Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

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methylcholanthrene, N-2-fluorenylacetamide, nickel subsulfide, radium, and ethiorene have been linked to the pathogenesis in some cases of uveal melanoma [3]. In rare cases, uveal melanomas can occur in families (see below). However, no locus for hereditary uveal melanoma has been identified. A number of congenital conditions that can predispose to uveal melanoma have been described, including ocular melanocytosis, neurofibromatosis I, and possibly familial multiple mole syndrome. The molecular and genetic changes leading to the development of uveal melanoma in these conditions are not understood [2].

The histologic origin of uveal melanoma is not understood. One school of thought, based on clinical and histopathological studies, holds that the majority of uveal melanomas appear to develop from benign nevi. Yanoff and Zimmerman [4] found spindle-shaped nevus cells at the periphery of approximately three-quarters of these tumors. Furthermore, additional separate nevi were found to be common in eyes with uveal melanomas. However, given the relative abundance of ocular nevi in the population (3–7%) and the rarity of the disease (1400–2000 new cases in the United States each year), only a small minority of nevi (1:5000–15,000) are thought to transform to malignant melanoma per year [5]. This process is well recognized in the development of skin melanomas, where certain nevi may become dysplastic, leading to hyperplasia, in situ carcinoma, and subsequent invasion and metastasis [6]. Others dispute these ideas, however, and suggest that the nevus-like cells found at the base of many uveal melanomas may represent a local mechanical compressive effect on the cells of the choroid [7,8]. Furthermore, de novo formation of a uveal melanoma in the absence of pre-existing nevi has been documented [9]. An alternative hypothesis is that an oncogenic agent might produce both uveal melanoma as well as excessive number of benign melanocytic tumors [10]. A third, lesser-known hypothesis that uveal melanomas derive from Schwann cells from the sheaths of ciliary nerves that transverse the tumor was postulated by DvorakTheobald in 1937 [11] and later supported by data from Vogel [12] (1970). Currently, it is not known what the genetic basis is for the changes from uveal melanocyte (to benign nevus?) to malignant melanoma.

One of the prognostic factors for outcome of uveal melanoma is the type of cells that make up the tumor. Most iris melanomas consist of spindle A cells and these almost never show mitotic figures. Uveal melanomas can consist of spindle (A or B), mixed spindle (mostly B), and epithelioid or epithelioid cell types. The most common type of uveal melanoma is the mixed cell tumor. Spindle B cells rarely show mitotic figures, whereas epithelioid cells frequently do [13]. A larger component of epithelioid cells is linked to poorer outcome. At this time the genetic basis underlying these different uveal melanoma cell types is not known. It is also unclear whether spindle-type cells are in the same pathway as epithelioid cells in the progression of uveal melanoma or whether these cell types represent divergent pathways of uveal melanoma cells. Furthermore, iris melanomas seem to behave differently from other uveal melanomas. It has been shown that there are cytogenetic differences between iris melanomas and choroidal and ciliary body melanomas [14,15].

A great deal of research is currently being conducted in the cancer field to identify the molecular changes between nontransformed cells and primary tumors and between primary tumors and their metastases. Techniques such as microarray analysis, differential display, suppression subtractive hybridization (SSH), and serial analysis of gene expression (SAGE) are being used for these analyses. A number of

these techniques are currently being applied to uveal melanomas to answer some of the questions raised above.

II.FAMILIAL UVEAL MELANOMA

Cases of familial uveal melanoma (FUM) are rare and comprise only 0.6% of all uveal melanoma cases; they have been postulated to follow an autosomal dominant mode of inheritance [16]. At least 53 families have been described with uveal melanoma. FUM most often affects first-degree relatives and rarely affects more than two people in a family [17]. However, in the largest family group, 7 family members over four generations had eyes enucleated, 5 with histologically proven choroidal or ciliary body melanomas [18–20]. In addition, these authors found that patients with FUM were four times as likely to have a second primary malignant neoplasm, suggesting that FUM may confer a generalized inherited predisposition to cancer. No linkage analysis has been reported for any of the reported families with uveal melanoma.

