Current Eye Research, 33:177–184, 2008Copyright ©c Informa Healthcare USA, Inc.ISSN: 0271-3683 print / 1460-2202 onlineDOI: 10.1080/02713680701867908

Expression of COX-2 and PrognosticOutcome in Uveal Melanoma

Lorna M. Cryan∗

School of Medicine and MedicalScience, UCD Conway Institute ofBiomolecular and BiomedicalResearch, University College Dublin,Dublin, Ireland

Luminita Paraoan∗

and Paul HiscottSchool of Clinical Sciences, Unit ofOphthalmology, University ofLiverpool, Liverpool, United Kingdom

Bertil E. DamatoSt Pauls Eye Unit, Royal LiverpoolUniversity Hospital, Liverpool,United Kingdom

Ian Grierson and Donna GraySchool of Clinical Sciences, Unit ofOphthalmology, University ofLiverpool, Liverpool, United Kingdom

Michael FarrellDepartment of Neuroscience,Beaumont Hospital, RCSI, Dublin,Ireland

Glen A. Doherty andDesmond J. FitzgeraldSchool of Medicine and MedicalScience, UCD Conway Institute ofBiomolecular and BiomedicalResearch, University College Dublin,Dublin, Ireland

Colm O’BrienSchool of Medicine and MedicalScience, UCD Conway Institute ofBiomolecular and BiomedicalResearch, University College Dublin,Dublin, Ireland, and Department ofOphthalmology, Mater MisericordiaeHospital, Dublin, Ireland

ABSTRACT Purpose: The expression of cyclooxygenase-2 (COX-2) and its prog-nostic value in uveal melanoma was examined. Methods: Paraffin-embeddedsections from 32 clinicopathologically well-characterized cases of primary uvealmelanoma were immunohistochemically stained for COX-2. COX-2 expressionwas evaluated in terms of both the intensity and the extent of staining for eachtumor. A COX-2 score encompassing both intensity and extent was also calcu-lated for each specimen. Results: 29 specimens (90.6%) contained moderate orintense positive immunoreactivity for COX-2. A statistically significant associa-tion (p < 0.05) between COX-2 expression (intensity and score) and metastaticdeath was established. Conclusion: Upregulation of COX-2 expression appearsto be associated with poor prognosis in uveal melanoma.

KEYWORDS eicosanoids; immunohistochemistry; tumor; uveal melanoma

INTRODUCTIONRecently, there has been increased awareness that the cyclooxygenase en-

zymes may play an important role in tumorigenesis. A reduction in the incidenceof colon cancer in individuals regularly taking non-steroidal anti-inflammatorydrugs (NSAIDs) such as salicylic acid was reported.1 Salicylic acid (aspirin)inhibits both forms of the cyclooxygenase enzyme, COX-1 and COX-2. Thecyclooxygenase isozymes constitute the rate-limiting step in the formation ofthe prostaglandins and thromboxane, with COX-1 being expressed in most tis-sues and COX-2 being the inducible isoform. COX-2 is frequently expressed inneoplastic cells of stromal and epithelial origin and in the vasculature of tumors,with little or no expression in adjacent normal tissue.2 NSAIDs are believed toreduce the incidence of colon cancer primarily via inhibition of COX-2, leadingto a reduction in COX-2-derived prostanoids. In a number of animal models ofcarcinogenesis, selective pharmacological inhibition of COX-2 attenuates tumorgrowth.2–6 Prostaglandins derived from COX-2 may promote tumor formationby providing mitogenic stimulation for tumor cells,7,8 and increasing the bloodsupply via angiogenesis.9 Prostaglandins also increase the migratory and invasiveproperties of tumor cells,8,10 thereby promoting metastasis.

In adults, uveal melanoma is the most common malignant intraoculartumor.11 The incidence of uveal melanoma is estimated to be approximately5 cases per million per year. Treatment of the intraocular tumor may involve

Received 23 October 2007Accepted 13 December 2007

∗Both authors contributed equally to this study.

