Karen Sisley

Abnormalities of chromosomes 3 and 8 in posterior uveal melanoma correlate with prognosis

Posterior uveal melanomas have nonrandom alterations affecting chromosomes 3, 6, and 8. Loss of chromosome 3 in uveal melanoma has been shown to act as a predictor of disease‐free and overall survival. To confirm the significance of chromosome 3 loss and to extend the observations to include those of the associated alterations of chromosome 8, we have conducted a cytogenetic analysis on a series of 42 tumours from patients with primary uveal melanoma who were followed up for a median of 31 months (range = 8‐96 months). Abnormalities of chromosomes 3 and 8 were the commonest changes and were confirmed in 10 tumours using flourescence in situ hybridization. Monosomy of chromosome 3 was found in 21 (50%) of the tumours, and 23 (54%) tumours had additional copies of 8q. Alterations of chromosomes 3 and 8 were found occurring together in 19 (45%) of the tumours and were significantly associated with a ciliary body component (P < 0.0001). Prognostic indicators and changes of chromosomes 3 and 8 were analysed for correlation with patient survival. Of the chosen parameters, only ciliary body involvement (P = 0.003), monosomy of chromosome 3 (P = 0.0007), and additional copies of 8q (P = 0.003) correlated with reducted survival. Evaluation of the dosage effect of additional copies of chromosome arm 8q showed a significant association with reduced survival (P = 0.0001), which was also predictive of a decreased disease‐free interval (P = 0.01). Thus, the cytogenetic analysis of uveal melanoma may provide a valuable predictor of prognosis. Genes Chromosom. Cancer 19:22–28, 1997.

Prediction of prognosis in patients with uveal melanoma using fluorescence in situ hybridisation

BACKGROUND/AIMS Uveal melanoma is the commonest malignancy of the eye, with a high proportion of patients dying of metastatic disease. Tumours showing a loss of chromosome 3 and gains of chromosome 8 are associated with a worse prognosis. The efficiency of fluorescence in situ hybridisation (FISH) in determining copy numbers of these chromosomes was assessed in individual tumours and related to patient survival. METHODS 33 fresh frozen samples were analysed with centromeric probes for chromosomes 3 and 8. Patient outcomes were divided into two groups: (1) absence of genetic abnormalities (no genetic imbalance) and (2) presence of genetic abnormalities (genetic imbalance). The log rank test was used to compare survival, which was represented by Kaplan-Meier survival curves. RESULTS Of the 33 tumours analysed, 16 showed evidence of genetic imbalances. Of these 16 tumours, 14 patients had died by the end of the study, with 10 having died of liver metastases. Of the tumours without evidence of genetic imbalances, five patients had died by the end of the study, although none had died as a result of either liver metastases or from the primary uveal melanoma. The difference in survival between the two groups was highly significant (p<0.0001). CONCLUSION The authors have shown that FISH analysis for chromosome 3 and 8 is a reliable and efficient technique in the analysis of fresh frozen tumour specimens and is valuable in the prediction of prognosis in individuals with uveal melanomas.

A critical appraisal of the prognostic and predictive factors for uveal malignant melanoma

The intraocular uveal tract comprises the iris, ciliary body and choroid. It contains a well-characterized population of melanocytes, from which uveal malignant melanoma takes origin. In adults uveal melanoma is the commonest primary intraocular malignant tumour. The estimated annual incidence ranges from 7.9 cases per million per year in the USA to 10 cases per million per year in Europe. Uveal melanoma is commoner in Caucasian males, with a median age of presentation of around 60 years. The incidence amongst whites is eight times greater than in blacks. The cause of uveal malignant melanoma is unknown, but several risk factors have been associated with disease development. Some personal characteristics, such as fair complexion, light irides, uveal naevi, dysplastic naevus syndrome, oculodermal and ocular melanocytosis and neurofibromatosis type 1 (NF1) have been associated with an increased risk of uveal melanoma. Furthermore, familial uveal melanoma is recognized and accounts for 0.6% of all uveal melanoma cases, with a proposed autosomal dominant mode of inheritance. The mortality due to uveal melanoma has remained relatively unchanged, despite earlier detection and consequently smaller primary tumour burdens. Metastasis occurs via the blood, classically to the liver, and around 1% of patients have clinically demonstrable liver metastases at presentation. Ultimately, 40% of patients will go on to develop liver metastases, which occur, on average, 10 years after diagnosis and treatment of the primary neoplasm. This probably reflects subclinical hepatic metastasis at the time of the initial diagnosis. Once liver metastases are clinically apparent, the outlook is poor (survival on average 5–9 months).

Loss of heterozygosity of the thyroid hormone receptor B in posterior uveal melanoma.

