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Anatomy and Pathology Analysis of Inflammatory Cells in Uveal Melanoma after Prior Irradiation T. H. Khanh Vu,1 Inge H. G. Bronkhorst,1 Mieke Versluis,1 Marina Marinkovic,1 Sjoerd G. van Duinen,2 Johannes Vrolijk,3 Gregorius P. M. Luyten,1 and Martine J. Jager1 PURPOSE. Primary uveal melanomas with a poor prognosis contain high numbers of infiltrating macrophages, especially of the M2 phenotype, as well as lymphocytes. We wondered whether local inflammatory responses were affected by irradiation a
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  Anatomy and Pathology  Analysis of Inflammatory Cells in Uveal Melanoma after Prior Irradiation  T. H. Khanh Vu, 1  Inge H. G. Bronkhorst, 1  Mieke Versluis, 1  Marina Marinkovic, 1 Sjoerd G. van Duinen, 2   Johannes Vrolijk, 3 Gregorius P. M. Luyten, 1 and Martine J. Jager  1 P  URPOSE . Primary uveal melanomas with a poor prognosiscontain high numbers of infiltrating macrophages, especially of the M2 phenotype, as well as lymphocytes. We wondered whether local inflammatory responses were affected by irradiation and therefore determined the presence of inflam-matory cells in uveal melanomas enucleated after prior irradiation. M ETHODS . We analyzed 46 uveal melanoma–containing eyesthat had to be enucleated due to nonresponsiveness, tumor recurrence, or complications. Immunofluorescent staining wasperformed to determine the presence of CD68 þ andCD68 þ CD163 þ macrophages, and of CD4 þ , CD8 þ , and Foxp3 þ regulatory T lymphocytes. Outcomes were compared with clinical and histologic parameters. R  ESULTS . Numbers of CD68 þ and CD68 þ CD163 þ macrophagesin secondarily enucleated eyes varied widely, but did not differ from primarily enucleated eyes and were not related to thereason for enucleation. Similarly, the number of CD4 þ , CD8 þ ,and Foxp3 þ T lymphocytes showed great variability. Tumors with epithelioid cells showed significantly more lymphocytesthan spindle cell tumors. In the first 2 years after enucleation,previously irradiated tumors showed increased numbers of lymphocytes compared with primarily enucleated eyes. C ONCLUSIONS . Numbers of infiltrating T lymphocytes andmacrophages varied widely between tumors, but tumors with high numbers of macrophages also contained more lympho-cytes. Irradiation had no effect on the number and type of macrophages, but led to an increased amount of T lymphocytesup to 24 months postirradiation. Because the presence of infiltrating cells was related to the tumor cell type, it isconceivable that the presence of an infiltrate is especially aconsequence of the primary tumor characteristics beforeirradiation. (   Invest Ophthalmol Vis Sci. 2013;54:360–369)DOI:10.1167/iovs.12-9424 M alignant melanoma of the uvea is the most commonprimary intraocular neoplasm, with an annual incidence varying from two to eight cases per million per year inEuropean countries. 1 Over the past decades, patient survivalhas not improved, despite progress in the diagnosis of melanocytic lesions and successful treatment of the intraocular melanoma. 2–4 The 5-year mortality rate after diagnosis of auveal melanoma is approximately 30% due to metastaticdisease. 2,5 Until the 1970s, the traditional treatment of uveal melano-ma was enucleation. 6 However, when eye-preserving tech-niques became available, and after the Collaborative Ocular Melanoma Study revealed no survival difference in medium-sized melanoma after irradiation versus enucleation, 2–4,6–8 ashift to more eye-saving approaches occurred. 2–5,7,9 Localradiotherapy is often able to preserve the eye and sight, and isless mutilating than enucleation. 6,10  While eye retentionfollowing local treatment is usually achieved in more than80% of cases after 5 years, 8,10,11 secondary enucleation may berequired when failure of local tumor control occurs. This may be inadequate tumor regression or local (contiguous andnoncontiguous) recurrence. 