Correspondence: Colm O’Brien, Institute ofOphthalmology, Mater Misericordiae Hospital,60 Eccles Street, Dublin 7, Ireland. E-mail:cobrien@mater.ie

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enucleation or a variety of treatments aimed at con-servation of the eye and as much vision as possible,including local resection, phototherapy, and radiother-apy. However, despite successful treatment of the pri-mary tumor, about 40% of patients go on to developliver metastases.11 Once liver metastases are clinicallyapparent the outlook is poor, with an average survivalof just 5–9 months.12,13

A recent study investigated the expression of COX-2 in mixed-cell uveal melanomas and identified pos-itive staining for COX-2 in 58% of cases studied.14

COX-2 was largely expressed in the tumor cells andinfiltrating macrophages; also expression of COX-2correlated with markers of poor prognosis, includingepithelioid cell type, the presence of vascular closedloops, and the presence of infiltrating lymphocytes. Inrecent preclinical studies, administration of selectiveCOX-2 inhibitors reduced both experimental metas-tases and metastases arising after surgical removal ofprimary breast tumors.15–17 Specifically, treatment ofmice in a colorectal cancer model with a selectiveCOX-2 inhibitor, NS-398, delays the formation of livermetastases,17 the most frequent cause of cancer-relateddeath in patients with uveal melanoma. Consideringthat selective COX-2 inhibitors are approved for clini-cal usage and are known to be anti-carcinogenic, theymay represent a realistic adjuvant therapy for uvealmelanoma. The present study is the first to directly in-vestigate the potential association of COX-2 expressionwith patient survival and metastasis in uveal melanoma.

METHODSTumor Samples

Tumor samples were obtained from patients under-going enucleation for the primary treatment of uvealmelanoma at the Ocular Oncology Centre, St Paul’s EyeUnit, Royal Liverpool University Hospital. The studywas approved by the local Ethics Committee. The tu-mors were fixed in formalin for 48 hr and subsequentlyprocessed and paraffin-embedded for routine patholog-ical studies. Histological data was collected for thesetumors, including the presence or absence of periodicacid-Schiff (PAS) stained loops, largest basal diameter,tumor thickness, and cell type. Clinical survival datawas also collected for all of these patients.

Uveal (choroidal) melanomas were obtained from 32patients, comprised of 15 males and 17 females, withan average age of 69.0 years (range 36–87). The largest

basal tumor diameter averaged 13.5 mm (range 9–18),and the tumor thickness averaged 8.7 mm (range 2–15). Of the 20 tumors assessed for PAS-stained loops, 6were positive. Of the 32 uveal melanomas analysed forCOX-2 staining, 4 were of epithelioid cell type, 11 wereof spindle cell type, and 17 were of mixed cell types.

The median follow-up time of non-deceased patientswas 12.8 years. Twenty-two patients died, twelve frommetastatic disease. Ten patients died of other causes,which included pneumonia, cerebrovascular disease, is-chemic heart disease, and “old age.” Time to death wasmeasured from the date of the initial ocular treatmentfor uveal melanoma to the date of death. Date and causeof death were provided by the National Cancer Registry.

