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**Chapter 5**

**Diagnosis, Histopathologic and Genetic Classification**

Uveal melanoma (UM) is the most common cause of primary eye cancer in the western world. During embryogenesis neural crest cells migrate to the neural tract where they devel‐ op into melanocytes. Melanomas of the uvea are derived from these melanocytes. UM may arise in the iris (5%), ciliary body (23%) or choroid (72%). Choroidal melanomas are the most common and usually display a discoid, dome-shaped or mushroom shaped growth pattern. Approximately 80% of the primary intraocular tumours are diagnosed as UM in patients above the age of 20 years, with a mean age of 60 years (Singh & Topham, 2003). Despite a shift towards more conservative eye treatments, survival has not improved during 1973 to 2008 (Singh et al, 2011). Growth of the primary tumour is related with histopathological fea‐ tures, as well as the genetic changes within these tumours. In this chapter we will not dis‐ cuss iris melanoma, as this shows a different clinical and genetic behaviour, compared to ciliary body and choroidal melanoma. The clinical features, histopathological profile and ge‐ netic alterations of UM, as well as therapeutic options for primary tumours and metastases

The incidence of UM ranges from 4.3 to 10.9 per million (Singh et al, 2009). For the past fifty years, the incidence has remained stable, unlike trends indicating a higher incidence of cuta‐ neous melanoma. The incidence in Europe and United States is comparable to that in Aus‐ tralia and New Zealand. In Europe, a lower incidence is reported in Spain and the south of

and reproduction in any medium, provided the original work is properly cited.

© 2013 van Beek et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**of Uveal Melanoma**

http://dx.doi.org/10.5772/53631

**1. Introduction**

will be discussed.

**2. Epidemiology**

N.C. Naus, A. de Klein and E. Kilic

J.G.M. van Beek, A.E. Koopmans, R.M. Verdijk,

Additional information is available at the end of the chapter

## **Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma**

J.G.M. van Beek, A.E. Koopmans, R.M. Verdijk, N.C. Naus, A. de Klein and E. Kilic

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53631

## **1. Introduction**

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[217] Penna E, Orso F, Cimino D, Tenaglia E, Lembo A, Quaglino E, et al. microRNA-214 Contributes to Melanoma Tumour Progression Through Suppression of TFAP2C. The

[218] Chan E, Patel R, Nallur S, Ratner E, Bacchiocchi A, Hoyt K, et al. MicroRNA Signatures

[219] Hanna JA, Hahn L, Agarwal S, Rimm DL. In Situ Measurement of miR-205 in Malignant Melanoma Tissue Supports its Role as a Tumor Suppressor microRNA. Laboratory

Differentiate Melanoma Subtypes. Cell Cycle 2011;10(11):1845-1852.

melanoma. The Journal of Investigative Dermatology 2010;130(8):2062-2070.

Lines. Cancer Research 2007;67(6):2456-2468.

Research 2010;70(10):4163–4173.

Research 2010;16(5):1577-1586.

EMBO Journal 2011;30(10):1990-2007.

Investigation 2012, doi: 10.1038/labinvest.2012.119.

2010;12(5):e9685.

136 Melanoma - From Early Detection to Treatment

553-561.

1751.

Uveal melanoma (UM) is the most common cause of primary eye cancer in the western world. During embryogenesis neural crest cells migrate to the neural tract where they devel‐ op into melanocytes. Melanomas of the uvea are derived from these melanocytes. UM may arise in the iris (5%), ciliary body (23%) or choroid (72%). Choroidal melanomas are the most common and usually display a discoid, dome-shaped or mushroom shaped growth pattern. Approximately 80% of the primary intraocular tumours are diagnosed as UM in patients above the age of 20 years, with a mean age of 60 years (Singh & Topham, 2003). Despite a shift towards more conservative eye treatments, survival has not improved during 1973 to 2008 (Singh et al, 2011). Growth of the primary tumour is related with histopathological fea‐ tures, as well as the genetic changes within these tumours. In this chapter we will not dis‐ cuss iris melanoma, as this shows a different clinical and genetic behaviour, compared to ciliary body and choroidal melanoma. The clinical features, histopathological profile and ge‐ netic alterations of UM, as well as therapeutic options for primary tumours and metastases will be discussed.

