the prognostic value of cyclin d1, p53, and mdm2 protein expression in uveal melanoma

Original Paper

The prognostic value of cyclin D1, p53, and MDM2protein expression in uveal melanoma

Sarah E. Coupland1*, Gerasimos Anastassiou2, Andreas Stang3, Harald Schilling2, Ioannis Anagnostopoulos1,

Norbert Bornfeld2 and Harald Stein1

1 Department of Pathology, University Hospital Benjamin Franklin, Free University, Berlin, Germany2 Department of Ophthalmology, University Hospital Essen, Germany3 Institute for Medical Informatics, Biometry and Epidemiology, University Hospital Essen, Germany

*Correspondence to:Dr S. E. Coupland,Institute of Pathology, UKBF,Hindenburgdamm 30,D-12203 Berlin, Germany.E-mail:[email protected]

Received: 21 July 1999

Revised: 27 September 1999

Accepted: 29 November 1999

Abstract

Malignant uveal melanoma is the commonest primary intraocular tumour in adults. It

metastasizes frequently and 50% of patients die within 10 years of diagnosis. The expression of

cyclin D1, p53, and MDM2 in uveal melanoma and their relationship to metastasis-free 5-year

survival was determined, in order to investigate whether these proteins help to distinguish those

patients with a favourable prognosis from those with a poorer one. Ninety-six eyes enucleated for

uveal melanomas were immunohistochemically analysed for the protein expression of cyclin D1

and related cell-cycle markers, p53 and MDM2. The evaluation of the specimens was undertaken

by two independent pathologists without knowledge of the outcome. Statistical analysis of clinical,

morphological, and immunohistological features was performed. A `favourable outcome' was

de®ned as survival of at least 5 years after diagnosis, without metastases (n=57). An

`unfavourable outcome' was de®ned as death from metastases within the ®rst 5 years after

diagnosis of uveal melanoma (n=39). Cyclin D1 positivity (>15% positive tumour cells) as well

as p53 positivity (>15% positive tumour cells) was associated with an unfavourable outcome (for

cyclin D1: odds ratio=4.2, 95% con®dence interval 1.5±11.8, p=0.006; for p53: odds ratio=3.2,

95% con®dence interval 1.1±9.3, p=0.03). In addition, cyclin D1 positivity was associated with

the presence of extraocular extension of the tumour ( p=0.01), with the mixed or epithelioid cell

type (p=0.02), and with the tumour cell MIB-1 positivity ( p=0.0001). MDM2 immunoreactivity

of the tumour cells showed a potential correlation with clinical outcome (odds ratio=2.1, 95%

con®dence interval 0.8±5.8, p=0.13). Multiple logistic regression models showed that cyclin D1

positivity is an independent prognostic factor after control for other prognostic markers. The

expression of cyclin D1 in uveal melanoma is associated with a more aggressive course and

histologically unfavourable disease. This could serve as a further independent prognostic factor in

uveal melanoma. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords: cyclin D1; p53; MDM2; uveal melanoma; prognosis

Introduction

Uveal melanoma is the commonest primary intraoculartumour in white adults, with an incidence rate of 0.7per 100 000 [1]. At the time of initial diagnosis ofuveal melanoma, most patients have no demonstrableevidence of metastatic disease. However, metastasesappear within 5 years in 19±35% of patients and theaverage survival rate following the diagnosis of meta-static disease, usually in the liver, is between 2 and 7months [2,3]. Newer and differing treatment optionsare being investigated for metastatic disease, includingsurgery, systemic chemotherapy, intra-arterial chemo-therapy, chemoembolization, and chemoimmuno-therapy [4]. As surgery and systemic chemotherapyoffer minimal bene®ts for these patients, interest hasbeen shown in the results of ongoing multicentre trialsinvestigating the use of agents such intra-arterialfotemustine and chemoimmunotherapy. Such trialsemphasize the need for the identi®cation of prognostic

markers via histological or molecular biological analy-sis for the selection of `high-risk' patients, who mightbe eligible for adjuvant therapy. Certain prognosticfactors for tumour metastasis and patient survival arealready available in uveal melanoma [5], but it isapparent that new markers are required to predict theclinical behaviour in individual patients.