III.PREDISPOSING CONGENITAL CONDITIONS

Ocular melanocytosis is a mostly unilateral congenital condition characterized by hypermethylation of the uveal tract and the episclera. In cases where the eyelid or scalp is affected as well, the condition is called oculodermal melanocytosis (nevus of Ota). It is estimated that 2% or less of all uveal melanomas are associated with oculo (dermal) melanocytosis [21]. The lifetime risk for developing uveal melanoma is 1:400 for a patient with ocular melanocytosis. Remarkable in this regard is that, unlike normal eyes, eyes in this condition, with increased pigmentation, show an increased incidence of uveal melanoma [22].

Neurofibromatosis type I is primarily a disorder of neural crest–derived cells, characterized by congenital hyperchromia and an excessive number of melanocytic nevi in the uveal tract. In rare cases, these patients develop uveal melanoma [23]. This disease is inherited in an autosomal dominant fashion.

Familial atypical mole and melanoma syndrome may be associated with an increased risk of uveal melanoma. However, this link is controversial, since contradictory results have been obtained in different studies [24].

IV. SOMATIC MUTATIONS

A.Cytogenetics

In recent years a host of new techniques have become available to do karyotype analysis on tumor cells. Conventional banding techniques usually require cells in culture to obtain metaphase spreads. More recent techniques, such as fluorescence in situ hybridization (FISH), employ fluorescent probes to look for structural changes in tissue specimens, allowing detection of deletions, translocations, and amplifications. Comparative genomic hybridization (CGH) is a whole-genome screen for DNA copy number alterations. Furthermore, spectral karyotyping (SKY) analysis

employing whole-chromosome specific fluorescent labeling can be used to refine analysis of complex genomic alterations involving marker chromosomes and various translocations. With the advent of these techniques, it is possible to study the karyotypes of uveal melanomas and to relate structural changes in the genome with certain aspects of the disease.

A number of chromosomal abnormalities have been identified that occur nonrandomly in uveal melanomas such as monosomy of 3, gain of 6p, loss of 6q, gain of 8q, and deletion of 1p. The specific oncogenes or tumor suppressor genes within these structural changes that are crucial for certain stages of tumor development are mostly unknown at this time.

1.Monosomy of 3

Several groups have identified monosomy of 3 as a nonrandom cytogenetic aberration that is specific to uveal melanomas [25–31]. CGH studies of copy number abnormalities in these tumors have confirmed these findings [32–34]. These studies showed that monosomy of 3 occurred in about 50% of the cases. Furthermore, cases have been reported with isodisomy of 3, whereby loss of heterozygosity is found even in the presence of two chromosomes 3, resulting in a functional monosomy [35]. The majority of tumors showed a complete loss of chromosome 3. However, in a more recent study, a few patients with uveal melanoma had a partial deletion of chromosome 3 [36]. Among these patients, the smallest region of overlap (SRO) spans 3q24–q26, with a second SRO of about 2.5Mb on 3p25. This suggests the presence of two tumor suppressor genes on chromosome 3. Mutation or deletion of the other allele at each of these locations may have led to gene inactivation, although this has not been detected by CGH or karyotyping. The well-known tumor suppressor gene VHL in this region on 3p is not located in the SRO at 3p25. Since the SROs are on different arms of chromosome 3, these findings may help explain the loss of the entire chromosome 3 in uveal melanomas. Other regions of 3p may also be important in uveal melanomas: a case was reported with the only structural rearrangement being a translocation involving chromosomes 3 and 22, with a breakpoint at 3p13 [37].

Loss of chromosome 3 correlates strongly with metastatic disease [38]. Longterm studies have shown that 4 years after diagnosis, about 70% of the patients with monosomy of 3 in their primary tumor had died of metastasis. Tumors with two copies of chromosome 3 rarely develop metastases [39]. Furthermore, monosomy of 3 has also been associated by some with ciliary body involvement of the tumor [40– 42]. Therefore the basis of poor prognosis in patients with ciliary body tumors may be genetic. In fact, a patient was reported with a ciliary body uveal melanoma that had monosomy of 3 as the only visible cytogenetic abnormality [30]. The development of vascular networks is also associated with ciliary body involvement. This might suggest that monosomy of 3 has a role in vascular network formation, although local environment in the ciliary body could also explain this vascular behavior [43]. Local environment could also have a role in selecting for genetic changes required for tumor progression [44]. An important caveat in many of the correlations between cytogenetic abnormalities and clinical characteristics is the nature of the tumor population that is used. Results may vary depending on the size of the tumors used in the analyses. The advent of alternative therapies to

enucleations might also change the type of tumors available for cytogenetic analysis, especially with regard to tumor size.

