL-12 (p70), and IL-13 were below detection limits in all samples from the three groups. The concentrations of TNF-a, TNF-b, IFN-g, IL-1b, IL-2, IL-4, IL-5, and IL-17 were rarely positive and showed borderline
concentrations when compared with the lower detectable limit.
Correlations AVRL Group Statistically significant positive correlations were observed between G-CSF and IL-6 (r = 0.7, p50.001, r2 = 0.5), and IL-1ra and IL-6 (r = 0.7, p50.001, r2 = 0.5) (Figure 2). IVRL Group Analysis of the data showed positive correlations between MCP-1 and IL-12p40 (r = 0.9, p50.001, r2 = 0.8), IL-8 and IL-12p40 (r = 0.7, p50.05, r2 = 0.5), IL-8 and IL-6 (r = 0.7, p50.05, r2 = 0.5). Uveitis Group This group showed positive correlations between MCP-1 and IL-12p40 (r = 0.8, p50.05, r2 = 0.6), MCP-1 and IL-1ra (r = 0.8, p50.05, r2 = 0.6), MCP-1 and IL-10 (r = 0.8, p50.05, r2 = 0.6), IL-8 and IL-12p40 (r = 0.7, p50.05, r2 = 0.5), IL-10 and IL-6 (r = 0.7, p50.05, r2 = 0.5) (Figure 3). No other significant correlation could be detected for analyzed cytokines in aqueous humor of patients with AVRL, IVRL, or uveitis.
Effect of Treatment in Cytokine Profile We previously reported the IL-10 concentration in aqueous humor in 3 cases with diagnosis of vitreoretinal lymphoma to serial treatment with methotrexate (MTX) and rituximab (RTX) administered by intraocular injections. Derived from those results, we concluded that IL-10 levels in aqueous humor correlated with disease activity and could possibly be used as monitoring instrument for therapy response20. In this study we used those 3 patients and analyzed the longitudinal behavior of the cytokines in relation to treatment administration and disease activity. In one of the patients we observed that aqueous humor IL-6 and IL-8 were correlated with treatment (Figure 4), but this finding wasn’t consistent in the other patients. Even though some changes were observed in the aqueous concentration of cytokines in relation to treatment administration, those changes were variable in each patient and, as opposed to IL-10 levels, there was no definite pattern of response with treatment.
DISCUSSION Due to the challenge that diagnosis of vitreoretinal lymphoma represents and its high rate of relapse, Ocular Immunology & Inflammation
!
2014 Informa Healthcare USA, Inc.
51.75 55 55 51.67 41215 55 55 51.67 51.67 50.56 50.56 50.56 51.67 51.67 50.56 50.56 50.56 51.67 51.67 51.67
0.42 1.00 0.47 0.54 0.55 0.50 0.62 0.62 0.62 0.21 0.76 0.80 0.18 0.07 0.23 0.23 0.56 1.00 1.00 0.21
p Value 51.75 55 55 8.84 165.7 55 55 51.67 4.43 50.56 50.56 50.56 88.19 2.37 1.29 9.21 1.09 51.67 51.67 51.67
Median 51.75–318.49 – 55–32.53 2.69–39.37 9.2–41215 55–11.48 55–5.04 51.67–2.08 51.67– 883.08 50.56–4.28 50.56–1.56 50.56–1.05 51.67–41215 51.67–7.2 50.56–17.88 50.56–381.44 50.56–14.89 – – 51.67–2.98
Range
Cytokine concentrations are expressed in picogram/milliliter (pg/ml).