We recently demonstrated the expression of cyclinD1 protein in uveal melanoma by immunohisto-chemistry, with a positive correlation between cyclinD1 cell positivity and tumour cell type, location, andgrowth fraction [6]. Alterations in cyclin D1 andrelated cell-cycle proteins have also been reported inuveal melanoma cell lines [7], indirectly supporting our®ndings. Cyclin D1 is a critical regulator for cell-cycleprogression through the G1 to the S-phase. The role ofcyclin D1 activity appears to be mediated through thecyclin-dependent kinase (Cdk)/cyclin D1 complex,which induces the phosphorylation of the retinoblas-toma gene protein (pRb) [8,9]. Phosphorylation of

Journal of PathologyJ Pathol 2000; 191: 120±126.

Copyright # 2000 John Wiley & Sons, Ltd.

pRB results in the release of transcription factorsbound to pRB, such as E2F, and ultimate cell-cycle

progression from G1 to the S-phase. An ampli®cation

of the cyclin D1 gene, CCND1, located on chromo-

some 11, with resulting overexpression of the protein,

has been implicated in the development of a number of

malignancies, including cutaneous melanoma, mantle

cell lymphoma, and breast cancer [10]. Tumour cell

positivity for cyclin D1 was found to be detrimental in

the overall survival of patients surgically treated for

oesophageal cancer [11,12], breast cancer [13±15], and

papillary cancer of the thyroid [16].In addition to cyclin D1, other cell-cycle regulatory

molecules may participate in the development and

progression of human tumours. Abnormalities of

the p53 tumour suppressor gene (also known as

TP53) are among the most frequent molecular events

in human neoplasia. The p53 protein initiates either G1arrest of cells (and therefore indirectly antagonizes

the effect of cyclin D1 in cell progression), or

apoptosis, in response to physiological stress such as

DNA damage. The murine double minute 2 (MDM2)

oncoprotein plays a central role in regulating p53.

MDM2 is, in turn, a proto-oncogene and is over-

expressed in many human tumours [17,18]; this onco-

genic potential of MDM2 is considered to be

independent of p53 [19]. Although a number of studies

have investigated the status of p53 in uveal melanoma,

there are no investigations to date regarding MDM2 in

these tumours.In the present study, we undertook to determine the

prognostic value of cyclin D1, p53, and MDM2

protein expression in a clinically well-de®ned collection

of uveal melanomas.

Materials and methods

Patients with primary uveal melanoma (iris melanoma

excluded) who survived the ®rst 5 years after diagnosis

without metastases were de®ned as those with a

`favourable outcome'. An ùnfavourable outcome'

included patients with primary uveal melanoma (iris

melanoma excluded) with metastases within the ®rst

5 years after diagnosis. All patients were diagnosed

between 1972 and 1997 and the primary therapy was

enucleation. Both `favourable' and ùnfavourable'

cases were selected from the medical archives of the

Department of Ophthalmology, University of Essen,

before the morphological and immunohistological

characteristics of the tumours were collected. Consec-

utive patients with known outcome status and avail-

able tissue blocks were selected for this study in

alphabetical order until 39 eligible ùnfavourable'

(mean age at diagnosis 62 years, range 15±84 years)and 57 `favourable' cases (mean age at diagnosis 59

years, range 21±81 years) were recruited. Due to time

and ®nancial restrictions, the recruitment of cases was

stopped at this point.

Conventional histology and immunohistology

All enucleated eyes were ®xed in 4% formalin andembedded in paraf®n. Conventional histological stainsof the uveal melanomas were assessed for tumourlocation (in particular, involvement of the ciliarybody), tumour size (¡10 and >10 mm), extension ofthe tumour (scleral in®ltration or extraocular growth),and tumour cell type [20]. For the statistical analysis,the tumours were divided into two groups according tocell type: `spindle' and `non-spindle' type, the latterincluding mixed tumours with epithelioid cell pre-dominance and pure epithelioid cell tumours.