Immunohistochemical Detectionof COX-2

Paraffin-embedded uveal melanoma sections(4 μm) were stained for COX-2 using a biotin-streptavidin-based immunoperoxidase technique.Paraffin-embedded sections from normal human eyeswere also stained for COX-2 as a positive control.The expression of COX-2 in the normal humaneye was previously described.18–20 Tissue sectionswere deparaffinized and rehydrated prior to antigenretrieval. The antigen retrieval step involved microwavetreatment of the sections in sodium citrate buffer for5 min, which were then left at room temperature fora further 30 min. Endogenous peroxidase activity wasquenched in the tissue sections by incubation with0.3% H2O2 in 100% methanol for 30 min. Slides werethen washed 3 × 5 min in 1 × phosphate bufferedsaline (PBS) at 50 RPM. Non-specific binding wasblocked by incubation of the tissue sections in 1.5%normal goat serum provided with the Vectastain Elitekit (Vector Laboratories, Burlingame, CA, USA) for 1hr diluted in 1 × PBS. The tissue sections were thenincubated with a 1 in 200 dilution of a Cayman rabbitpolyclonal anti-human COX-2 antibody (SPI-BIO,Montigny le Bretonneux, France) in 1.5 % normal goatserum for 2 hr. Some sections were incubated withnormal goat serum in place of the primary antibodyas a negative control. Sections were washed again, 3× 5 min in 1 × PBS, and then incubated with thebiotinylated anti-rabbit secondary antibody, providedwith the Vectastain kit, for 30 min at room temperature.The tissue was washed 3 × 5 min in 1 × PBS andincubated with the ABC complex from the kit for

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30 min at room temperature. Slides were then washedas before and exposed to NovaRED©R chromagen(Vector Laboratories, Burlingame, CA, USA) for 3min. Sections were counterstained with haematoxylin(Sigma Aldrich, Dublin, Ireland) for 30 sec and washedin tap water for 5 min. The slides were rinsed withmethanol, cleared with xylene, mounted using DPXmounting media (BDH Laboratory Supplies, Poole,England), and subsequently viewed using standardlight microscopy.

Analysis of StainingAnalysis of COX-2 staining in uveal melanoma sec-

tions was carried out at two centers of pathology withthe requisite experience in tumor pathology, namelythe Department of Neuropathology, Beaumont Hospi-tal, Dublin, and the Unit of Ophthalmology, School ofClinical Sciences, University of Liverpool, Liverpool,by three independent assessors. Staining was graded interms of intensity and the percentage cells of each tu-mor with positive reactivity. The intensity of stainingin these tumors was categorized as (−) = absent (back-ground), (+) = slight to moderate (between − and ++),or (++) = intense (equating to that of positive con-trol tissue, see RESULTS). The extent of COX-2 im-munoreactivity was further categorized as ‘high’ (pos-itivity within >50% of the tumor cells) or ‘low’ im-munoreactivity (positive staining within 50% or less ofthe tumor cells). Grading of all uveal melanoma sectionsfor immunohistochemical COX-2 staining was carriedout in a masked and randomized manner. A COX-2score, incorporating both the maximum intensity andthe percentage of cells staining for COX-2, was also cal-culated for each tumor. To calculate the COX-2 score,the percentage of cells stained was multiplied by an in-tensity factor (for an intensity of 0—multiplied by 1,for an intensity of 1—multiplied by 2, and for an inten-sity of 2—multiplied by 4) in a similar manner to thatpreviously described.21

Statistical AnalysisKaplan Meier survival curves were used to display

and calculate percentage survival over time in humanuveal melanoma donors, and the Logrank test was usedto compare survival curves. The exact Fischer test wasused to analyse data within contingency tables wheretwo variables were analyzed and both were subdividedby outcome. Spearman’s rank correlation coefficient

was used to examine how consistently two variablesvaried together. The statistical packages GraphPad In-Stat and GraphPad Prism were used for these analyses.Statistical significance was set at a p value of less than0.05.

RESULTSCOX-2 Staining of Uveal Melanomas

Normal eyes were included as positive controls andthese displayed typical expression of COX-2 in the opticnerve, ciliary muscle, and retina, as previously described(data not shown).18–20 Of the 32 tumors analysed, just3 were completely negative for COX-2. The extent ofCOX-2 staining in uveal melanoma tumors was vari-able, ranging from isolated positive cells throughoutthe tumor to patches of positive cells, both of epithe-lioid and spindle type, in a localized area (Fig. 1 andTable 1). Staining of the vascular endothelium withinthese tumors was not apparent, although melanophagessometimes did label for COX-2.