G-CSF GM-CSF IFN-g VEGF MCP-1 TNF-a TNF-b IL-1b IL-1ra IL-2 IL-4 IL-5 IL-6 IL-7 IL-8 IL-10 IL-12(p40) IL-12(p70) IL-13 IL-17
Detection limit
AVRL
20.27 ± 74.6 – 2.68 ± 7.75 14.9 ± 11.4 259.38 ± 280.41 1.17 ± 3.25 0.29 ± 1.18 0.12 ± 0.48 69.06 ± 208.23 0.39 ± 1.04 0.09 ± 0.36 0.5 ± 1.73 206.77 ± 337.08 2.1 ± 1.95 2.88 ± 5.26 42.29 ± 92.59 2.89 ± 4.34 – – 0.65 ± 1.05
Mean ± SD 51.75 55 55 13.07 153.1 55 55 51.67 3.77 50.56 50.56 50.56 8.77 3.95 1.5 50.56 1.4 51.67 51.67 51.67
Median 51.75–2.99 – – 5.39–27.95 22.36–257.16 – – – 51.67–106.53 – – 50.56–1.06 1.94–356.58 51.67–5.98 50.56–5.25 50.56–9.59 50.56–3.62 – – 51.67–2.11
Range
IVRL
0.67 ± 1.17 – – 15.57 ± 7.61 158.82 ± 76.89 – – – 18.2 ± 33.96 – – 0.13 ± 0.35 84.61 ± 135.62 3.67 ± 1.85 1.7 ± 1.59 1.19 ± 3.17 1.87 ± 1.15 – – 0.51 ± 0.89
Mean ± SD 14.72 55 55 18.28 236.1 55 55 51.67 9.25 50.56 50.56 50.56 232.16 1.92 2.94 50.56 3.4 51.67 51.67 51.67
Median
51.75–353.67 – 55–32.49 51.67–63.6 19.86–650.34 55–7.57 – – 51.67–62.25 50.56–2.78 50.56–0.58 50.56–1.05 28.33–936.24 51.67–3.99 0.69–22.98 50.56–4.84 50.56–9.37 – – –
Range
Uveitis
51.48 ± 107.99 – 4.8 ± 10.34 20.83 ± 17.48 273.31 ± 208.81 1.59 ± 2.99 – – 15.95 ± 18.29 0.88 ± 1.25 0.06 ± 0.18 0.4 ± 0.46 373.67 ± 351.14 1.54 ± 1.53 6.0 ± 6.7 1.03 ± 1.61 3.78 ± 2.85 – – –
Mean ± SD
TABLE 1. Detection limit, p value, median, range, mean and standard deviation (SD) of cytokines concentration in aqueous humor samples of active vitreoretinal lymphoma (AVRL) group, inactive vitreoretinal lymophoma (IVRL) group and uveitis group.
Vitreoretinal Lymphoma versus Uveitis 37
38 J. N. Caraballo et al.
FIGURE 1. Boxplot graphs showing aqueous humor concentrations of (A) IL-1ra, (B) IL-8, (C) VEGF, and (D) G-CSF in patients with uveitis, active vitroretinal lymphoma (AVRL), and inactive vitreoretinal lymphoma (IVRL). X’s are outliers.
FIGURE 2. Dispersion graphs showing aqueous humor concentration of (A) G-CSF versus IL-6 and (B) IL-1ra versus IL-6 in patients with active vitreoretinal lymphoma.
significant effort has gone to develop more specific and sensitive diagnostic tools, as well as disease monitoring methods. To our knowledge, only one study analyzing aqueous concentration of cytokines other than IL-10 and IL-6 in vitreoretinal lymphoma has been published, and that was during the revision phase of this article.23 As opposed to our study, these authors evaluated the cytokines in the vitreous at the time of diagnosis. They had 21 Asian patients and 27 controls. Their controls were patients that had
structural disease in the retina and did not have uveitis, and thus no cellular infiltration of the vitreous. Interestingly, they found elevation of cytokine levels similar to ours, but since they did not have a uveitis control group, they could only state that there was a difference compared to their nonuveitis group. While their data are important, our study compares the levels to those in patients with uveitis, since it is this group that is most often confused with VRL. Ocular Immunology & Inflammation
Vitreoretinal Lymphoma versus Uveitis
39
FIGURE 3. Dispersion graphs showing AqH concentration of (A) MCP-1 versus IL-12p40, (B) MCP-1 versus IL-1ra, (C) MCP-1 versus IL-10, (D) IL-8 versus IL-12p40, and (E) IL-10 versus IL-6 in patients with uveitis.
FIGURE 4. Longitudinal measurements of IL-10, IL-8, and IL-6 in one case with vitreoretinal lymphoma. Time of administration of intraocular methotrexate and rituximab in indicated (arrows).