Additional slides were stained for immunohistochem-ical studies using several monoclonal and polyclonalantibodies which are reactive in paraf®n-embeddedtissues. An antigen retrieval method using a pressurecooker was performed before immunohistochemicalstaining [21]. The staining consisted of a ®rst-stageincubation with the following primary monoclonalantibodies: cyclin D1 (clone P2D11F11; Novocastra,Germany); p53 (clone DO7; DAKO, Denmark), MDM2(clone Ab-1; Calbiochem, Germany) and MIB-1 (direc-ted against the Ki-67 antigen). The antibodies were madevisible with an indirect immunoperoxidase method forp53 and MDM2, whereas the alkaline phosphatase anti-alkaline phosphatase (APAAP) method [22] was used todemonstrate the binding of cyclin D1 protein. In heavilypigmented tumours, the sections were placed in H2O2 for18 h to remove melanin pigmentation from the tumourcells prior to coverslipping the slides, as previouslydescribed [23].

Cells were considered positive for cyclin D1, p53,MDM2, and MIB-1 only when distinct nuclear stain-ing was identi®ed. The percentage of immunoreactivenuclei in the uveal melanoma was evaluated by count-ing at least 5r100 cells using the 40x objective(Olympus, BH2). Positive controls included cases ofmantle cell lymphoma for cyclin D1, tonsil for p53,and colon carcinoma for MDM2. Negative controlswere obtained by omitting the primary monoclonalantibodies.

Statistical analysis

The risk of metastasis within the ®rst 5 years ofdiagnosis was estimated for the histopathologicalcharacteristics and immunohistochemical factors usingodds ratio (OR) estimates, 95% con®dence intervals(95% CIs), and p values calculated from logisticregression models. Potentially confounding factorsexamined were extraocular tumour growth, scleralin®ltration, ciliary body involvement, histologicaltype, and greatest tumour dimension [24]. We assessedpotential interaction between the expression of p53 andMDM2 on an additive scale. The Fisher's exact test(two-tailed) was used to assess the association betweenthe conventional histological and immunohistochem-ical characteristics. All analyses were performed withSAS [25].

Cyclin D1 in the prognosis of uveal melanoma 121

Copyright # 2000 John Wiley & Sons, Ltd. J Pathol 2000; 191: 120±126.

Results

Conventional and immunohistology

Routine histology of the 96 specimens enrolled in the

present study showed a tumour dimension greater than

10 mm in 77 uveal melanomas (80%) (Table 1). The

uveal melanomas consisted of 49 (51%) spindle B, 35

(36%) mixed tumours, and 12 (13%) pure epithelioid

tumours (Figure 1A). Thirty-six of the tumours (38%)

demonstrated ciliary body involvement. Extraocular

extension of the tumour was present in 7 (7%) cases

and scleral in®ltration in 29 (30%) (Figure 1B). The

distribution of the tumours within the `favourable' and

ùnfavourable' groups is summarized in Table 1.

The results of the immunohistochemical staining aresummarized in Table 2. Due to technical problems(®xation and heavy pigmentation), the staining forcyclin D1, p53, and MIB-1 was unsuccessful in sevenùnfavourable' and seven `favourable' cases; similarly,MDM2 staining was unsuccessful in seven ùnfavour-able' and six `favourable' tumours. Consequently, thesepatients were excluded from subsequent statisticalanalyses. It is unlikely that unsuccessful stainingmight have biased our results, because personsinvolved in the staining were masked to the outcome(i.e. favourable or unfavourable). Speci®c nuclearstaining for cyclin D1 was seen in all cases (n=82),with the number of positive cells varying between 1and 30% (Figure 1C). p53 staining was also positive inall tumours (n=82), ranging between 1 and 50%(Figure 1D). MDM2 was negative in two tumours;the remaining cases (n=81), however, demonstrateda range in immunoreactivity between 1 and 20%(Figure 1E). The number of cells positive for MIB-1varied between 2 and 20%, mean 6%.

Associations between cell-cycle proteins andhistological features

The univariate analyses demonstrated a correlationbetween cyclin D1 positivity and extraocular growth ofthe tumour, as well as between cyclin D1-positivity andhistological cell type. The presence of more than 15%cyclin D1-positive cells was associated with extraocularextension of the tumour ( p=0.01). In addition, cyclinD1 positivity (with more than 15% positive cells) wasassociated with mixed or epithelioid cell type(p=0.02). Furthermore, a strong association was seenbetween cyclin D1 positivity (>15%) and the percen-tage of tumour cells expressing MIB-1 (p=0.0001). Noassociation was found between p53 or MDM2 stainingand the histological features of uveal melanoma.

