Clinical and Experimental Ophthalmology

2003;

31

: 505–508

Original Article

_____________________________________

Original Article

Serum melatonin levels following enucleation and transpupillary thermotherapy in patients with choroidal melanoma

Hayyam Kiratli

MD

,

1

Sansal Gedik

MD

,

1

Dürdal Us

MD

2

and Sevgül Bilgiç

MD

1

1

Ocular Oncology Service, Department of Ophthalmology, and

2

Department of Clinical Biochemistry, Hacettepe University School of Medicine, Ankara, Turkey

A

BSTRACT

Purpose:

To study serum melatonin levels in patients withchoroidal melanoma preoperatively and following enuclea-tion and transpupillary thermotherapy.

Methods:

Forty patients with preoperative choroidalmelanoma were included in the study. Of these, 15 patientsunderwent enucleation (group 1) and 25 patients receivedtranspupillary thermotherapy (group 2). A further 20 patientswithout melanoma acted as age-matched controls. Serummelatonin levels were determined 1 day preoperatively,and at the first and sixth months postoperatively usingELISA.

Results:

Preoperative serum melatonin levels were highestin group 2 followed by group 1, but this was not statisticallysignificant compared to controls. Postoperatively, a con-stant decrease of serum melatonin was observed in bothgroups after the first month, which reached statistical signif-icance at the sixth month (

P

= 0.003). Also, a significantregression in tumour thickness was documented in group 2(

P

= 0.003). Three patients in group 1 developed hepaticmetastases.

Conclusions:

Patients with choroidal melanomas had higherlevels of serum melatonin compared to individuals withouttumours. Smaller tumours were associated with moreelevated melatonin levels compared to larger tumours.Serum melatonin levels fell considerably following enuclea-tion and transpupillary thermotherapy.

Key words:

choroidal melanoma, enucleation, melatonin,transpupillary thermotherapy.

I

NTRODUCTION

Melatonin, or N-acetyl-5-hydroxytyrptamine, is a neuro-hormone secreted by the pineal gland, colon, retina and theciliary body.

1

Melatonin is 60–80% plasma protein boundand serum levels follow a circadian rhythm, reaching amaximum between 02:00 and 04:00 hours, then droppinggradually during the day.

2

This hormone participates in theregulation of various biological physiological rhythmsincluding induction of sleep, signalling the solar day, andreproductive behaviour.

3,4

It also plays a role in thermo-regulation and modulation of immune reactions.

3

Because ofits highly lipophilic molecular structure, it serves as an extraand intracellular free radical scavenger, notably for thehydroxyl radicals.

3

In the retina, melatonin is locally produced by photorecep-tors and inner nuclear layers and is involved in photoreceptordisc shedding and phagocytosis, cone retinomotor movements,melanosome aggregation in the apical processes of the retinalpigment epithelia, and dark adaptation.

5,6

Similar to serumlevels, melatonin peaks during the night in the retina but theproduction is not dependent on the pineal gland.

7

Also, bloodand aqueous melatonin levels do not correlate.

8

The oncostatic effects of melatonin have been recog-nized for more than two decades. Pineal glands of patientswho had died of cancer were found to have greater weightthan those who had died from other causes.

9

Serum mela-tonin levels were significantly higher in patients withvarious kinds of advanced cancer than control groups.

10

In the present study, serum melatonin levels were deter-mined in patients with choroidal melanoma and the effectsof organ removal and an eye preserving treatment on theselevels were evaluated.

M

ETHODS

This prospective study involved 40 patients with choroidalmelanoma, of whom 15 were managed with enucleation and

�

Correspondence:

Associate Professor Hayyam Kiratli, Department of Ophthalmology, Hacettepe Hastanesi Goz ABD, Sihhiye 06100, Ankara, Turkey.

Email: hkiratli@hacettepe.edu.tr

506 Kiratli

et al

.