2.Gain of 8q

Uveal melanomas show a gain of chromosome 8q in about 50–60% of the tumors [45]. In about 45% of the cases, this gain coincides with monosomy of 3 [42]. Abnormalities of 8q in the form of translocations occur in 24% of the cases with disomy of 3. On the other hand, 82% of the cases with monosomy of 3 showed multiplication of 8q and the majority of these presented isochromosomes of 8q [26]. Since isochromosomes, not only of 8q but also of 6p, are almost exclusively found in the presence of monosomy of 3, it has been suggested that the loss of chromosome 3 may be associated with isochromosome formation itself. Several tumor suppressor genes residing on chromosome 3 may be involved in regulating the centromere or mitotic division, and loss of these genes may result in isochromosome formation [26,38].

There is also a correlation between gain of 8q and ciliary body location and poor prognosis, as is the case with monosomy of 3. The disease-free interval decreases with increased copy number of 8q [42]. This suggests that overexpression of a gene or genes on chromosome 8q is linked to development of the metastatic phenotype. This trend was also found when 8q gains were compared between nonmetastasizing primary tumors, metastasizing primary tumors, and metastases [38]. Gains of 8q were present in 14, 53, and 100% of these tumors, respectively.

Comparisons among tumors have narrowed the amplified region down to 8q23–24-qter [26,32–34]. This region contains the c-myc gene, which is frequently amplified as a later event during tumor progression in many different cancers. Detailed studies in uveal melanomas revealed that c-myc was amplified in 70% of the tumors. In about 43% of these tumors, c-myc amplification could not be explained by simple 8q abnormalities such as trisomy of 8 and isochromosome of 8q. In these tumors, c-myc amplification may be a result of intrachromosomal rearrangement or translocation of a small region of 8q [46]. Alternatively, c-myc amplification may occur as extrachromosomal amplification of c-myc on double minutes, as frequently occurs in other tumors like myeloid leukemias. Both the size, ranging into submicroscopic, and instability in culture might contribute to the apparent absence of double minutes in these tumors.

Since all tumors with c-myc amplification had monosomy of 3 but only 50% of the tumors with monosomy of 3 had c-myc amplification, this latter event may follow loss of a gene or genes on chromosome 3.

3.Chromosome 6 Abnormalities

Chromosome 6 anomalies are found in about 40% of uveal melanomas [33,41,44,47]. These anomalies occur as gains of 6p and/or losses of 6q. In fact, SKY analysis of uveal melanomas and melanoma cell lines suggests that abnormalities involving chromosome 6 may be more frequent than reported by cytogenetic analysis [48]. The majority of anomalies of chromosome 6 are associated with choroidal melanomas only and not with those of the ciliary body [26,28,29,31,44,49,50]. They occur mostly in the absence of abnormalities of 3 and 8, which are associated with ciliary body uveal melanomas [49] although tumors of mixed location show aberrations from

both locations [41,44]. Prognosis is generally better even in the presence of abnormalities of 3 and 8 [41]. Alterations of chromosome 6 in uveal melanoma are similar to those in cutaneous melanoma [51–53], although this still has to be proven at the gene level. Parrella et al. [45] have suggested a model with a bifurcated progression of the tumor with monosomy of 3 and gain of 6p as separate pathways, both followed by the loss of 8q. Losses of 6q are significantly higher in metastasizing primary uveal tumors and metastases than in nonmetastasizing primary tumors [38]. The smallest region for deletion of 6q is 6q22-qter [33].

Gains of 6p on the other hand, were found to occur in only slightly higher numbers in the nonmetastasizing primary uveal melanomas than metastasizing primary tumors and metastases [38]. The SOR on 6p is 6p21-pter [33]. A gene(s) that is the crucial target for this abnormality has not been identified. However, p21 has been mapped to 6p and is thought to be involved in cutaneous melanoma as well as the HLA class I genes, whose expression has been found to be altered in uveal melanomas [54]. Interestingly, it has also been reported that uveal melanomas show frequent loss of heterozygosity at 6p [55].