When determining the association among the immu-nohistochemical markers investigated, the univariateanalyses demonstrated positive associations among thepositivities of all three parameters: cyclin D1 withMDM2 ( p=0.04, OR=3.4), cyclin D1 with p53(p=0.001, OR=6.6), and MDM2 with p53(p=0.001, OR=18.2).

Predictive value of the investigated parametersfor the development of metastases

The univariate analysis demonstrated that the risk ofdeveloping metastases depended on tumour positivityfor cyclin D1 (>15% positive cells, OR=4.2, 95% CI1.5±11.8, p=0.006), for p53 (>15% positive cells,OR=3.2, 95% CI 1.1±9.3, p=0.03), the presence ofextraocular growth, in®ltration of the sclera, the celltype (mixed and epithelioid), and ciliary body involve-ment. The greatest tumour diameter (>10 mm) andMDM2 immunoreactivity showed odds ratios of 2.2and 2.1, respectively, although these estimates wereimprecise (Table 1). The assessment of interaction

Table 1. Univariate analysis estimating the risk fordeveloping metastases

Ùnfavourable'

cases*

`Favourable'

cases* Odds ratio(95% CI),

p valueN % N %

Extraocular growth

No 33 85 56 98

Yes 6 15 1 2 10.2

(1.7±62.4), 0.01

Scleral in®ltration

No 20 51 47 82

Yes 19 49 10 18 4.5(1.8±11.0), 0.001

Cyclin D1 expressionNegative (<15%) 19 59 43 86

Positive (15±30%) 13 41 7 14 4.2

(1.5±11.8), 0.006

Ciliary body involvement

No 17 44 43 75

Yes 22 56 14 25 4.0

(1.7±9.4), 0.002

p53 expression

Negative (<15%) 21 66 43 86

Positive (15±50%) 11 34 7 14 3.2(1.1±9.3), 0.03

Cell typeSpindle cells only 15 38 34 60

Mixed or

epithelioid cells

24 62 23 40 2.4

(1.0±5.4), 0.04

Greatest tumour dimension

¡10 mm 5 13 14 25

>10 mm 34 87 43 75 2.2(0.7±6.7), 0.16

MDM2 expression<10% 21 66 41 80

10±20% 11 34 10 20 2.1

(0.8±5.8), 0.13

*Due to technical problems (®xation and heavy pigmentation), the staining

for cyclin D1, p53, and MDM2 was unsuccessful for a few patients; the

study sizes are therefore not the same across all analyses.

122 S. E. Coupland et al.


between p53 and MDM2 demonstrated neither a

synergistic nor an antagonistic effect (data not shown).Due to the relatively small study size, we limited the

number of independent variables in the models to

three. We did not include the variable extraocular

growth in the models, since the number of patients

with extraocular growth was too small (n=7).

Although based on small numbers, the independent

effects of the immunohistochemical factors were

assessed by including all three factors in the regression

model. This analysis showed that cyclin D1 positivity

remained an independent risk factor for metastasis(OR=3.2; 95% CI 1.0±10.0). Positivity of p53 might

also be a potential independent risk factor (OR=2.0;

95% CI 0.5±7.9), whereas immunoreactivity for

MDM2 (OR=1.1; 95% CI 0.3±4.0) cannot be judged,

as the precision of the estimate is too low. After

controlling for the presence of scleral in®ltration and

ciliary body involvement, cyclin D1 and p53 positivityshowed a clear association with the outcome, although

A B

C D

E

Figure 1. (A) Epithelioid cell uveal melanoma (haematoxylin and eosin staining; x40 objective). (B) Intrascleral tumour in®ltration byan epithelioid cell uveal melanoma (haematoxylin and eosin staining; x20 objective). S=sclera; C=choroid. (C) Cyclin D1 positivityin uveal melanoma (APAAP, x20 objective). (D) p53 positivity in uveal melanoma (PAP, x20 objective): the arrow indicates obviousp53 nuclear expression; the dotted staining was considered to be non-speci®c background staining. (E) MDM2 staining in uvealmelanoma (PAP, x20 objective)



the estimate for p53 positivity was less precise(Table 3).

