Abstract
Purpose.:
Monosomy 3 (M3) and abnormalities of chromosome 8 associate with poor prognosis in uveal melanomas (UM). Although M3 has been the subject of more in-depth studies, none have intensively focused on chromosome 8. To elucidate the potential role of chromosome 8 abnormalities, array comparative genomic hybridization (aCGH) was performed on primary UM.
Methods.:
A specifically-designed custom high-resolution array was developed focusing on changes most implicated in UM. Probes for chromosome 8 had a mean spacing of 2.3 kb while chromosomes infrequently affected had a mean spacing of 36.6 kb. A series of 75 UM, including one formalin-fixed paraffin sample were analyzed, and where possible control DNA extracted from the patient's own peripheral blood was used.
Results.:
The most common copy number abnormalities were chromosome 8 (75%) and M3 (51%), with M3 and gain of the long arm of chromosome 8 (8q+) associated in 41% of cases. Also identified were partial deletions of chromosome 3 (3%) and regional 8q+ (23%), and the intensive coverage of chromosome 8 revealed small focal deletions and amplifications affecting both arms. The most significant predictor of prognosis was M3/8q+ having a hazard ratio of 10.1 (P < 0.0001).
Conclusions.:
Neither 8p deletion nor focal changes affecting chromosome 8 were linked to outcome. The most significant indicator was M3/8q, and multiple 8q+ associated with shorter survival. Studying UM with this technology provides a powerful robust tool for predicting prognosis while considering other genetic changes, allowing the future incorporation of such data as it becomes clinically significant.
Uveal melanoma (UM) is the most common primary intraocular tumor of adults, with a highly aggressive nature. Approximately 50% of affected patients die from their disease.
1 The survival of patients with UM is almost entirely dependent on whether they develop liver metastases because once these are detected the prognosis is extremely poor (averaging 6 months), reflecting the multifocal and highly chemo-resistant nature of these lesions, which renders them inoperable and unresponsive to current therapies.
2 New therapies such as the use of mitogen-activated protein kinase kinase (MEK) inhibitors may however prove of value in the future and the area is the subject of intense research.
3 Approximately 500 new cases are diagnosed in the United Kingdom each year and Sheffield, as a national center for the treatment of UM, sees approximately 150 cases annually.
Previous research has shown that UM are characterized by changes of chromosomes 1, 3, 6, and 8, and a relationship has been identified between abnormalities of chromosomes 3 and 8 and a poor prognosis.
4–8 Changes of chromosome 1 also relate to poor prognosis, while those of chromosomes 6 and 11 indicate patients with a better outcome or associate with a less aggressive cell type.
4,5,8 Although loss of a complete copy of chromosome 3 (monosomy or M3), detected by a variety of methods, has long been associated with a worse prognosis in UM, this characteristic alone has not always proved sufficient in predicting which patients are likely to develop metastases. More recently, there has been speculation as to the genes involved in metastatic progression, and although the
BAP1 gene is potentially targeted through M3 and is an interesting candidate,
9,10 little is still known about the genetic drivers of metastasis in UM. Evidence however suggests that changes of chromosome 8 are subsequent to M3 and may therefore facilitate the process.
11,12
An additional gain of 8q, particularly with the concomitant loss of 8p, has proved to be a good predictor of poor prognosis, specifically when considered in combination with M3.
5,12,13 The incidence of 8q gain ranges from 55% to 75% of primary UM depending on the technique used.
14–18 The entire q arm is often amplified and cytogenetic studies initially defined a minimal regional of gain as 8q21-qter,
19 which was subsequently refined by spectral karyotyping and comparative genomic hybridization (CGH) to suggest that two distinct regions of 8q may be amplified, at 8q21.1-21.2 and 8q23-24.
16,18,20,21 Genetic studies of the metastases of UM have suggested gain of 8q may be particularly related to their development, since 8q+ is a consistent finding, whereas other changes, such as the loss of chromosome 3, are not always present. In earlier reports of 16 metastatic lesions of UM, gain of all or part of 8q was found in all but one instance, while abnormalities of chromosome 3 were less common and found in only 10 of 16 cases.
6,22–24 Furthermore, in a recent bacterial artificial chromosome (BAC)-based array CGH study, gains of 8q, often at a high level, were the most common abnormality found in the 66 UM liver metastases studied, 89% compared with M3 in 73%.
12 It is also important to consider the manner in which the gain of 8q arises, mostly in the form of an isochromosome and in a recent single nucleotide polymorphism (SNP)–based study, 8p loss (and M3) remained an independent predictor of poor metastatic outcome after adjusting for the effects of all other variables.