We measured and compared the concentrations of other known cytokines in the aqueous humor of patients with vitreoretinal lymphoma and uveitis where we had also obtained IL-10 and IL-6 levels. No statistically significant differences in the aqueous concentration of these additional cytokines were found when comparing the 3 groups (active and inactive vitreoretinal lymphoma and uveitis). Our data showed that measurement of these cytokines was not sufficient to differentiate lymphomatous from uveitic inflammation in the eye. However, we previously observed there was a difference in median aqueous concentration of IL-6 and IL-10 when comparing the active vitreoretinal lymphoma group with the uveitis group (manuscript submitted). We found a positive correlation of IL-10 with IL-6 and MCP-1 in the uveitis group. IL-6 showed a positive correlation with G-CSF and IL-1ra in the active !
2014 Informa Healthcare USA, Inc.
vitreoretinal lymphoma group, with IL-8 in the inactive vitreoretinal lymphoma group, and with IL-10 in the uveitis group. Alternatively, none of the cytokines analyzed showed changes related to treatment response, being inadequate for monitoring disease activity, opposed to what we have previously observed with IL-10 concentrations in aqueous humor. Measurements of IL-10 and IL-6 in the aqueous humor are good complementary tools in the diagnostic workup of PVRL.18,24 IL-10 shows higher concentrations in active vitreoretinal lymphoma.25 Conversely, IL-6 shows higher concentrations in uveitis.13,26 Even though in our previous study we did not find any statistically significant difference between the groups, median concentrations of IL-10 and IL-6 were remarkably different when we compared the AVRL group and the uveitis group. Similar
40 J. N. Caraballo et al. behavior to IL-6 was observed in the concentrations of G-CSF, which was higher in the uveitis group. G-CSF is a growth factor that stimulates bone marrow and proliferation of granulocytes and it is supposed to be increased in inflammatory responses like uveitis. Alternatively, IL-8 is a major proinflammatory cytokine and constitutes a general marker of active uveitis; we detected IL-8 in higher concentrations in the uveitis group. These results correlate with those described by Valentinincic et al.,26 who determined several interleukin levels in serum and aqueous humor of patients with different uveitis entities and found increased IL-8 and IL-6 levels in uveitis group when compared to control groups. The concentration of VEGF-A was higher in the uveitis group, with no statistical difference noted between the three groups. VEGF, through its promotion of angiogenesis and increased vascular permeability, plays an important role in the pathogenesis of uveitis and uveitic complications, such as cystoid macular edema (CMO), choroidal neovascularization (CNV), and retinal neovascularization (RNV).27 In uveitis, there is a major inflammatory process with expression of multiple cytokines, including IL-6, which can upregulate the expression of VEGF through various molecular pathways.28 This would explain why the concentration of VEGF is higher in the uveitis group, in comparison to the AVRL group, where the IL-6 concentration is lower due to prevalence of an anti-inflammatory status. The concentrations of VEGF-A found in our study were lower than those reported in age-related macular degeneration29,30 and diabetic retinopathy (nonproliferative and proliferative).31,32 In the AVRL group, IL-10 did not appear to be directly or indirectly associated with other cytokines, but it showed positive association with IL-6 and MCP1 in the uveitis group. MCP-1 is a chemoattractant cytokine that has been found to be elevated in aqueous humor in active stages of anterior uveitis.33 Some studies have hypothesized that the increase of IL-10 in uveitis is a response to control the inflammation.34 The levels of IL-10 in vitreous and aqueous humor samples have been shown to be correlated with the active vitreoretinal lymphoma.35,36,25 Sou et al.35 reported the vitreous concentration of IL-10 in 6 patients (10 eyes) who underwent intravitreal injections with MTX and noted that once chemotherapy was completed with clinical remission of PVRL, the concentrations of IL-10 in the vitreous fluid reached barely detectable levels. We previously analyzed the IL-10 levels in aqueous humor in 3 patients with PVRL receiving intraocular chemotherapy with MTX and RTX and observed that IL-10 aqueous levels correlate quite well with disease activity (reflected by number of cells in vitreous found in ophthalmologic examination). This could possibly be used as a monitoring
instrument for treatment response (manuscript submitted). In this study we analyzed the longitudinal measurements of multiple cytokines in those 3 patients. The changes observed in the different cytokine concentrations in the aqueous humor in response to treatment administration were not consistent in every patient. No other cytokine was found to have similar response to treatment as does IL-10. The analysis of cytokines in aqueous humor of patients with suspected PVRL is a relatively simple process that can provide us with useful information. The study of immunologic markers like cytokines allows us to better understand the pathogenic features of PVRL, and this study shows that IL-10 and IL-6 are the best immunologic markers to be used as complementary diagnostic and monitoring tools for vitreoretinal lymphoma. PVRL is an uncommon malignancy, which makes its study very difficult. This is why the majority of the studies, including ours, have small sample sizes. As shown recently by Usui et al. in an Asian cohort, though, it appears that some of the cytokines aside from IL-10 might be increased in VRL as compared to controls without cellular infiltration of the vitreous. Only IL-10 levels appear to be important as a way to follow treatment and to help with the diagnosis of VRL.23 Larger collaborative series with longitudinal measurement of aqueous humor interleukin concentrations and their response to treatment are necessary to verify these findings.