25 received transpupillary thermotherapy (TTT). Twentyage-matched individuals presenting consecutively for refrac-tive errors only were selected as controls. Exclusion criteriaincluded obesity, history of a chronic disease, neoplasia andchronic drug intake, particularly hormone therapy.

In addition to a complete ophthalmic examination,patients with choroidal melanomas were initially worked-upwith thoracal and abdominal computed tomography, liverultrasonography, and renal and hepatic function tests in aneffort to detect possible metastases.

Enucleation was performed for tumours larger than11 mm in thickness and with extensive exudative retinaldetachments (group 1). Posterior choroidal melanomasincluding juxtapapillary tumours having ultrasonographicthickness of less than 4.5 mm received TTT through a slit-lamp mounted 810 nm infrared diode laser (group 2).Depending on the size, location and pigmentation of thetumour, 0.5–3.0 mm spot sizes each of 1 min duration at150–550 mW power settings were applied. Treatment wasrepeated at 8–10 week intervals until a flat amelanoticscar was obtained. Following initial experiences, TTT wasfurther limited to tumours having thickness less than 3.5 mm.Patients falling between the two groups were managed witheither I

125

plaque brachytherapy or linear accelerator basedstereotactic radiosurgery. However, because of their insuffi-cient numbers during the study period, these patients werenot included into the final analysis.

Blood samples were collected from a forearm vein theday before the operation, and at the second and sixthmonths postoperatively, always at 14:00 hours. This timewas chosen arbitrarily based on the knowledge that serummelatonin levels are highest during the night and fall duringdaytime. Serum samples were kept at –70

°

C until assayswere performed. Melatonin levels were determined with acommercially supplied ELISA kit (IBL, Hamburg, Germany).First, 50

µ

L of extracted standard, control and samples wereput into the appropriate wells. To these, 50

µ

L of melatoninbiotin and 50

µ

L of antiserum were added and the plate wascarefully shaken. The plate was then sealed with adhesivefoil and incubated for 20 h at 4

°

C. Next day, each well waswashed three times with assay buffer and 150

µ

L of enzymeconjugate was added. The plate was sealed again and incu-bated for 120 min at room temperature in an orbital shakerat 500 r.p.m. After this procedure, each well was washedthree times with assay buffer and 200

µ

L para-nitrophenylphosphate (PNPP) substrate solution was added. The platewas further incubated for 30 min at room temperature in anorbital shaker. The substrate reaction was stopped byadding 50

µ

L PNPP stop solution into the wells. Theabsorbance values were measured by a spectrophotometerat 405 nm.

The preoperative, early postoperative and late post-operative serum melatonin measurements were comparedbetween groups using independent samples

t

-test. Tumourregression in group 2, measured by change in ultrasono-graphic tumour thickness, was analysed using Wilcoxonsigned rank test.

R

ESULTS

The mean ages were 51.2 (range 40–69) years in patientsundergoing enucleation (group 1), 52.9 (range 31–79) yearsin patients receiving TTT (group 2), and 51.4 (range 30–76)years in the control group. The mean preoperative ultra-sonographic tumour thickness was 12.4 (range 11.0–17.2)mm in group 1, and 3.9 (range 3.1–4.5) mm in group 2. Atsixth months postoperatively, the mean tumour thicknesswas 2.1 (range 1.4–3.6) mm, showing significant regressionfollowing TTT (

P

= 0.008).The preoperative serum melatonin levels were apprecia-

bly higher in patients with choroidal melanomas comparedto the controls but this did not reach statistical significance(Table 1). Highest values were obtained from group 2patients. A decrease in serum melatonin levels was docu-mented in all patients 1 month later regardless of the proce-dure performed (Fig. 1) but this was not statisticallysignificant (

P

= 0.142). At the sixth postoperative month, asignificant decrease was noted in both groups (

P

= 0.003).Three patients from group 1 developed hepatic metastasesand these patients had the lowest preoperative and post-operative serum melatonin levels among patients withchoroidal melanomas.

Table 1.