25
Besides being the most frequent observation amongst metastatic UM lesions, evidence suggests that the greater the number of copies of amplified 8q, the shorter the disease-free interval,
5 and in a recent gene expression study, large regions of 8q were shown to be overexpressed in those cases with a short disease free survival.
26 Taken together, these findings suggest that genes targeted by gain of 8q are important to the progression and development of metastases by UM.
Uveal melanomas have been studied previously by array CGH, but at a relatively low resolution of up to 40 kb median probe spacing.
12,13,21 In this context, we have designed a high-resolution CGH array in order to specifically investigate abnormalities of chromosome 8, while also covering other abnormalities related to UM at a higher resolution.
The series comprises 75 primary UM from patients treated at the Royal Hallamshire Hospital (Sheffield, UK) during the period 1994 to 2014. All patients underwent enucleation. Immediately following the procedure in theater, samples were placed in cryovials, collected into liquid nitrogen and stored at −80°C until required. Informed consent was obtained from all UM patients with ethical approval (SSREC 94/247 and 09/H1008/141) and the procedures adhered to the tenets of the Declaration of Helsinki. One sample was obtained from archival formalin-fixed paraffin (FFPE) material. Within the series, approximately one-half the patients had a long follow up/known outcome in combination with cytogenetic analysis. The remainder were analyzed either prospectively or selected on the basis that good quality frozen samples were available.
In the series as a whole, 43 patients were male and 32 were female. There were 44 cases from the choroid, 10 from the ciliary body and 21 from a combined location. Forty were of mixed cell type, 27 were spindle cell, and 8 epithelioid. Twenty-six cases had developed distant metastases. The average median tumor diameter was 14.7 mm (smallest 6.3, largest 22.2). This data is available in
Supplementary Table S1.
The mean age at surgery was 61 years, with the youngest aged 22 and the oldest 87. The mean survival following surgery was 48 months (shortest 2, longest 201).
Design.
A CGH microarray was designed using the eArray tool from Agilent (Santa Clara, CA, USA). For each chromosome, probes were selected to ensure an even coverage. Different densities of probes were chosen, so that those chromosomes previously identified as significant in uveal melanoma were covered with a greater number of probes.
For chromosome 8, the mean interprobe spacing was 2.3 kb, 14.5 kb for chromosomes 1, 3, 6 and 11, and 36.6 kb for the remainder, apart from Y, where the density was approximately one probe every 1443.5 kb. A comparable “off the shelf” design from Agilent (e.g., G4449A has a 13-kb overall median probe spacing [11 kb in RefSeq genes]).
An additional 11,539 probes were included as controls and replicates by the manufacturer and the arrays were provided in a 4 × 180 k format.
Sample Processing and Microarray Procedure.
Analysis.
Seventy-four of 75 cases showed copy number changes, with considerable variation in the levels of amplification found at different chromosomal loci. In most cases, DNA from the patient's own circulating lymphocytes was used as the reference. This offered the considerable advantage of controlling for copy number variation, ensuring that any copy number changes found were likely to be accurate and genuinely associated with the disease. The array had been designed specifically with UM in mind and was concentrated on known associated chromosome changes (1, 3, 6, 8, and 11). Although a wealth of data exists on the relevance of these changes, the most consistently linked with prognosis are those of 3 and 8, which often occur together.
In this series the frequency of changes affecting the most commonly altered chromosomes was as follows 1p-/q+ (40%), M3 (51%), 6p+ (40%), 8q+ (75%), 8p− (28%). The most frequent alteration 8q+, affecting 56 cases (75%), comprised UM with whole-arm gains and also those with only a partial gain of material on 8q. Monosomy 3 was found in 38 cases (51%) and in most instances was associated with a gain of at least one copy of 8q (31 cases). In only one UM was a partial gain of 8q associated with M3 and the most common relationship was between 8p loss and UM cases with both M3 and 8q gain, corresponding to the previously observed close association between M3 and isochromosome 8.
5,28
Breaking this series into those cases with entire or partial gains of 8q the largest group, 39 cases (52%), had gained at least one whole extra copy of 8q, and in nine cases this corresponded to gain of an entire chromosome 8 (simultaneous gain of 8p and 8q). For the remaining cases where only 8q was gained, approximately one-half had multiple gains, with nine cases having two extra whole copies, while five cases had three or more. The gain of 8q was accompanied by a loss of 8p in 21 cases (28%), but no losses of 8p were found without a concomitant whole gain of 8q. For the second group, cases with only partial gain of 8q (17 cases, 23%), there was a considerable range of partial gains affecting the long arm (
Fig. 1). Furthermore all cases with regional loss of chromosome 3 were associated with partial 8q gain. In one instance, this involved both regional loss and gain of chromosome 3 (loss from 3pter to 3p24.1 and a contiguous gain from 3p24.1 to 3p14.1). Abnormalities of chromosome 6 (38 cases, 51%) mainly involved the gain of material from 6p (40%) and 30 cases (40%) had abnormalities of chromosome 1, of which the overwhelming majority showed loss of material from the short arm. There was general agreement between the results of aCGH and previous cytogenetic analysis (see
Supplementary Table S1).