Cytogenetic Investigations of posterior uveal melanomas have Identified non-random changes of chromosomes 3, 6, and 8. Monosomy of chromosome 3 with additional copies of 8q occur principally in the subgroup of uveal melanomas arising from the ciliary body. To determine whether ciliary body and choroidal melanomas maintain submlcroscoplc differences in their genetic aetiology, studies of the loss of heterozygosity (LOH) of THRB (ERBA β) on 3p were undertaken. DNA from the blood and tumours of 19 patients with uveal melanoma were analysed for LOH of two restriction fragment length polymorphisms (RFLP). Approximately 60% of the uveal melanomas demonstrated allelic loss of THRB. Although the majority of tumours with allelic loss originated wholly, or In part, from the ciliary body, choroidal melanomas also demonstrated allelic loss of THRB. These results suggest that deletions of chromosome 3 occur in the absence of visible cytogenetic changes and may imply that alterations of chromosome 3 are integral to the tumourlgenesis of both sets of uveal melanomas.

Clinical applications of chromosome analysis, from fine needle aspiration biopsies, of posterior uveal melanomas

Purpose An accurate assessment of prognosis is essential to the clinical assessment of malignancy. In posterior uveal melanoma specific chromosome alterations have been shown to correlate significantly with prognosis; but the procedure is restricted to patients treated surgically, and in consequence has been limited mainly to large tumours. Fine needle aspiration biopsy (FNAB) may provide sufficient material to perform this technique, and allow its use in the in situ assessment of tumours, including small lesions. To determine the feasibility of this approach we have conducted a pilot study using enucleated tumours.Methods Ten cases of posterior uveal melanoma were studied. In each instance both a test FNAB and a standard tissue preparation were conducted, and the results compared. FNABs were obtained from enucleated tumours by aspirating cells using a 5 ml syringe with a .25 gauge needle; cells were injected into phosphate-buffered saline, spun down and established in vitro. Conventional short-term cultures were established from tumour tissue samples, which were minced prior to the establishment of cultures. Cytogenetic analysis was performed following standard protocols.Results Of the 10 cases examined, full chromosome analysis was obtainable from all standard tissue short-term cultures. Cytogenetics was successful from cultures of 6 FNAB, with 2 further FNAB producing partial analyses. No major clonal differences were determined between the two procedures.Conclusions Cytogenetic analysis of FNAB appears to be entirely feasible for posterior uveal melanomas, and may permit an accurate in situ assessment of tumours, including small lesions.

Reduced expression of autotaxin predicts survival in uveal melanoma.

Aim: In an effort to identify patients with uveal melanoma at high risk of metastasis, the authors undertook correlation of gene expression profiles with histopathology data and tumour-related mortality. Methods: The RNA was isolated from 27 samples of uveal melanoma from patients who had consented to undergo enucleation, and transcripts profiled using a cDNA array comprised of sequence-verified cDNA clones representing approximately 4000 genes implicated in cancer development. Two multivariate data mining techniques—hierarchical cluster analysis and multidimensional scaling—were used to investigate the grouping structure in the gene expression data. Cluster analysis was performed with a subset of 10 000 randomly selected genes and the cumulative contribution of all the genes in making the correct grouping was recorded. Results: Hierarchical cluster analysis and multidimensional scaling revealed two distinct classes. When correlated with the data on metastasis, the two molecular classes corresponded very well to the survival data for the 27 patients. Thirty two discrete genes (corresponding to 44 probe sets) that correctly defined the molecular classes were selected. A single gene (ectonucleotide pyrophosphatase/phosphodiesterase 2; autotaxin) could classify the molecular types. The expression pattern was confirmed using real-time quantitative PCR. Conclusions: Gene expression profiling identifies two distinct prognostic classes of uveal melanoma. Underexpression of autotaxin in class 2 uveal melanoma with a poor prognosis needs to be explored further.

Expression of PAX 3 alternatively spliced transcripts and identification of two new isoforms in human tumors of neural crest origin

The developmental gene PAX 3 is expressed in the early embryo in developing muscle and elements of the nervous system, including the brain. Since no one has investigated the expression of the isoforms of PAX 3 in the neuroectodermal tumors melanoma and small cell lung cancer (SCLC), we have carried out a comprehensive screening for the expression of the isoforms PAX 3a–e using RT‐PCR in human melanoma cell lines, primary human ocular and secondary cutaneous melanomas. We have identified 2 new isoforms of PAX 3, g and h, which we have isolated, cloned and sequenced. Sets of primers for each isoform were designed and their specificity was confirmed by sequence analysis of the products. The isoforms PAX 3a–e were detected in all human cutaneous melanoma cell lines (8/8), but only PAX 3c (1/2) and PAX 3d (2/2) in ocular melanoma cell lines. The same PAX 3 isoforms were detected in more than 80% of human cutaneous melanomas: PAX 3a and b (15/17), PAX 3c (14/17), PAX 3d (16/17) and PAX 3e (15/17). In contrast the results for 7 SCLC cell lines were PAX 3a (0/7), PAX 3b (1/7), PAX 3c (3/7), PAX 3d (6/7), PAX 3e (2/7); 8/8 cutaneous melanoma cell lines and 8/8 ocular melanoma tissues, together with 14/17 cutaneous melanoma tissues screened, expressed the new isoform PAX 3g. All 8 cutaneous melanoma cell lines expressed PAX 3h, but it was not detectable in any of the tumor tissues (0/20). Neither of the 2 ocular melanoma cell lines expressed the 2 new isoforms. Comparison of the different amplicon staining intensities on a gel suggests that PAX 3c and PAX 3d are the predominant transcripts expressed, with relatively low expression of PAX 3e and PAX 3h. We propose that these and the 2 new isoforms we have discovered may be important in oncogenesis and differential diagnosis of melanomas or SCLC.