Discussion

As new therapies emerge for metastatic disease in uvealmelanoma, a better understanding of the mechanismsof development and progression is essential in order toimprove the management of patients and, in particular,to identify so-called `high-risk' patients. Several prog-nostic factors have been identi®ed from previousstudies. These include morphological features of uvealmelanoma such as tumour size, location, intrascleraland extrascleral growth, tumour cell type, mitoticcount rate, size of tumour cell nucleoli, tumour vesselpattern and density, lymphocyte in®ltration, and HLAstatus, as well as genotypical features such as monos-omy 3 [26±32]. In the present study, some of the above

morphological characteristics were evaluated and inagreement with these larger series, those tumourcharacteristics associated with an increased risk ofmetastasis included the presence of extraocular growth,scleral in®ltration, the cell type (mixed and epithelioid),and ciliary body involvement. More recent series haveevaluated tumour cell proliferation via immunohisto-chemistry and this has been described as an importantprognostic factor [33,34]. We [6] and others [7] haverecently described a dysregulation of proteins involvedin G1±S cell-cycle progression in uveal melanoma. Thepurpose of this immunohistochemical examination wasto investigate the prognostic value of some of thesecell-cycle markers, in particular, cyclin D1 protein.

In the present study, speci®c nuclear staining forcyclin D1 was seen on immunohistochemical examina-tion in all uveal melanomas, with variable numbers ofpositive cells ranging between 1 and 30%. These resultsdiffer slightly from those in our original series of 66uveal melanomas from Berlin, where 85% of tumoursdemonstrated a range of cyclin D1 positivity between 2and 55% [6]. This difference between the Essen andBerlin series can be explained by one uveal melanomawhich demonstrated a particularly high percentage oftumour cell positivity; on exclusion of this one tumour,the two series demonstrate similar ranges. The `Berlincollection' was not included in the present investiga-tion, due to its shorter clinical follow-up period. Athreshold of 15% positive cells was selected to de®necyclin D1 positivity, in order not to interpret a tumouras cyclin D1-positive because of only a few scatteredstained cells, which may not be representative. Thisapproach has been used by other investigators examin-ing the prognostic value of cyclin D1 expression inother tumours [35,36]. In the present series, 62 (75.6%)of the 82 uveal melanomas investigated were `cyclinD1-negative tumours', demonstrating less than 15%positivity for this marker. Twenty uveal melanomas(24.4%), in contrast, demonstrated 15±30% cyclin D1-positive tumour cells and were thus considered `cyclinD1 positive tumours'. Statistical analysis of morpho-

Table 3. Multiple logistic regression analysis*

Statistical models{ Odds ratio 95% CI p

Model 1

Cyclin D1 3.2 1.0±10.0 0.04p53 2.0 0.5±7.9 0.34

MDM2 1.1 0.3±4.0 0.87

Model 2Scleral in®ltration 4.1 1.3±12.6 0.01

Ciliary body involvement 3.6 1.3±10.1 0.02

Cyclin D1 positivity 3.5 1.1±11.5 0.03

Model 3

Scleral in®ltration 3.9 1.3±11.9 0.02

Ciliary body involvement 3.9 1.4±10.8 0.01p53 positivity 2.7 0.8±9.1 0.11

*Positivity for cyclin D1 15±30%, for p53 15±50% of positive tumour cells,and for MDM2 10±20% of positive tumour cells.

{Reference groups for the independent variables are cyclin D1: <15% of

positive tumour cells, p53: <15% of positive tumour cells; MDM2: <10%

of positive tumour cells; scleral in®ltration: no; ciliary body involvement: no.CI=con®dence interval.