12–17 In addition, secondary enu-cleation is sometimes necessary in case of radiation-relatedocular side effects, such as neovascular glaucoma, persistenthemorrhage, or exudative retinal detachment. 14 The histopathologic findings of uveal melanoma previously treated with different eye-conserving therapies are quitesimilar, regardless of the difference in physical properties of the various radiation sources used. 15–17 These findings include: vacuolization with balloon cell degeneration, tumor cellnecrosis surrounding vascular sclerosis, vascular damage, andfibrosis of the tumor stroma with frequent accumulation of pigmented macrophages. 15–17 Several studies have revealed the presence of tumor-infiltrating macrophages and lymphocytes in primarily enucle-ated as well as irradiated and secondarily enucleated eyes with uveal melanoma. 18–25 High numbers of tumor-infiltratingmacrophages in primarily enucleated eyes are related to anunfavorable prognosis and are associated with the presence of epithelioid cells, increased microvascular density, and mono-somy of chromosome 3. 18,21 Such intratumoral macrophages were mainly of the tumor-promoting M2 phenotype, harboringanti-inflammatory and proangiogenic functions. 26 Moreover,tumors containing higher numbers of lymphocytes wereassociated with a bad prognosis. 25,27 Histologic studies after transpupillary thermotherapy (TTT) or transscleral thermo-therapy (TSTT) showed an influx of macrophages after localtherapy. 28,29 It is feasible that macrophages play an importantscavenger role in removing debris after irradiation. Based onthese findings, we hypothesize that the combination of irradiation and thermotherapy will induce an influx of macrophages that should be noticeable in uveal melanomaenucleated after prior radiotherapy. In addition, becauseprimarily enucleated uveal melanomas that have monosomy 3 From the Departments of  1 Ophthalmology, 2 Pathology, and 3 Molecular Cell Biology, Leiden University Medical Center, Leiden,The Netherlands.Supported by the Board of Directors Leiden University MedicalCenter (IHGB, PhD position recipient), Rotterdamse VerenigingBlindenbelangen, Landelijke Stichting voor Blinden en Slechtzien-den, and Stichting Glaucoomfonds, Nederlands OogheelkundigOnderzoek, Oogfonds, Prins Bernhard Cultuurfonds (THKV).Submitted for publication January 3, 2012; revised September 5and November 20, 2012; accepted November 20, 2012.Disclosure: T.H.K. Vu , None; I.H.G. Bronkhorst  , None; M. Versluis , None; M. Marinkovic , None; S.G. van Duinen  , None; J. Vrolijk  , None; G.P.M. Luyten  , None; M.J. Jager  , NoneCorresponding author: Martine J. Jager, Department of Oph-thalmology, Leiden University Medical Center, PO Box 9600, 2300RC, Leiden, The Netherlands; m.j.jager@lumc.nl. Investigative Ophthalmology & Visual Science, January 2013, Vol. 54, No. 1 360 Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc.  contain more inflammatory infiltrate, 20,21,27 and tumor recur-rence may perhaps especially occur in tumors that havesimilarly lost one copy of chromosome 3, irradiated uvealmelanomas enucleated due to failure of local tumor control canbe expected to contain more inflammatory cells; therefore, wedetermined the number and subtype of tumor-infiltratingmacrophages and lymphocytes in uveal melanoma–containingeyes enucleated after irradiation and compared results to prior studies on primarily enucleated eyes. M  ATERIALS AND M ETHODS Eye-Preserving Radiotherapy  The primary choice of conservative treatment at the Department of Ophthalmology of the Leiden University Medical Center (LUMC), TheNetherlands,isbrachytherapy,deliveredwithruthenium-106applicators.Between 1995 and 2008, plaque treatment was frequently combined with adjuvant TTT. 30 Proton beam radiotherapy was indicated for largeand highly prominent melanomas, or in tumors located close to or incontact withthe optic disc. 6,11 For this treatment, patientswere referredto the Hˆopital Ophthalmique Jules