Aldehyde dehydrogenase activity selects for the holoclone phenotype in prostate cancer cells

Aldehyde dehydrogenase 1 (ALDH) activity is considered to be a marker of cancer stem cells (CSCs) in many tumour models, since these cells are more proliferative and tumourigenic than ALDH(Lo) cells in experimental models. However it is unclear whether all CSC-like cells are within the ALDH(Hi) population, or whether all ALDH(Hi) cells are highly proliferative and tumourigenic. The ability to establish a stem cell hierarchy in vitro, whereby sub-populations of cells have differing proliferative and differentiation capacities, is an alternate indication of the presence of stem cell-like populations within cell lines. In this study, we have examined the interaction between ALDH status and the ability to establish a stem cell hierarchy in PC3 prostate cancer cells. We demonstrate that PC3 cells contain a stem cell hierarchy, and isolation of ALDH(Hi) cells enriches for the most primitive holoclone population, however holoclone formation is not restricted to ALDH(Hi) cells. In addition, we show that ALDH activity undergoes phenotypic plasticity, since the ALDH(Lo) population can develop ALDH(Hi) populations comparable to parental cells within 2 weeks in culture. Furthermore, we show that the majority of ALDH(Hi) cells are found within the least primitive paraclone population, which is circumvented by culturing PC3 cells as spheroids in defined medium favouring stem cell characteristics. Although ALDH(Hi) status enriches for holoclone formation, this activity may be mediated by a minority of ALDH(Hi) cells.

Reduced expression of TAP-1 and TAP-2 in posterior uveal melanoma is associated with progression to metastatic disease.

In order to determine the effects of the loss or reduced expression of molecules associated with antigen presentation (transporter associated with antigen presentation [TAP]-1, TAP-2, low molecular weight protein [LMP]-2 and LMP-7), we examined the expression of these molecules in primary uveal melanoma lesions. Paraffin-embedded sections from 29 primary uveal melanoma lesions were analysed for expression of TAP-1, TAP-2, LMP-2 and LMP-7 using specific primary antibodies followed by a three-stage immunoperoxidase technique. Microscopic examination was undertaken to determine differences in expression of these molecules on the tumour and the surrounding stroma. Overall, 72% (21 out of 29) of the tumours showed some loss or reduced expression of TAP-1, TAP-2, LMP-2 and/or LMP-7. Statistical analysis of these results showed that progression to metastatic disease was strongly associated with reduced expression of TAP-1 (P<0.05) and TAP-2 (P <0.01), taking patient age, tumour site and histology into account. We conclude that the reduced expression of molecules important in eliciting an immune response, such as TAP-1 and TAP-2, may facilitate the metastatic spread of uveal melanoma lesions and may have important implications for prospective immunotherapy.

Expression of integrins, degradative enzymes and their inhibitors in uveal melanoma: differences between in vitro and in vivo expression.

Posterior uveal melanoma is the most common intraocular malignancy in adults. Metastasis occurs in approximately 40% of all cases and spread is primarily to the liver. Once secondary hepatic disease has developed the prognosis is poor. Metastasis involves a series of adhesion and de-adhesion events, coupled with regulated tissue degradation to facilitate tumour cell invasion and spread to both local and distant sites. These processes are assisted by the expression of integrins and degradative enzymes by both tumour and host cells. Using a series of 10 uveal melanomas, we investigated the expression of a panel of integrins, degradative enzymes and their inhibitors that have been shown to be associated with metastasis. In addition, we undertook to establish if there might be differential expression in response to growth under artificial conditions. All the tumours expressed matrix metalloproteinases (MMP)-2 and-9, tissue inhibitor of metalloproteases (TIMP)-2, urokinase plasminogen activator (u-PA), plasminogen activator inhibitor (PAI)-1 and PAI-2. Differences in the expression of the integrins α1β1, α2β1 and α6β1 were observed; in particular, these differences appeared to relate to expression as a consequence of growth in culture. In summary, uveal melanoma cells express both degradative enzymes and their respective inhibitors, which are important in metastasis. It would appear that differential expression of integrins is present, probably as a response to in vitro stimulation.