Association of Staining with SurvivalFor each specimen, at least three masked evaluations

were made (L.P., P.H., and M.F.). The summary resultsfor each tumor are displayed in Table 1.

Kaplan-Meier survival curves of both metastasis-related death (Fig. 2 A–C) and overall survival overtime were constructed for high versus low COX-2 ex-pression. The tumors were classified as ‘high’ or ‘low’COX-2 expression in terms of the percentage of cellsstained within the tumor (Fig. 2A), the maximum in-tensity of staining (Fig. 2B), and the COX-2 score(Fig. 2C).

There was a trend toward an association of im-munoreactivity for COX-2 within >50% (high extent)of the tumor with reduced cancer specific survival(P = 0.07) in comparison with immunoreactivity within≤50% of the tumor (low extent) (Fig. 2A). A maximumintensity of staining of ++ (high COX-2 intensity) wasstrongly associated with reduced metastasis-specific sur-vival in donors, compared with the remaining donorswith a maximum intensity of + or − (low COX-2 inten-sity) (p = 0.009) (Fig. 2B). A high COX-2 score (above150) was also associated with reduced cancer specificsurvival, in comparison with patients with a low COX-2 score (150 or less) (p = 0.036) (Fig. 2C).

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FIGURE 1 Expression of COX-2 in uveal melanoma as detected by immunohistochemistry. The images are from uveal melanoma

tumors immunohistochemically stained for COX-2 (red color) and are at a magnification of 25× (A) and 40× (B, C, and D). (A)

Retina/choriocapillaries (R) and inner part of a choroidal melanoma (M). The retina (internal positive control) is gliotic and partly degen-

erated and stains intensely for COX-2. Tumor staining at + level. Fibrotic choriocapillaries do not stain for COX-2. (B) Heavily pigmented

epithelioid melanoma in which most cells have COX-2 staining at + level, but some cells (arrows) exhibit ++ staining. (C) Choroidal

melanoma tumor with most cells positive for COX-2, including spindle cells (arrow). (D) Negative area for COX-2 immunoreactivity within

a mixed cell type choroidal melanoma.

Association of Stainingwith Histological and Clinical

Prognostic Markers

Twenty of the 32 tumors analyzed for the presenceof COX-2 staining had also been analyzed for thepresence of closed periodic acid Schiff (PAS) stainedloops. Closed PAS positive loops were detected in 6tumors (33%). The presence of closed PAS stainedloops was not associated with the maximum intensityof COX-2 staining or the percentage of cells staining forCOX-2.

For each tumor, data on the largest basal diameter(LBD) (average 13.5 ± 0.4 mm) and tumor thickness(average 8.7 ± 0.5 mm) was available. There was nosignificant association of LBD with the percentage of

cells immunoreactive for COX-2 (Spearman rank cor-relation r = 0.165, p = 0.37, n = 32). Also, there wasno significant correlation between tumor thickness andthe extent of COX-2 staining (Spearman rank correla-tion r = –0.10, p = 0.610, n = 32). Classification ofuveal melanomas as small, medium, or large in termsof size has previously been adopted for evaluation.22

The tumors assessed in this study were either medium(45.2%) or large (54.8%), according to this classifica-tion. There was no significant association between theextent of COX-2 staining and tumor size as categorisedabove (p = 0.28, Fischer’s exact test, n = 31).

