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For uveal melanoma it has been shown that nuclear DNA content (ploidy) abnormalities correlate closely with survival,1-4 although conflicting results have been reported for older archival material,5 fresh tissue,6 and differences in DNA quantification techniques.7 A significant correlation between aneuploidy and epithelioid cell type has been reported2 3 8; other studies indicate similar findings,9 or contradict these findings.4 In this issue of the BJO (p 1433) Toti et al report conflicting results on the prognostic value of aneuploidy and association of aneuploidy with epithelioid cell type in uveal melanoma. Furthermore, they found a remarkably high incidence of hypodiploidy.
Methods for ploidy analysis include flow cytometry (FCM), static image analysis (IA), and—more recently—the cytogenetic approach. Each ploidy analysis has its own advantages and disadvantages, many of which reflect the reported conflicting results.10 11 Ploidy analysis by FCM and IA has been reported for uveal melanoma.1-9 12 13 Both methods plot the cells at the different stages of the cell cycle on a histogram. FCM is a widely accessible and fast method, quantifying fluorescent nuclear material in suspension, requiring a relatively large sample size (>5000 cells). IA measures the quantity of light (pixels) in a Feulgen stained specimen requiring fewer cells (100–500 cells), and is more suitable in detecting rare events. The coefficient of variation (CV), reflecting the width of the peak, hence the accuracy of measurements, is usually larger in IA than in FCM. IA is particularly useful in cell recognition and classification, and measurements of cell components. Comparing IA and FCM on uveal melanomas, tetraploidy was detected in tumours that were diploid by FCM.12 By IA it was found that all spindle A cells were diploid,7 12 and most tetraploid peaks were formed by epithelioid cells.12 The lack of association of aneuploidy with epithelioid cell type may partly be explained by the application of a modified (five categories) classification (see Totiet al). Simplification (into three categories) has improved histological correlation of uveal melanoma with malignancy,14 but is still subject to variations in interpretation.
The principal problems with classification of tumour histograms on FCM occur when the deviation from the normal histogram pattern of controls is subtle.10 Near diploid, near tetraploid, and small non-diploid cell populations can be observed in histograms from fresh frozen tissue by FCM, using normal tissue component as the normal counterpart.11 When using paraffin embedded material, no samples are classifiable as hypodiploid (DI <0.95), as by definition the first G0/G1 peak appearing in the histogram is considered to be diploid.11 15 Fixation artefacts may arise particularly in the generation of near diploid DNA aneuploid tumours, but also in the diploid standard.11 Because of the above named restrictions, the recommended FCM ploidy classification on paraffin embedded tissue from solid tumours is diploid versus non-diploid (aneuploid).11 Conflicting results on the prognostic value of DNA aneuploidy can be attributed to differences in methods6 7 and definition of aneuploidy (Toti et al). Difficult areas in flow histograms, specifically high CV, high G2M phase, as well as near diploid aneuploidy and hypodiploidy can be clarified by IA,16 or in frozen tissue by FCM.11
Hypodiploidy has been investigated in IA studies of several solid tumours (frozen tissue and paraffin embedded tissue),17-19 and by FCM on frozen tumour tissue.15 20 Schilling et al 8performed IA on paraffin embedded irradiated and non-irradiated uveal melanomas. They found hypodiploidy exclusively in irradiated melanomas. This is in sharp contrast with the unusual high percentage of non-irradiated, hypodiploid uveal melanomas reported by Toti et al in this issue. Nevertheless, the aggressive behaviour of hypodiploid tumours is an intriguing concept, because loss of one chromosome may result in a hypodiploid DNA pattern. Interestingly, loss of chromosome 3 is an important prognostic factor in uveal melanoma.21 22 However, additional copies of chromosome 8q, which have an inverse dosage effect on survival,22 may balance this. Furthermore, considerable discrepancies have been found between flow cytometric and cytogenetic studies in the detection of aneuploidy in human solid tumours.23
In recent years, fluorescent in situ hybridisation (FISH) has been established as a valuable tool for the detection of the chromosomal aberrations and aneuploidy in a variety of human solid tumours,24 including uveal melanoma.25 The sensitivity of FISH for the detection of aneuploidy is dependent mainly on the number of analysed chromosomes per specimen. Comparative genomic hybridisation (CGH) is based on dual colour FISH between patient tumour DNA and normal reference DNA, and is applicable on paraffin embedded tissue. CGH on uveal melanoma revealed non-random loss of chromosome 3, gain of 6p and loss of 6q, and gain of 8q.26
In summary, discordant results between the different methods of ploidy analysis, different methods of handling tissue, and technical factors most probably represent intrinsic limitations of the different methods, in addition to intratumour ploidy heterogeneity.