Gonin, Lausanne, Switzerland. Patients and Specimens Tissue specimens were obtained from 69 consecutive eyes with uvealmelanomas that had undergone a secondary enucleation after prior irradiation between 1996 and 2010 at the LUMC. Only formalin-fixedparaffin-embedded specimens from which enough tumor material waspresent for histopathologic analysis were selected from the archives of the Pathology and Ophthalmology Departments, leaving 46 eligiblespecimens for inclusion. Reasons for enucleation included thefollowing: failure of local tumor control, defined as (1) contiguoustumor progression after partial regression due to nonresponsiveness toprior treatment; (2) intraocular tumor recurrence after total tumor regression; or (3) radiation-related ocular complications, such aspersistent hemorrhage, exudative retinal detachment, or a blindpainful eye due to neovascular glaucoma. Clinical histories wereanalyzed by a clinician, using fluorescein angiographic and ultrasono-graphic (including A and B scans) images, to accurately divide eyes intothree subgroups based on the reason for enucleation. Clinical data fromthe 46 enrolled cases were collected from the clinical records.Clinical, histopathologic, and inflammatory parameters on a set of 43 primarily enucleated nonirradiated tumors have been describedpreviously  20,21,27 and were used for the comparison with irradiated,secondarily enucleated eyes. All patientswere informedregarding the use of their eye for research purposesandsignedaninformedconsentform.TheuseoftumormaterialforresearchfollowedthecurrentrevisionofthetenetsoftheDeclarationof Helsinki (World Medical Association Declaration of Helsinki 1964;Ethical principles for medical research involving human subjects). Histopathologic Data   After enucleation, eyes were fixed in 4% neutral-buffered formalin for 48 hours and embedded in paraffin. Two ocular pathologists analyzedthe hematoxylin and eosin–stained (H&E) 4- l m sections for thepathologic diagnosis, tumor location, ciliary body involvement(presence or absence), tumor cell type (spindle or nonspindle), largestbasal diameter (LBD, in millimeters), prominence (in millimeters), andthe presence of necrosis. Immunostaining Protocol for Tumor-InfiltratingMacrophages and Lymphocytes Immunostaining was performed using double- and triple-immunofluo-rescence (IF) staining as described earlier, 20,31 to identify the differentsubsets of tumor-infiltrating leucocytes. In short, 4- l m sections werecut from paraffin-embedded tumor blocks and deparaffinized. Antigenretrieval was performed by a 10-minute incubation in boiling Tris-EDTA buffer, at pH 9.0.The primary antibodies used to characterize the macrophagephenotype were mouse anti-human CD68 mAb (1:50, clone 514H12,ab49777; Abcam, Cambridge, UK) as a marker for total macrophages,and mouse anti-human CD163 mAb (1:100, clone 10D6, NCL-CD163;Novocastra, Newcastle upon Tyne, UK) as a marker for M2macrophages.Therabbitanti-humanCD3pAb(1:100,IgG,cloneab828;Abcam)wasused to label CD3 þ T cells, and the mouse anti-human CD8 mAb (1:100,IgG2b,clone4B11;Novocastra)wasusedtolabelCD8 þ Tcells.Themouseanti-human Foxp3 (forkhead box p3) mAb (1:200, IgG1, clone 236A/E7; Abcam) was used as a nuclear marker for regulatory T cells (Tregs).Secondary antibodies were IgG2a (AlexaFluor 488) goat-anti-mousefor CD68, IgG1 (546) goat-anti-mouse for CD163, IgG (546) goat-anti-rabbit for CD3, IgG2b (647) goat-anti-mouse for CD8, and IgG1 (488)goat-anti-mouse for Foxp3 (all antibodies from Invitrogen/Molecular Probes, Eugene, OR). Detection of Immunostaining Images of the stained sections were captured with a confocal laser-scanning microscope (LSM510; Carl Zeiss Meditec, Jena, Germany) in amultitrack setting and H&E-stained sections were used for orientationand location of the scans. Each scan represented one square opticalfield (area, 0.137 mm 2  ). All images for the tumor-infiltrating lymphocytes were 1024 3 1024pixels and for the macrophages 512 3 512 pixels with stack size 368.5 3 368.5 l m. A PH2 Plan-NEOFluar 25 3  /0.80 Imm Korr objective (CarlZeiss Meditec) was used.The fluorochrome signals in the slides were visualized with anartificial color: red for both AlexaFluor 546 antibodies (CD163, CD3),blue for AlexaFluor 647 (CD8), and green for both AlexaFluor 488antibodies (CD68, Foxp3). Images were viewed as an overlay, as a set of two (for macrophages) or three divided panels (for lymphocytes).  