4.Loss of 1p

It has been reported that loss of chromosome 1p occurs in about 25% of uveal melanomas [34,42,45]. Losses on chromosome 1p were found in primary uveal melanomas that have metastasized and metastases but not in nonmetastasizing primary tumors. This suggested that this region could harbor a tumor suppressor gene important for tumor progression [38]. The SRO was at 1p21-23 and was detected only in metastasizing tumors. Loss of 1p36 is a recurrent event in about 35% these tumors [48], a region that is also frequently deleted in metastatic skin melanomas [56–58]. In many other tumors, loss of 1p is also a late event in tumor progression [59]. Structural abnormalities of 1 are most frequently observed for ciliary body melanomas and those of mixed location (ciliary body and choroid) and correlate with poor prognosis [44]. Translocations involving 1p consistently yielded deletion of 1p. Furthermore, translocation partners for chromosome 1 seem to be nonrandom, with preference for chromosome 8, 13, and 15 involving similar breakpoints. We have karyotyped an epithelioid melanoma cell line (Mel290) and found the only visible cytogenetic abnormality to be duplication of the short arm of chromosome 1 from 1p13-32 and its translocation to chromosome 8q. In addition there is an internal deletion of 1p32-36. This karyotype has remained relatively stable for over 60 passages in culture [60].

5.Other Chromosomal Abnormalities

Uveal melanomas often have minimal cytogenetic changes as compared to many other solid tumors, although the numerical changes (but not the structural changes) increase as tumor size increases. In addition to the frequent karyotypic changes described above, a number of other recurrent changes like trisomy of 21 (often associated with ciliary body tumors), rearrangements of 11 (often associated with choroidal tumors), and loss of the Y chromosome (more frequent as the tumor progresses) have been reported. Loss of heterozygosity of chromosome 9p21, which is the locus of the p16 gene, has been reported in about 28% of uveal melanomas, although mutation of the p16 gene is rarely observed [50,61,62].

B.Alterations in Gene Expression

In addition to nonrandom structural changes in uveal melanoma, a large number of genes have been studied that may have a role in some stage of the development of these tumors. In the next section these genes are roughly divided into categories representing essential alterations for cells to manifest malignant growth. For most of these genes, the basis for their overexpression is not known and changes in expression may be a result of a direct mutation in the gene or a mutational event in a pathway upstream of the gene. This may lead to altered patterns of gene expression in different signal transduction pathways.

1.Cell Cycle Regulation/Proliferation

A critical stage for cells to monitor their environment and to sense signals—which determine whether the cell will proliferate or become quiescent or postmitotic—is the G1 phase of the growth cycle. The retinoblastoma protein (pRb) and other proteins in the pRb pathway have a central role in this decision point [reviewed in Ref. 63]. When hypophosphorylated, pRb blocks cell cycle progression by preventing the transcription factor E2F from transactivating genes important for G1-S phase progression. Cell cycle progression proceeds when pRb is phosphorylated by the cyclin dependent kinases cdk2 and cdk4/6. These kinases are bound to regulatory cyclin units to form active kinase complexes. Upstream of these kinases are inhibitory factors such as p16, p21, and p27. Another factor upstream of pRb is transforming growth factor beta (TGFb), which can prevent phosphorylation of pRb by upregulation of p15 and p21 and suppression of c-myc expression [64,65]. The Ras protein is a positive regulator upstream of cyclin D. Many of the positive and negative effectors of this pathway have been shown to be altered in different cancers.