DECLARATION OF INTEREST This study was performed at the Mayo Clinic, Rochester, MN and was supported in part by an unrestricted grant from Research to Prevent Blindness, Inc, NY, and from the Mayo Cancer Center. The authors have no proprietary interests in this work.
REFERENCES 1. Coupland SE, Damato B. Understanding intraocular lymphomas. Clin Exp Ophthalmol. 2008;36:564–578. 2. Grimm SA, Pulido JS, Jahnke K, et al. Primary intraocular lymphoma: an International Primary Central Nervous System Lymphoma Collaborative Group report. Ann Oncol. 2007;18:1851–1855. 3. Choi JY, Kafkala C, Foster CS. Primary intraocular lymphoma: a review. Semin Ophthalmol. 2006;21:125–133. 4. Chan CC, Rubenstein JL, Coupland SE, et al. Primary vitreoretinal lymphoma: a report from an International Primary Central Nervous System Lymphoma Collaborative Group symposium. Oncologist. 2011;16: 1589–1599. 5. Akpek EK, Ahmed I, Hochberg FH, et al. Intraocularcentral nervous system lymphoma: clinical features, diagnosis, and outcomes. Ophthalmology. 1999;106:1805–1810. Ocular Immunology & Inflammation
Vitreoretinal Lymphoma versus Uveitis 6. Bardenstein DS. Intraocular lymphoma. Cancer Control. 1998;5:317–325. 7. Schabet M. Epidemiology of primary CNS lymphoma. J Neurooncol. 1999;43:199–201. 8. Corn BW, Marcus SM, Topham A, et al. Will primary central nervous system lymphoma be the most frequent brain tumor diagnosed in the year 2000? Cancer. 1997;79: 2409–2413. 9. Rothova A, Ooijman F, Kerkhoff F, et al. Uveitis masquerade syndromes. Ophthalmology. 2001;108:386–399. 10. Merle-Beral H, Davi F, Cassoux N, et al. Biological diagnosis of primary intraocular lymphoma. Br J Haematol. 2004;124:469–473. 11. Davis JL. Diagnosis of intraocular lymphoma. Ocul Immunol Inflamm. 2004;12:7–16. 12. Coupland SE, Bechrakis NE, Anastassiou G, et al. Evaluation of vitrectomy specimens and chorioretinal biopsies in the diagnosis of primary intraocular lymphoma in patients with masquerade syndrome. Graefes Arch Clin Exp Ophthalmol. 2003;241:860–870. Epub 2003 Sep 30. 13. Chan CC, Wallace DJ. Intraocular lymphoma: update on diagnosis and management. Cancer Control. 2004;11: 285–295. 14. Cassoux N, Merle-Beral H, Leblond V, et al. Ocular and central nervous system lymphoma: clinical features and diagnosis. Ocul Immunol Inflamm. 2000;8:243–250. 15. Buggage RR, Whitcup SM, Nussenblatt RB, et al. Using interleukin 10 to interleukin 6 ratio to distinguish primary intraocular lymphoma and uveitis. Invest Ophthalmol Vis Sci. 1999;40:2462–2463. 16. Chan CC, Gonzalez JA. Primary Intraocular Lymphoma. Singapore: World Scientific; 2007:1–267. 17. Chan CC. Molecular pathology of primary intraocular lymphoma. Trans Am Ophthalmol Soc. 2003;101:275–292. 18. Cassoux N, Giron A, Bodaghi B, et al. IL-10 measurement in aqueous humor for screening patients with suspicion of primary intraocular lymphoma. Invest Ophthalmol Vis Sci. 2007;48:3253–3259. 19. Akpek EK, Maca SM, Christen WG, et al. Elevated vitreous interleukin-10 level is not diagnostic of intraocular-central nervous system lymphoma. Ophthalmology. 1999;106: 2291–2295. 20. Raja H, Snyder MR, Johnston PB, et al. Effect of intravitreal methotrexate and rituximab on interleukin-10 levels in aqueous humor of treated eyes with vitreoretinal lymphoma. PLoS One. 2013 Jun 4;8(6):e65627. 21. Ooi KG, Galatowicz G, Calder VL, et al. Cytokines and chemokines in uveitis: is there a correlation with clinical phenotype? Clin Med Res. 2006;4:294–309.
