Cancer Genetics and Cytogenetics 127 (2001) 181–183

0165-4608/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved.PII: S0165-4608(00)00441-6

Short communication

A novel method for eliminating the melanin pigments from melanoma cells undergoing cytogenetic analysis in cases of uveal melanoma

Debashish Das*, Ata-Ur-Rasheed

Hyderabad Eye Research Foundation, L.V. Prasad Eye Institute, L.V. Prasad Marg, Banjara Hills Road #2, Hyderabad, 500 034, India

Received 23 August 2000; received in revised form 22 November 2000; accepted 24 November 2000

Abstract

A method has been developed to eliminate melanin pigments from chromosomal plates of uvealmelanoma tumors. On preparing the chromosomal plates of tumor masses of posterior uveal mel-anoma for cytogenetic analysis in intermediate pigmented population, it was found that the mela-nin pigments obscured the cells and thereby the chromosomes. Thus, cytogenetic analysis couldnot be carried out. Hence, a method was developed in our laboratory to eliminate these obscuringpigments. Several different attempts were made to eliminate the melanin pigments. Finally, wash-ing the cells with phosphate buffered saline before the harvesting stage served the purpose. Afterharvesting and preparing the chromosomal plates, the cells were found to be devoid of melaninpigments. The protocol would help researchers trying to carry out cytogenetic analysis on mela-noma tumor masses in populations with intermediate to dark pigmentation. © 2001 Elsevier Sci-

ence Inc. All rights reserved.

1. Introduction

Uveal melanoma is the most common intra-ocular malig-nant tumor in the adults [1–4], which arises from the ciliarybody and choroid and constitutes so called posterior uvealmelanoma (PUM) [2]. Cytogenetic studies have revealed aseries of genomic alterations in these cases, which provideclues to the cascade of events leading to the tumorigenesis.Uveal melanoma usually occurs sporadically. Rarely, theremay be genetically predisposing factors. Reproducible chro-mosomal changes in uveal melanoma have been reported [5].

Cases of clinically diagnosed posterior uveal melanoma con-stitute the study group. The information given by the patientsrevealed that they were exposed to one of the risk factors citedin the literature, which might have predisposed to the develop-ment of the malignancy. All these cases were from India and allof them had an intermediate pigmentation of their skin. The his-tory of all the patients along with their pedigrees was collected.The pedigree analysis revealed that the cases were isolated.

On following the procedure of Mandhal [6] for preparingthe cells for cytogenetic analysis, it was observed that the

melanin pigment present in the tumor completely obscuredthe chromosomes (Fig. 1). No protocol is currently avail-able to eliminate the melanin pigments from the tumor cellsin an Asian population for additional cytogenetic analysis.Sisley et al. [1] in Sheffield, UK, were contacted for a suit-able protocol to eliminate the melanin pigments. The grouphas personally communicated that they could not suggest aprotocol as they did not face a problem of this sort. This ob-scuring by the melanin pigment appears to be peculiar to theAsian population with intermediate pigmentation. Thus, inan effort to eliminate the melanin pigments, we made sev-eral attempts using different methods: (i) bleaching tech-nique (using 0.25% KMnO

4

and 5% Oxalic acid); (ii) wash-ing the cells with RPMI-1640 without fetal calf serum(FCS) before setting up the culture; (iii) washing the cellswith phosphate buffered saline (PBS) before setting up theculture; (iv) washing of the cells with PBS before culture aswell as before the harvesting stage; and (v) washing thecells with RPMI without FCS before harvesting.

None of these methods eliminated the melanin pigments.When the cells were washed before setting up the culture,no growth was observed, as they stayed nutritionally defi-cient for a long time (from the time of enucleation to thetime of culture set up).

Finally a modified procedure of Mandahl [6], as de-scribed in the following, eliminated the melanin pigments.

* Corresponding author. Developmental Neurobiology, Max-DelbruckCenter for Molecular Medicine, Robert Rossle St. 10, 13125 Berlin, Ger-many.

E-mail address

: d.das@mdc-berlin.de (D. Das).

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D. Das, A.-U. Rasheed / Cancer Genetics and Cytogenetics 127 (2001) 181–183

Fig. 1. Melanin pigments masking the cells and chromosomes (before washing). Magnification �40.

Fig. 2. The cells and chromosomes after washing with PBS. Magnification �40.

D. Das, A.-U. Rasheed / Cancer Genetics and Cytogenetics 127 (2001) 181–183

183

2. Method for eliminating the melanin pigments

The melanoma tissue was removed from the eyeball andwas finely minced under sterile conditions with a scalpel andscissors in a petri plate containing 2 ml of PBS, pH 6.8. Then,the sample was incubated with RPMI-1640 medium contain-

ing 20% FCS at 37

�

C in a 5% CO

2

incubator in tissue cultureflasks. The time of incubation varied based on the type of cul-ture; Direct culture preparation (12 h), short-term culture (5days), and long-term culture (15 days). Before starting theharvesting stage, 10

�

l of (0.2%) ethidium bromide and 10

�

lof (0.02%) colchicine were added to the tissue culture flasks.These were additionally incubated for 90 min at 37

�

C in a 5%CO

2

incubator. Then, the finely minced tissue was washedwith PBS five times (each time mixing the minced tissue vig-orously and then centrifuging at 2000 rpm for 10 min). Thecells were exposed to hypotonic treatment for 30 min with0.075 M (0.56%) KCl. This was followed by washing thecells with Carnoy’s fixative (methanol:acetic acid: 3:1) forfive times. After leaving the cells at 4

�

C for 24 h, chromo-somal plates were prepared by cold treatment. On observingthe slides under microscope it was seen that the cells were de-void of the melanin pigments. This led to the clear visualisa-tion of the cells and chromosomes (Fig. 2).

The cytogenetic analysis of uveal melanoma provides avaluable predictor for prognosis. All the cases in the studygroup were of the intermediate pigmentation. During cyto-genetic analysis the melanin pigments obscured the chro-mosomes. Several different attempts were made including

the most commonly used bleaching technique but the mela-nin pigments could not be eliminated.

The melanin pigments were finally eliminated by themethod described here. The methodology is especially rele-vant to researchers and cytogenetists working in the field ofmelanoma in a population of intermediate to high pigmenta-tions, particularly such as the Asian population.

Acknowledgments

The authors would like to thank Dr. G. Kumarmanick-avel, Senior Genetic Scientist, Vision Research Foundation,Chennai, India, for all his kind help and guidance in carry-ing out this study.

References

[1] Sisley K, Curtis D, Rennie IG, Rees RC. Loss of heterozygosity of thethyroid hormone receptor B in posterior uveal melanoma. MelanomaRes 1993;3:456–61.

[2] Rennie IG. The Ashton lecture. Uveal melanoma, the past, present andfuture. Eye 1997;11:255–64.

[3] Singh AD, Sheilds CL, Sheilds JA, DePotter P. Diagnosis and treat-ment of uveal melanoma. Semin Oncol 1996;23:763–7.

[4] Guttof RF, Chumbley LC. Uveal malignant melanoma. Cur Opin Oph-thalmol 1991;2:250–8.

[5] Seregard S. Posterior uveal melanoma the Swedish perspective. ActaOphthalmol Scand 1996;74:315–29.

[6] Mandahl N. Methods in solid tumor cytogenetics: a practical approachseries. Vol. II. IRL Press, Oxford University Press, 1992.