The retinoblastoma protein itself is infrequently mutated in uveal melanomas. However, pRb was frequently phosphorylated on residues 807 and 811, which leads to inactivation of its tumor suppressor activity [66]. These residues are targets for cyclin D-cdk4/6 kinase activity. Since cyclin D1 was expressed in most tumors in this study and not in normal uveal melanocytes, it suggested a role for this cyclin-cdk complex in the inactivation of pRb. The reason for cyclin D overexpression is not clear in uveal melanomas although in other cancers this has been a result of gene amplification, translocation or disruption of upstream regulatory pathways. The c-myc proto-oncogene has been shown to induce expression of cyclin D, and since c-myc is frequently overexpressed in uveal melanomas, it may alter cyclin D levels [46]. Cyclin D1 expression has been positively associated with epithelioid cell type, anterior location, and extraocular extension and growth fraction and generally with unfavorable outcome in uveal melanomas [67,68]. Overexpression of c-myc demonstrated by immunohistochemistry and flow cytometry has been correlated both to worse prognosis [69] and better prognosis [70–72]. Studies of ras, a protooncogene upstream of cyclin D, revealed no activating mutations at codons 12, 13, and 61 in uveal melanomas [73,74].

Abnormalities of p16 are often associated with skin melanoma [75]. Although a significant amount of the uveal melanomas showed loss of heterozygosity (LOH) at 9p21, where the p16 gene resides, deletion mapping and mutation screening have not shown p16 inactivation in uveal melanoma [62]. Promoter hypermethylation of the

p16 promoter, however, occurs in 32% of primary uveal melanomas and 50% of uveal melanoma cell lines [76]. Expression of p16 could be restored in these cell lines by adding a demethylating agent, suggesting that hypermethylation caused the inactivation of the gene. Significantly, one cell line (Mel270) underwent growth arrest for a considerable time. Colonies that resumed growth were shown to have undergone hypermethylation of the promoter again. In addition to its important role in regulating the cell cycle, p16 may also prevent invasion and angiogenesis [77,78]. Others have described the lack of p16-cdk4 complexes in transformed cells in a small study comparing cultured uveal melanomas to uveal melanocytes [79]. Deficiency in p16 binding to cdk4 has also been found in some familial cutaneous melanomas [80]. TGF-b, like p16, is a negative factor upstream of cyclin D. TGF-b binding to the TGF-b receptor (TGF-bR2) can activate signal transduction pathways involving SMAD proteins, leading to upregulation of cdk inhibitors [81]. Normally TGF-b suppresses growth of human melanocytes but this response is lost in melanoma cells [82–84]. Abnormalities of the TGF-b pathway have been found in 61% of uveal melanomas, potentially occurring at the level of TGF-bR2 or the SMAD 2, 3, and 4 proteins. However, in another study, all uveal melanomas were found to stain positive for TGF-b [85]. Recently, reduced expression of myristoylated alanine-rich C kinase substrate (MARCKS) was found in the choroidal melanoma cell line OCM-1 as compared to primary cultures of choroidal melanocytes [86]. MARCKS levels sharply increase when 3T3 cells exit the cell cycle into G0 during serum starvation, suggesting a role in cell proliferation. Since MARCKS is a calmodulin binding protein, it may modulate calmodulin’s function in G1 progression and mitogenesis. Increasing levels of this protein in OCM-1 by transfection reduces cell proliferation.

Another control on unlimited cell proliferation is telomere erosion. This process leads to cell senescence and acts via p16. Telomerase activity, which maintains telomere length, is upregulated in all uveal melanomas [87].

2.Apoptosis

In addition to alterations in the proliferation, differentiation, and senescence pathways, tumor cells often also manifest alterations in the apoptotic pathway. A very common target for inactivation in cancers is the proapoptotic regulator p53 [88]. It senses DNA damage and other abnormalities, such as oncogene overexpression and hypoxia, and activates the apoptotic cascade via Bax. At the same time, p53 activates p21, which blocks the cell cycle. p53 forms an autoregulatory loop with MDM2, in which p53 positively regulates MDM2, which functionally inactivates p53 by binding to it and targeting it for degradation. MDM2 also has oncogenic potential independent of p53 [89].

Mutation of p53 is an uncommon event in uveal melanoma [72,90]. Tumor positivity of p53 in uveal melanomas varies among different studies from 0 to 100%, which may be the result of different fixation techniques, antibodies, and antigen retrieval techniques [68,72,90–92]. Results may also depend on the source of the melanoma cells, since p53 is rapidly upregulated when tumor cells are brought into culture. Mdm2 is upregulated in most uveal melanomas and is also correlated with poor clinical outcome [66,68]. The mechanism by which this occurs is as yet unclear. Bc1-2 is a survival factor; it can counteract Bax activity and can also block p53