!
2014 Informa Healthcare USA, Inc.
41
22. Wessa P. Free Statistic Software. Office for Research Development and Education, version 1.1.23-r7, 2012. 23. Usui Y, Wakabayashi Y, Okunuki Y, et al. Immune mediators in vitreous fluids from patients with vitreoretinal B-cell lymphoma. Invest Ophthalmol Vis Sci. 2012;53: 5395–5402. 24. Hirakata A, Inami T, Saito M, et al. Diagnostic value of interleukin-10 and interleukin-6 in the vitreous of intraocular malignant lymphoma patients. Nihon Ganka Gakkai Zasshi. 2004;108:359–367. 25. Chan CC, Whitcup SM, Solomon D, et al. Interleukin-10 in the vitreous of patients with primary intraocular lymphoma. Am J Ophthalmol. 1995;120:671–673. 26. Valentincic NV, de Groot-Mijnes JD, Kraut A, et al. Intraocular and serum cytokine profiles in patients with intermediate uveitis. Mol Vis. 2011;17:2003–2010. 27. Gulati N, Forooghian F, Lieberman R, et al. Vascular endothelial growth factor inhibition in uveitis: a systematic review. Br J Ophthalmol. 2011;95:162–165. 28. Cohen T, Nahari D, Cerem LW, et al. Interleukin 6 induces the expression of vascular endothelial growth factor. J Biol Chem. 1996 Jan 12;271(2):736–41. 29. Funk M, Karl D, Georgopoulos M, et al. Neovascular agerelated macular degeneration: intraocular cytokines and growth factors and the influence of therapy with ranibizumab. Ophthalmology. 2009;116:2393–2399. 30. Roh MI, Kim HS, Song JH, et al. Concentration of cytokines in the aqueous humor of patients with naive, recurrent and regressed CNV associated with amd after bevacizumab treatment. Retina. 2009;29:523–529. 31. Zhou L, Sun H, Xu J, et al. Level of vascular endothelial growth factor and interleukin-6 in aqueous humor in diabetic retinopathy patients. Yan Ke Xue Bao. 2010;25: 26–30. 32. Selim KM, Sahan D, Muhittin T, et al. Increased levels of vascular endothelial growth factor in the aqueous humor of patients with diabetic retinopathy. Indian J Ophthalmol. 2010;58:375–379. 33. Verma MJ, Lloyd A, Rager H, et al. Chemokines in acute anterior uveitis. Curr Eye Res. 1997;16:1202–1208. 34. Jabs DA, Nussenblatt RB, Rosenbaum JT. Standardization of uveitis nomenclature for reporting clinical data: results of the First International Workshop. Am J Ophthalmol. 2005; 140:509–516. 35. Sou R, Ohguro N, Maeda T, et al. Treatment of primary intraocular lymphoma with intravitreal methotrexate. Jpn J Ophthalmol. 2008;52:167–174. 36. Kawamura H, Yasuda N, Kakinoki M, et al. Interleukin-10 and interleukin-6 in aqueous humor during treatment of vitreoretinal lymphoma with intravitreally injected methotrexate. Ophthalmic Res. 2009;42:172–174.
Copyright of Ocular Immunology & Inflammation is the property of Taylor & Francis Ltd and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