Serum melatonin levels in patients with choroidalmelanoma who underwent enucleation (Group 1) or transpupillarythermotherapy (Group 2) compared to age-matched controls

Group Mean

±

SD serum melatonin level (pg/mL)

Preoperative 1 month postoperative

6 months postoperative

Group 1 160.11

±

146.73 73.32

±

102.92 55.85

±

78.90Group 2 182.59

±

159.11 132.01

±

125.78 80.51

±

86.28Control 89.12

±

107.90 – –

Figure 1.

Graphical representation of serum melatonin measure-ments in patients with choroidal melanoma who underwentenucleation (Group 1,

�

) or transpupillary thermotherapy (Group 2,

�

).

Melatonin in choroidal melanoma 507

D

ISCUSSION

Melatonin may have a direct cytotoxicity on cancer cells ormay induce mitotic arrest.

11,12

In hamsters, removal of thepineal gland resulted in an increase in growth and dissemi-nation of melanoma transplants.

9

In patients with breastcarcinoma, the average 24 h melatonin secretion and thenocturnal peaks of melatonin were significantly depressedand this was more pronounced as the tumour increased insize.

13

Based on these findings, Bartsch

et al.

hypothesizedthat the pineal gland and melatonin might have a role inimmune response modulation and that low melatonin levelsassociated with malignant tumours might induce insufficientimmune response favouring growth of the tumour.

13

Melatonin has a significant inhibitory effect on uvealmelanoma cell growth

in vitro

in a dose-dependent manner.

14

In hamster melanoma cells, melatonin inhibits cellular pro-liferation at physiological (low) concentrations whereas athigher concentrations, the induction of melanogenesis isinhibited.

15,16

In normal uveal melanocytes, there are at leastthree functional melatonin receptors (Mel 1a, 1b and 2) andit is believed that the inhibitory effect of melatonin on uvealmelanoma cells is related to the activation of Mel 1b mem-brane receptor.

3

Melatonin induces a significant reductionin forskolin-stimulated cAMP accumulation in melanomacells.

17

The capability of forskolin-stimulated cAMP accu-mulation is closely related to the metastatic ability ofmelanoma cells.

17

In pathological states like tumorigenesisand viral infections, the metabolic pathway producing mela-tonin from tryptophan is disrupted and trytophan is prefer-entially metabolized to kynurenine, which has no inhibitoryeffect on melanoma cells.

14

The secretion of melatonin increases in early malignancy.

12

Kerenyi

et al.

measured serum melatonin levels of 1.44–2.14 pg/mg in normal individuals against 9.70–20.86 pg/mgin patients with melanoma, representing a considerableincrease in the latter group.

18

A significant decline in serummelatonin levels was measured after the institution of chemo-therapy.

12

Our results are similar to these observations inthat patients with choroidal melanoma had elevated serummelatonin levels compared to controls, which then decreasedfollowing enucleation or TTT. Best prognosis was reportedin patients with the highest levels of serum melatonin.

10

Interestingly, patients who underwent TTT and hencehaving relatively smaller tumours had higher melatoninlevels compared to the enucleation group, who had largertumours. All three patients with systemic metastases occurredin the enucleation group with lowest melatonin values.

There are certain inherent difficulties in studying serummelatonin in uveal melanoma patients. Melatonin is notstored in the pineal gland and shortly after its synthesis, it isreleased into the blood.

19

Uveal melanoma cells can alsoproduce melatonin; however, aqueous melatonin concentra-tions in patients with uveal melanoma were not differentfrom normal controls.

8

This would suggest that the increaseof serum melatonin levels in our patients is unlikely to bedue to secretion from uveal melanomas. Otherwise, much

higher levels would be expected in patients with largetumours (group 1). With current techniques, the source ofserum melatonin (i.e. pineal gland

vs

intraocular) cannot bedetermined. Our finding of high serum melatonin levelsassociated with small choroidal melanomas may be an indi-rect evidence of the growth inhibitory effect of melatoninon human melanoma cells

in vivo

, which has already beenpreviously demonstrated

in vitro

.

R

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