Cell material from the peritoneal aspirations was subjected to routine diagnostic cytopathology protocols including Giemsa, Papanicolaou, Hematoxylin-Eosin (BDH-Chemicals) stains and immunocytochemistry using the melanocyte specific antibody S-100 (Dako). Immunocytochemical staining against S-100 was performed using Horse Radish Peroxidase (HRP) (Dako). Cell smears were re-hydrated, treated with 3% hydrogen peroxide for 15 minutes and rinsed with Tris Buffered Saline with 0.05% Tween 20 (Dako). After cooling for 20 min, sections were incubated with the primary antibody (rabbit antihuman monoclonal S-100 antibody, 1:400 dilution, Dako) for 1 hour at room temperature and then incubated for 45 min with an anti-mouse HRP labelled polymer (EnVision+System-HRP, Dako). Finally slides were treated with a diaminobenzidine (DAB) chromogenic substrate (Dako) for 10 min, counterstained with hematoxylin, dehydrated and coverslipped.

Malignant cells from two peritoneal aspirations were collected by centrifugation (10 min/1500 rpm/25°C). They were subsequently cultured in eight 25 cm² T-flasks at 37°C and 5% CO₂, in Dulbecco's Minimum Essential Medium supplemented with 10% fetal bovine serum, 0.08 mg/ml amphotericin, 25 units/ml penicillin, and 25 pg/ml of streptomycin (Invitrogen). When high mitotic index was reached, cells were exposed to colcemid (0.1 μg/ml) (Invitrogen) for 30 min, in 37°C, and harvested using trypsine (Invitrogen), after 0.075 KCL hypotonic treatment and Methanol/Acetic acid (BDH-Chemicals) fixation. For the construction of the representative karyotype, we combined G-Banding after Trypsine and Giemsa (GTG-Banding), inverted 4',6-diamidino-2-phenylindole (DAPI)-banding, subtelomeric FISH (TeloVysion-Vysis) and telomeric FISH (Dako), in a total of 400 metaphases from 8 short term monolayer cell cultures. For dual-color interphase or metaphase FISH we used satellite probes specific for chromosomes 7 and 17 (Cytocell). In brief, our general FISH protocol was based on pepsin pre-treatment, formamide or NaOH target denaturation, over-night hybridization and high stringency post hybridization washes. Telomere-specific Peptide Nucleic acid Analog (PNA) hybridizations were performed using a Cy3-(Indocarbocyanine)- conjugated (CCCTAA)₃ probe (Dako), according to manufacturer's instructions. All FISH preparations were mounted and counterstained with VectaShield antifade medium (Vector), containing 0.1 μg/ml DAPI (Sigma). GTG-Banding was performed after trypsine denaturation (Invitrogen) and Giemsa (BDH-Chemicals) staining. Digital images were captured in a Perceptive Systems Imaging, a Metasystems or an Applied Imaging molecular cytogenetics workstations equipped with fluorescent Zeiss, or Nikon microscopes. Quantification of telomeric PNA fluorescence was performed in 500 chromatids on DAPI counterstained metaphase preparations in a single hybridization experiment using the Isis software (Metasystems).

Two mononucleotide markers, BAT-25 and BAT-26 were tested for microsatellite instability by radioactive PCR after Polyacrylamide Gel Electrophoresis (PCR-PAGE) assay, using the following primers: BAT25.1 (5'-TCGCCTCCAAGAATGTAAGT-3'), BAT25.2 (5'-TCTGCATTTTAACTATGGCTC-3'), BAT26.1 (5'-TGACTACTTTTGACTTCAGCC-3') and BAT26.2 (5'-AACCATTCAACATTTTTAACCC-3'). Experiment was monitored by controls for human microsatellite stability (normal genomic and MIN DNA from a patient with human Hereditary Non-Polyposis Colon Cancer – HNPCC-).

Telomerase activity of cell lysates was analyzed by the telomeric repeat amplification protocol (TRAP) assay with a TRAPeze Telomerase Detection kit (Intergen) according to manufacturer's instructions. Approximately 10⁶ cells were harvested and lysed in 400 μl of 1× CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid, 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate) lysis buffer [Tris-HCl 10 mM, pH 7.5; 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM MgCl₂, 0.5% CHAPS 10% glycerol, DEPC (Diethylpyrocarbonate) treated water on ice for 30 min. Cell debris were spun down for 20 minutes at 12,000 r.p.m at 4°C. Each reaction was carried out by using 2 μl of supernatant, 1 μl of each primer, 0.5 μl of Taq-Polymerase (TAKARA), 10 μl of solution-Q (Qiagen), 5 μl of 10× buffer, 2 μl of dNTPs, in DEPC treated water in final volume of 50 μl. The primers used for the TRAP-assay PCR, were TS-5'-AATCCGTCGAGCAGAGTT-3' and Cxa-5'-GTGTAACCCTAACCCTAACCC-3'. The PCR program consisted first of an incubation at 30°C for 30 min and then in a thermocycler, 94°C for 2 min; 94°C for 30 s, 50°C for 25 s, 72°C for 30 s (33×); 72°C for 1 min. PCR products were electrophoresed in a 10% 19:1 acrylamide gel (Sigma)/0.5× TBE (Tris/Borate/EDTA) buffer using the mini protean II gel system (Biorad). Gels were stained with 2 μl of SYBR Green (Sigma) for 15 min at room temperature in 50 ml of TBE 0.5× buffer, and then exposed to UV light and visualized by a Kodak image acquisition station.