Of the 32 uveal melanomas analysed for COX-2staining, 11 were of spindle cell type, 4 were ofepithelioid cell type, and 17 were of mixed cell types.There was a significant association of the presence

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TABLE 1 Clinicopathological features and analysis of COX-2 staining for each tumor

SpecimenAge

(years) SexLBD

(mm)Tumor

thicknessTumor

sizeCelltype

ClosedPAS-

positiveloops

Survival(years)

Presentsurvival

Deathfrom

metastasis

%COX-2

positivecells

Max intensityof COX-2positive

cellsCOX-2score

1 36 F 14.5 9.7 L M + 1 Y 100 ++ 4002 55 F 16.6 3 L M − 1 Y >70 ++ 2803 71 M 15 15 L M 9 Y 60 ++ 2404 60 M 15 2 M M + 8 Y 50 ++ 2005 82 F 17 11 L M – 1 Y >50 ++ 2006 87 M 11 8 M S 5 >90 + 1807 77 F 13 10 L S 4 >90 + 1808 77 F 14 15 L S 13 Y >90 + 1809 64 M 15.9 8.7 L M + 2 Y >70 + 140

10 79 M 15 8 M S 2 >70 + 14011 74 M 9 8 M M – 12 Y >70 + 14012 69 F 14 6 M E + 1 Y 30 ++ 12013 82 F 10 8 M M – 5 50 + 10014 42 M 13 10 L M 13 Y 50 + 10015 80 F 15 7 M M – 1 20 ++ 8016 74 M 12 12 L E 6 <40 + 8017 65 F 18 9 L S – 12 Y 40 + 8018 63 M 12 6 M M 12 Y <40 + 8019 55 M 13 7 M M – 12 Y <10 ++ 4020 87 F 14.9 9.5 L E – 9 <20 + 4021 86 F 10 N/A N/A E 3 20 + 4022 46 F 13 7 M S 13 Y <20 + 4023 78 F 15 9 L M – 10 20 + 4024 66 F 12 10 L S 13 Y <20 + 4025 74 M 14 8 M S – 2 Y <10 + 2026 85 F 16 11 L M + 9 10 + 2027 57 M 13 10 L M – 11 Y 10 + 2028 73 M 15 11 L M + 1 Y 10 + 2029 63 M 16 10 L M – 7 Y <1 ++ 230 75 M 10.8 8.2 M S – 6 Y 0 – 031 71 F 9 4 M S – 12 Y 0 – 032 54 F 9 8 M S 14 Y 0 – 0

Sex: M = male, F = female. LBD = largest basal diameter. N/A = Not available. Tumor size: M = medium, L = large. Cell type: E = epithelioid, S =spindle, M = mixed. Closed PAS (Periodic Acid Schiff) positive loops: + (presence) or – (absence). Survival is in full years as in September 2007. Presentsurvival and death from metastasis: Y = yes. Max intensity: – = absent, + = slight to moderate, ++ = intense.

of epitheliod cells in melanomas with high intensity(++) of COX-2 staining (p = 0.01, Fischer’s exact test,n = 32).

DISCUSSIONThe major finding of the current study is that in-

creased expression of COX-2 in uveal melanoma is as-sociated with reduced survival rates for these patients.A strong statistically significant association was demon-strated between metastatic death and both the intensityof COX-2 staining and the COX-2 score (encompassingboth intensity and extent of staining).

The mechanisms involved in the genesis and progres-sion of uveal melanoma, the most common intraoculartumor in adults, have not yet been fully elucidated.Changes in the expression of a number of molecularmarkers, including increased expression of insulin-likegrowth factor-1 receptor (IGF-1R) and down-regulationof endothelin receptor B,23,24 have been linked withpoor prognosis in uveal melanoma.

In common with the previous study of COX-2 ex-pression in uveal melanoma,14 we found a significant as-sociation between COX-2 expression and the presenceof epithelioid cells within tumors. We did not observe

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FIGURE 2 Kaplan-Meier survival curves for high versus low COX-2 expression. COX-2 expression was assessed in terms of the per-

centage of tumor cells with positive immunoreactivity for COX-2 (‘low’ represents less than or equal to 50%; ‘high’ represents more than