Assessment of Immunostaining For characterization of the macrophage subtypes, five representativehigh-power fields (  3 250 magnification) per slide were randomly selected. Because of the polymorphic appearance of macrophages, it was difficult to count them. Instead, we calculated the amount of staining in pixels per mm 2 by using an image-analysis software program(Stacks; Department of Molecular Cell Biology, LUMC, Leiden, TheNetherlands). In the resulting binary images, the green and red pixelscorresponded to the anti-CD68 and anti-CD163 staining, respectively.The overlay of both colors was expressed as yellow pixels (Fig. 1).CD3 þ CD8 þ cells (purple signal) were considered to be CD8 þ Tcells, CD3 þ CD8 À cells (red signal) CD4 þ helper T cells, andCD3 þ CD8 À FoxP3 þ cells (red signal with a green center) Foxp3 þ Tregs(Fig. 1). Positive cells were counted in 10 randomly taken high-power fields (  3 250 magnification) by two independent observers masked tothe clinical outcome and the reason for enucleation. The mean of thetwo observers was calculated for each tumor and tumor-infiltratinglymphocyte cell counts were presented as the number of cells per mm 2 . Necrotic areas were not analyzed. Statistical Analyses  All analyses were performed with a statistical software program (SPSSfor Windows, version 17.0; SPSS Inc., Chicago, IL). The performed tests were two-sided and a value of  P  < 0.05 was considered as statistically significant. For the comparison of categorical data between two or moreindependentgroups,the v 2 testwasperformed,and for numericaldata the nonparametric Wilcoxon rank-sum test and the Kruskal–Wallistest were performed. Spearman’s rank correlation analysis (two-sided)  IOVS  , January 2013, Vol. 54, No. 1 Analysis of Inflammatory Cells in Uveal Melanoma 361   was performed to assess correlations between the different types of immune cells, and with time between irradiation and enucleation. R  ESULTS The numbers of infiltrating immune cells were determined in46 uveal melanomas that had been irradiated previously, andoften treated by TTT. We compared the cell counts with demographic and histopathologic patient and tumor charac-teristics, as summarized in Table 1.Of the 46 irradiated eyes with uveal melanoma, 12 had beentreated with proton beam radiotherapy, 4 with rutheniummonotherapy, and 30 with sandwich therapy (ruthenium-106brachytherapy with TTT). Given the fact that only four eyesreceived ruthenium-106 as monotherapy and no conclusion F IGURE 1. Detection of macrophages and lymphocytes in uveal melanomas by double- and triple-IF staining with antibodies directed againstspecific immune cells. 362 Vu et al. IOVS  , January 2013, Vol. 54, No. 1  can be drawn from such a small sample size, these cases werecombined with the sandwich therapy group for statisticalanalysis. The median interval between irradiation and enucle-ation was 14 months (range: 4–146 months) for patients whoreceived proton beam irradiation, and 22 months (range: 3–125 months) for ruthenium-106 brachytherapy (with or  without TTT).Causes for enucleation were: nonresponsiveness to prior irradiation in 15 (33%) cases, tumor recurrence after prior totalregression in 14 (30%) cases, and radiation-related ocular sideeffects in 17 (37%) cases. Of the 12 eyes that had to beenucleated following proton beam irradiation, one (8%) wasdue to nonresponsiveness, two (17%) showed a tumor recurrence, and nine (75%) had radiation-related complications(especially neovascular glaucoma). Of