In univariate analysis, only cell type and genetic status were identified as significant variables. Kaplan Meier survival curves for these variables are shown in
Figure 2.
The hazard ratios associated with different genomic abnormalities are shown in
Table 1.
Table 1 Hazard Ratio and Significance of Genetic Aberrations in a Series of 75 Cases of Uveal Melanoma
Table 1 Hazard Ratio and Significance of Genetic Aberrations in a Series of 75 Cases of Uveal Melanoma
Adding various tumor characteristics such as age at operation and sex of the patient into a Cox proportional hazards model of survival gave only genetic status as being significant. The combination of M3 and gain of at least one whole copy of chromosome 8q proved the most powerful, with a hazard ratio of 10.1 (
P < 0.0001). Overall, shorter survival and risk of metastasis appears to be predicated by the presence of M3 accompanied by at least one additional copy of 8q. Using these two abnormalities together as a “test” for metastatic potential had a sensitivity of 81% and a specificity of 80%. Employing either M3 or 8q+ alone gave a better sensitivity (89% and 85%, respectively) but a much worse specificity (68% and 64%). It is difficult to determine if partial 3 loss and associated partial 8q gain have accordingly higher risk as only four cases were identified, and although one patient had died another was alive at 111 months, with average follow up of 40 months, ranging from 4 to 111 months. Furthermore, in this cohort 16 of the 39 cases with 8q+ (21% of the series) had more than one extra copy of 8q. These latter cases had an overall mean survival of 31.9 months, compared with 41.1 months for those with only a single gain of 8q. Hazard ratios for differing copy numbers of 8q are shown in
Table 2, supporting the suggestion that 8q copy number has prognostic implications, and increased gain correlates with a reduced disease free interval following initial diagnosis.
Table 2 Hazard Ratios of Differing Copy Number Gains of 8q in 75 UM
Table 2 Hazard Ratios of Differing Copy Number Gains of 8q in 75 UM
Paying particular attention to alterations affecting chromosome 8, we observed that, in addition to whole arm–based abnormalities, a number of small focal amplifications and deletions were identified. On 8p, these focal events occurred at a number of recurring loci, whereas on 8q they were fewer and appeared to be widely scattered. The occurrence of focal imbalances in relation to other changes of chromosomes 3 and 8 is detailed in
Table 3.
Table 3 Association of 8p Imbalances with M3 and 8q in 75 Cases of UM
Table 3 Association of 8p Imbalances with M3 and 8q in 75 Cases of UM
Focal amplifications were observed in 21 cases (28% of series), and 19 cases had focal deletions of 8p (25% of series). The number of cases affected by specific regions of focal deletion and the genes within are detailed in
Table 4. Only one region was subjected to a higher incidence of focal amplification and is also detailed in
Table 4. The most commonly implicated region for both deletion and amplification was 8p21.3 with seven cases showing a defined focal deletion and interestingly 12 cases having focal amplifications. Of those cases with focal deletions, nine UM had more than one focal deletion and seven UM had both focal amplifications and deletions of 8p, although not affecting the same regions. One of these cases had both an amplification and deletion of 8p21.3, but affecting different regions, suggesting hypervariability in the region. No associations were found between survival, cell type, tumor location, or age with any of the loci identified and the focal abnormalities did not correlate specifically with other genetic imbalances. For example, the most frequent focal amplification of 8p affected 8p21.3 and specifically targeted the
EGR3 as the sole gene at this locus in 12 cases, but was found in UM with otherwise deleted 8p, and those with no deletions of 8p.
Table 4 Regions of Focal Deletion or Amplification of 8p Found in 21 Cases of UM
Table 4 Regions of Focal Deletion or Amplification of 8p Found in 21 Cases of UM
The authors thank Paul Rundle, Sachin Salvi, Lesley Hinchcliffe, and Hardeep Mudhar of the Sheffield Ocular Oncology Service for their clinical input in support of the research.
Supported by a grant from the Weston Park Hospital Cancer Charity (Sheffield, UK); general support provided by the Ocular Oncology Research Fund (Sheffield, UK), Sheffield Teaching Hospital (Sheffield, UK), the National Eye Research Centre (Bristol, UK), and King Fahad Medical City (NAS: Riyadh, Saudi Arabia).
Disclosure: D.W. Hammond, None; N.S.D. Al-Shammari, None; S. Danson, None; R. Jacques, None; I.G. Rennie, None; K. Sisley, None