Table 2. Immunohistochemical results of the uveal melanomas examined

Staining of immunohistochemical markers* Overall{ `Unfavourable' cases `Favourable' cases

Cyclin D1 p53 MDM2 N % N % N %

Neg Neg Neg 47 49 13 33 34 60

Neg Neg Pos 6 6 2 5 4 7Neg Pos Neg 2 2 1 3 1 2

Neg Pos Pos 6 6 3 8 3 5

Pos Neg Neg 8 8 5 13 3 5

Pos Neg Pos 2 2 1 3 1 2Pos Pos Neg 3 3 2 5 1 2

Pos Pos Pos 7 7 5 13 2 4

Missing Neg Neg 1 1 0 0 1 2Neg Missing Neg 1 1 0 0 1 2

Missing Missing Missing 13 14 7 18 6 11

*Neg: `negative tumour' for cyclin D1 is a tumour with 1±15% positivity, for p53 1±15%, and for MDM2 0±10% positivity; Pos: `positive tumour' for cyclin

D1 is a tumour with 15±30%, for p53 15±50%, and for MDM2 10±20% positivity; Missing: unsuccessful staining.

{Due to statistical rounding, some percentages do not add up to exactly 100%.



logical and immunohistochemical features with clinicalfollow-up demonstrated that cyclin D1 expression inmore than 15% of tumour cells in uveal melanoma wasassociated with mixed and epithelioid cell tumour type,extraocular growth, the percentage of tumour cellspositive for MIB-1, and an `unfavourable' outcome.Based on the multiple logistic regression models, cyclinD1 positivity was further shown to be an independentrisk factor for the development of metastases in uvealmelanoma.

Considerable variation exists between the previouslyreported investigations of p53 expression in uvealmelanoma [37±40], with tumour positivity rangingfrom 0% [40], through 54% [37] to 67% [38]. In thepresent investigation, p53 nuclear positivity wasobserved in all tumours, ranging between 1 and 50%.In addition, uveal melanoma cell positivity for p53(>15% positive cells) demonstrated a potential corre-lation with an `unfavourable' outcome. In the Berlinseries, 60% of the uveal melanomas were positive forthis marker, with a range between 2 and 30% oftumour cells [6]. The differences between the variousstudies are most likely due to different ®xationtechniques, different p53 antibodies, and differentantigen retrieval techniques. The pitfalls and problemsof p53 immunohistochemical investigations, includingthe attainment of false-negative and false-positiveresults, have been addressed in detail in other humancancers and the value of the immunohistochemicalanalysis alone as indicative of mutation in the p53 genehas been questioned [41]. Further biochemical exam-inations are required to determine the importance ofthe p53 gene in uveal melanoma. Nevertheless, theimmunohistochemical detection of p53 in conjunctionwith related cell-cycle markers, although certainly notbeing suf®cient to reveal mutations in the TP53 gene,does allow an insight into the regulatory mechanismsof tumours.

An association between p53 and MDM2 tumour cellpositivity was seen in the present series, the lattermarker demonstrating immunoreactivity in 1±20% ofcells in 81 tumours. This association between the twomarkers is understandable to some extent, as MDM2acts as a regulator of p53 activity. The transcription ofthe mdm2 oncogene is induced by p53 protein afterDNA damage and the MDM2 protein then binds top53, and blocks its activities as a tumour suppressor,and promotes its degradation. These two proteins thusform an autoregulatory feedback loop in which p53positively regulates MDM2 levels and MDM2 nega-tively regulates p53 levels and activity [19]. MDM2itself has oncogenic potential, which is considered to beindependent of p53 [19] and a trend was recentlyobserved between increased MDM2 expression anddisease progression in cutaneous melanoma [42].Although based on small numbers, MDM2 immuno-reactivity showed a potential association with thedevelopment of metastases in the present study. Theseresults, however, require con®rmation in the form ofadditional biochemical investigations. Some authors

describe an additive effect of co-overexpression of p53and MDM2 in tumours such as soft tissue sarcomas[43]; our limited analyses of potential interactionbetween these factors did not show relevant synergisticor antagonistic effects.

In conclusion, the immunohistochemical detection ofcyclin D1, and potentially of p53, might provide novelprognostic factors for uveal melanoma. The indepen-dent prognostic effects of these factors need to beinvestigated in larger studies that will allow for thesimultaneous control of several previously describedprognostic factors not examined in this study, such asmonosomy 3 and vascular patterns.

Acknowledgements

We sincerely thank Mrs Helga Zimmerman-HoÈffken and Mrs

Heidrun Protz for their technical assistance.

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the prognostic value of cyclin d1, p53, and mdm2 protein expression in uveal melanoma

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