the 34 eyes treated with ruthenium-106 brachytherapy, 14 (41%) had to be enucleateddue to nonresponsiveness, 12 (35%) due to tumor recurrence,and 8 (24%) due to complications. At the time of irradiation, the median age of the patients (31males, 15 females) was 60 years (range: 32–84 years), and by the time of enucleation it was 63 years (range: 38–85 years).The primarily enucleated eyes and the irradiated eyesdiffered significantly with regard to the American JointCommittee on Cancer–Union Internationale Contre le Cancer (AJCC–UICC) prognostic stage groups (   P  < 0.001), tumor prominence (   P  < 0.001), LBD (   P  ¼ 0.014), involvement of theciliary body (   P  ¼ 0.001), break through Bruch’s membrane (   P  ¼ 0.005), and the presence of necrosis (   P  ¼ 0.001): these variables, except for necrosis, were seen more often innonirradiated eyes.  Tumor-Infiltrating Macrophages Double-IF was performed on tumor sections of 46 irradiatedeyes to analyze the amount and phenotype of tumor-infiltratingmacrophages. In cases of severe tumor pigmentation, positivecells were easily recognized by IF staining with the confocalmicroscope.Because most of the CD68-positive cells were also CD163-positive, macrophages in irradiated uveal melanoma belongedmainly to the M2 phenotype. The amount of CD68 þ (   P  ¼ 0.80)and CD68 þ CD163 þ (   P  ¼ 0.44) staining was similar in irradiatedand primarily enucleated uveal melanoma (Table 2, Fig. 2A). Inaddition, there was no relation between the amount of CD68 þ (   P  ¼ 0.16) and CD68 þ CD163 þ (   P  ¼ 0.34) staining and the causeof enucleation in irradiated eyes (Table 2, Fig. 2A) or with thetype of irradiation (data not shown).However, the frequency of macrophages in secondarily enucleated uveal melanomas seemed to decrease slightly with increase in time interval between irradiation and enucleation(  r  : < À 0.402, P  < 0.03; Fig. 2B). Because the time betweenirradiation and enucleation varied broadly between patients,the data were normalized to account for time after irradiationby dividing the secondarily enucleated eyes into four time-interval categories: 0 to 12, 13 to 24, 25 to 36, and > 36months, and then to compare these categories with the  T  ABLE 1. Comparison of Clinical and Histologic Data of Nonirradiated and Irradiated Eyes with Uveal Melanoma Nonirradiated EyesIrradiated Eyes Total Nonresponsiveness Recurrence Complications Subjects, n 43 46 15 14 17Sex, male/female 23/20 31/15 8/7 9/5 14/3Eye, right/left 23/20 25/21 8/7 8/6 9/8Prognostic groupsStage I 3 17* 6 3 8Stage IIA 9 18 7 4 7Stage IIB 13 4 1 1 2Stage IIIA 15 5 1 4 0Stage IIIB 3 2 0 2 0Cell typeSpindle 11 10 5 1 4Mixed þ epithelioidCB involvement32 36 10 13 13Not present 25 41* 15 10 16Present 18 5 0 4 1†Bruch’s membraneBroken 33 23* 6 5 12Intact 4 18 9 5 4Not clear 6 5 0 4 1NecrosisNot present 36 22* 8 9 5Present 7 22 7 3 12Unclear 0 2 0 2 0Median prominence, mm (range) 8 (2–12) 8 (2–12) 4 (1–14)* 3 (1–8) 4 (1–14) 4 (1–11)Median LBD, mm (range) 13 (8–18) 12 (4–21)* 12 (6–20) 12 (4–21) 10 (5–15)Median age at irradiation (range) - 60 (32–84) 56 (42–84) 61 (32–78) 60 (42–70)Median age at enucleation (range) 63 (27–88) 63 (38–85) 57 (42–85) 69 (38–80) 61 (43–77)Prominence and LBD measurements were obtained from histologic examinations. Prognostic groups based on the 7th edition AJCC–UICCcriteria for T staging, including the anatomic extent of the tumor based on involvement of the ciliary body and extrascleral tissues. P  values for categorical parameters were obtained by the v 2 test, for the numerical data by Wilcoxon rank-sum test and Kruskal–Wallis test; all statistical tests were two-tailed.* Significant at P   0.05 between irradiated and nonirradiated eyes.† Significant at P   0.05 between secondarily enucleated eyes: nonresponsiveness, recurrence, and complications.  IOVS  , January 2013, Vol. 54, No. 1 Analysis of Inflammatory Cells in Uveal Melanoma 363
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