To measure cellular telomere length, short term cultured cells were hybridized in situ with a fluorescent telomere-specific peptide nucleic acid probe, according to manufacturer's protocol. Briefly, cells were washed in PBS, and re-suspended to 10⁵ cells/100 μl of a hybridization mixture (Dako) containing 70% formamide and a telomere-specific FITC (Fluorescein isothiocyanate)-conjugated PNA probe. Control samples were re-suspended in hybridization solution without probe to obtain background fluorescence values. After hybridization, cells were spun down and washed twice with 4 ml PBS (Phosphate Buffered Saline) at 40°C for 10 min and finally re-suspended in PBS containing 0.1% Bovine Serum Albumin, 10 μg/ml RNase A (Roche) and 0.1 μg/ml propidium iodide (Calbiochem-Novabiochem). Cells were analyzed on a FACScan flow cytometer (Becton Dickinson) or stored at 4°C before analysis.

Peritoneal fluid samples were obtained by two subsequent paracenteses (within a 12-day interval) of a 38-year-old woman, presented at the Department of Gynecology, Laikon Hospital, with ascites and solid structures at her ovaries as revealed by CT-scan. Two years ago the patient had a less than 1.5 cm large, cutaneous nevus excised from the anterior surface of her left hip. The primary tumor was characterized as a nodular melanoma, Clark's level 3, Breslow's depth 2.0 mm. One out of 14 inguinal nodes, excised in a subsequent operation, was found to be invaded. She received 6 cycles of chemotherapy (cis-platin-dacarbazine) and remained disease-free for 15 months. The cytologic examination of the ascitic aspiration confirmed the presence of malignant cells positive for the melanocyte specific antibody S-100 (Figure. 1). The patient refused to be operated, gave her written consent for further research on the specimens obtained, and expired 40 days after presentation.

G-Banding analysis (according to ISCN 1995) 12 from 8 short-term cell cultures of two peritoneal aspirations taken in an interval of 12 days, showed a 46,XX,del(6)(q23?qter),del(9)(p10pter),der(10)t(7;10)(q31.3qter::p13)del(10)(p14?pter),der(11)t(5;11)(q22.3qter;q23)del(11)(q24?qter),i(17q) pseudodiploid karyotype, in 94–96% of 200 mitoses examined (Figure. 2A). Endoreduplication was observed in 4–6% of the malignant cells leading to a 92,XXXX,idemx2 karyotype. Subtelomeric FISH specific for all human telomeres except for chromosomes 16, 19, 20 and the short arms of acrocentric chromosomes, was used to assist in the description of marker chromosomes identified by G-Banding (Figure. 2A), and to verify deletions spanning up to the end of rearranged chromosomes. To examine if this remarkable karyotypic stability was not confined only to dividing mitotic cells, we performed dual color interphase FISH with probes specific for centromeres 7 and 17, in 200 interphase nuclei obtained from 2 short-term cell cultures from both aspirations. Centromeres 7 and 17 showed notable numerical stability in these populations. The rates of whole genome endoreduplication were similar to those of the karyotyped mitotic cells (Figure. 2B).

In an attempt to attribute the karyotypic stability of this metastatic melanoma to measurable parameters related to chromosome stability in the context of neoplastic continuous growth, we examined microsatellite instability (MIN) and telomerase activity. Microsatellite unstable tumors show a significantly lower rate of chromosomal instability as compared to the MIN negative 3. To rule out underlying microsatellite genomic instability in this metastatic melanoma, we tested by PCR-PAGE the robust mononucleotide repeat markers BAT-25 and BAT-26. Both loci have been shown to be sensitive markers of MIN 13. Compared to positive and negative controls, this metastatic melanoma displayed no micro-satellite instability (Figure. 3A). Ectopic expression of telomerase in normal fibroblasts has been connected to karyotypic stability 14. We conducted a TRAP assay to test telomerase activity in cultured cells from 2 sub-cultures from both peritoneal aspirations. In both samples this assay was negative (Figure. 3B). To examine if these melanoma cells followed the ALT-pathway of telomere maintenance 11 we compared the relative telomeric length of our specimen by Quantitative-PNA-Flow-FISH 15 with the pseudodiploid human acute T cell leukemia JURKAT cell line, normal human fibroblasts and an ALT-positive cell line 16. Cell material for this test was obtained from a short-term subculture that was previously karyotyped and found to be composed exclusively from chromosomally abnormal mitotic cells. This comparison revealed that the melanoma cells had relatively short telomeres (Figure. 3C). PNA-telomeric FISH on 500 chromatids from 10 randomly picked metaphase spreads showed that most of the 46 chromosomes of this metastatic melanoma were uniformly capped with telomeric repeats (Figure. 3D) and no signs of structural chromosome instability attributed to telomere dysfunction such as end-to-end fusions and dicentric chromosomes were evident.