Identification of monosomy 3 in choroidal melanoma by chromosome in situ hybridisation

Abstract

Background/aims: In uveal melanoma monosomy 3 is emerging as a significant indicator of a poor prognosis. To date most cytogenetic studies of uveal melanoma have utilised fresh tissue or DNA extracted from tissue sections. In this study chromosome in situ hybridisation (CISH) was used to study monosomy 3 in tissue sections. The copy number of chromosome 3 was determined and related to patient survival.

Methods: Archival glutaraldehyde or formalin fixed, paraffin embedded material was obtained from 30 metastasising and 26 non-metastasising choroidal melanomas. Hybridisations were performed using centromere specific probes to chromosomes 3 and 18. Chromosome 18 was included as a control as previous abnormalities in uveal melanoma have not been described. Chromosomal imbalance was defined on the basis of changes in both chromosome index and signal distribution.

Results: CISH was successfully performed on both glutaraldehyde and formalin fixed tissue. Four cases were unsuccessful because of extensive tumour necrosis. All cases were balanced for chromosome 18. Monosomy 3 was detected in 15 of the 26 cases of metastasising melanoma; the 26 non-metastasising tumours were all balanced for chromosome 3. Monosomy 3 was significantly associated with metastases related death.

Conclusion: CISH can successfully identify monosomy 3 in archival glutaraldehyde or formalin fixed, paraffin embedded tissue sections. Similar to previous studies monosomy 3 is a significant predictor of metastases related death.

Figure 1

Scattergram showing distribution of chromosome index (CI) for chromosomes 3 (A) and 18 (B) in skin (solid diamond), retina (solid square), non-metastasising melanoma (NMM solid triangle); formalin fixed (FF, open triangle), glutaraldehyde fixed (GF, open square), and metastasising melanoma (MM solid circle; FF, open circle; GF, open triangle). The lines denotes the mean CI (solid line) (SD 3) (black broken lines) or 2.58 SD (grey broken lines) for retina. Values falling 3 SD below the mean were classified as chromosome loss.

Figure 2

Chromosome in situ hybridisation (CISH). (A) Glutaraldehyde fixed normal retina, hybridised with chromosome 18, showing two copies (double arrow) in most cells of the outer nuclear layer (onl). The photoreceptors (pr) are towards the bottom of the picture. (B) Glutaraldehyde fixed, metastasising epithelioid (e) melanoma, hybridised with chromosome 18, showing two copies (double arrow) in most cells. (C) Formalin fixed, non-metastasising epithelioid (e) melanoma, hybridised with chromosome 3, showing two copies (double arrow) in most cells. (D) Glutaraldehyde fixed, metastasising spindle (s) melanoma with moderate pigmentation (p), hybridised with chromosome 3, showing one copy in most cells (single arrow) (magnification ×1000).

Figure 3

Kaplan-Meier survival curve comparing survival with copy number for chromosome 3 as defined by agreement in both chromosome index and signal distribution.