50 %). (A) The maximum intensity of COX-2 staining in each tumor (‘low’ refers to – or + staining; ‘high’ refers to ++ staining), (B) and the

score, incorporating both percentage cells staining and intensity (‘low’ represents less than 150, ‘high’ represents greater than or equal

to 150).(C) The survival curves illustrated were generated for cancer-specific survival rather than total survival. There was a significant

association between the intensity of COX-2 staining and reduced cancer-specific survival (p = 0.009) and between the COX-2 score and

reduced cancer-specific survival (p = 0.036).

an association of COX-2 staining with the presence ofPAS positive vascular loops, as had been previously re-ported, but only a subset of melanomas in the currentstudy had been analyzed for PAS positive loops. Aninvestigation of the association of COX-2 expressionwith both survival and prognostic markers across an ex-

tended number of uveal melanoma specimens may bewarranted.

The association between COX-2 expression and un-favorable patient survival found in uveal melanomamirrors the association previously described for othermalignancies.25–27 Of note, increased expression of

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COX-2 is associated with poor survival and more ad-vanced stages of colon cancer,27 where NSAIDs areknown to be preventative.1 Expression of COX-2 inprimary tumor cells is also significantly associated withmetastasis in a number of tumors.28,29 Metastatic cellsoriginating from primary tumors with high levels ofCOX-2 also express elevated levels of the enzyme.30

Selective pharmacological inhibition of COX-2 attenu-ates metastasis in various preclinical models,15–17 andcould potentially represent a realistic adjuvant therapyfor uveal melanoma, where metastasis of the primarytumor is a major concern. A recent study showed that atopically applied COX-2 inhibitor could delay the rateof metastasis in a xenograft rabbit model.31

A review of published microarray studies of uvealmelanomas did not reveal any association of COX-2 with metastasis or monosomy 3, although these in-vestigations typically only reveal a subset of the morehighly differentially expressed genes.32,33 Thus, we ex-amined our own dataset from aggressive (monosomy3) versus non-aggressive (disomy 3) uveal melanomasanalyzed by Affymetrix U133A arrays, but did not de-tect differential expression of PGST2 (coding for cy-clooxygenase 2 = prostaglandin-endoperoxide synthase2, with chromosome localization on 1q25.2-q25.3) be-tween tumors with different cytogenetical and clinico-pathological characteristics at the mRNA level (datanot shown). These arrays previously led to the iden-tification and validation of, among others, PERP asa molecular determinant of apoptosis downregulatedin aggressive uveal melanoma.34 However, microarraydata addresses the totality of the tumor rather than in-dividual or groups of cells and may mask more subtlechanges in mRNA expression. A combination of ap-proaches may be preferential to reveal molecular alter-ations, while subsequent investigation of expression atthe protein level remains an important validation stepfor these types of experiments. Moreover, the expres-sion of COX-2 can be regulated at a post-transcriptionallevel resulting in increased protein expression of COX-2without alteration of mRNA levels.35

Further studies of COX-2 in the progression ofuveal melanoma could address the potential prolif-erative, invasive, angiogenic, and metastatic roles ofthis isozyme, all of which have been described forother neoplasms.7–10 The other cyclooxygenase en-zyme, COX-1, is overexpressed in a subset of cancers,including breast, ovarian, and testicular tumors.36–38

An investigation of the expression of COX-1 in uveal

melanoma might also be considered, particularly inlight of the recent concerns raised about the cardio-vascular safety of selective COX-2 inhibitors39 and theestablished chemopreventative effects of non-selectiveCOX inhibitors against colon cancer.1

In summary, the results presented here support thehypothesis that increased expression of COX-2 is asso-ciated with reduced survival in uveal melanoma. Fur-ther investigation into the precise role of COX-2 inmetastatic uveal melanoma is required before COX-2inhibitors and NSAIDs can be considered as a possibleadjuvant therapy for this disease.

ACKNOWLEDGMENTSThis study was supported by funding from Higher

Education Authority of Ireland’s Programme for Re-search in Third Level Institutions and the Health Re-search Board.

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