## **2. Epidemiology**

The incidence of UM ranges from 4.3 to 10.9 per million (Singh et al, 2009). For the past fifty years, the incidence has remained stable, unlike trends indicating a higher incidence of cuta‐ neous melanoma. The incidence in Europe and United States is comparable to that in Aus‐ tralia and New Zealand. In Europe, a lower incidence is reported in Spain and the south of

© 2013 van Beek et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Italy, about 2 per million, whereas registries in France, the Netherlands, Switzerland and Germany has intermediate values around 4 to 5 per million. The United Kingdom registered over 6 per million, and the highest incidence is up to > 8 per million in Norway and Den‐ mark (Virgili et al, 2007).

## **3. Predisposing factors**

Men and women with UM are more or less affected equally (Damato & Coupland, 2012; Singh et al, 2011). Iris melanoma is more common in women than in men (Damato & Coup‐ land, 2012). Several phenotypes, like blue or grey eyes and fair skin have been suggested to predispose for UM (Schmidt-Pokrzywniak et al, 2009). This might explain why Caucasians are approximately 150 times more frequently affected than Africans (Margo et al, 1998; Singh et al, 2005a). In Asians UM is less common (Biswas et al, 2002).

**Figure 1.** A large amelanotic uveal melanoma leads to a visual field defect.

when in doubt, an intraocular biopsy is taken of the tumour.

mimicking lesion (Eskelin & Kivelä, 2002; Khan & Damato, 2007).

Diagnosis of UM is based on a combination of clinical examination with slit lamp biomicro‐ scopy, indirect ophthalmoscopy (figure 1, 2a, 3a) and ultrasonography (US) (figure 2b, 3b). Iris melanomas are readily detectable by slit lamp biomicroscopy, whereas ciliary body tu‐ mours are hidden behind the iris and can be visualized by US. Choroidal tumours, depend‐ ing on their location, are diagnosed by dilated indirect ophthalmoscopy and US. In suspect cases of intravenous fluorescein angiography can be helpful in differentiating melanomas from other diagnoses. Also optical coherence tomography (OCT) and autofluorescence can provide additional information (Lavinsky et al, 2007; Shields et al, 2008). In selected cases,

Diagnosis, Histopathologic and Genetic Classification of Uveal Melanoma

http://dx.doi.org/10.5772/53631

139

Indirect ophthalmoscopy through a dilated pupil provides a correct diagnosis in more than 95% of the cases (Char et al, 1980). Accuracy of the right diagnosis is established to be over 99% by experienced clinicians with US, ophthalmoscopy, and fluorescein angiography and confirmed by histopathology (Collaborative Ocular Melanoma Study Group, 1990). The abil‐ ity to differentiate melanoma from other lesions has improved over the last decades. When comparing studies of 1964 and 1973, in 19% of the enucleated patients with the clinical diag‐ nosis melanoma no histopathological evidence of a melanoma was found (Ferry, 1964; Shields, 1973). The accuracy in diagnosing medium to small sized tumours is quite challeng‐ ing. Nine percent of presumed melanomas are found to have another diagnosis by fine nee‐ dle aspiration biopsy (Char & Miller, 1995). Most important is to minimise the delay in referring patients with melanoma to a specialised centre. It is reported that in 29% of the pa‐ tients a melanoma is missed during the first visit by an ophthalmologist, and that 31.5% of the patients referred to an oncology centre with the diagnosis of melanoma actually had a

**5. Diagnosis**

From all the parts of the uvea the iris is most exposed to ultraviolet light, because of filtering effects of the lens and retinal pigment epithelium (RPE), the choroid receives less light (Singh et al, 2004). Although several epidemiologic and case control studies have been per‐ formed to investigate the influence of sunlight exposure on UM, the results are not conclu‐ sive (Guenel et al, 2001; Holly et al, 1990; Pane & Hirst, 2000; Shah et al, 2005; Vajdic et al, 2002). UM may occur as a part of familial syndromes, like xeroderma pigmentosa, Li-Frau‐ meni syndrome and familial breast and ovarian cancer. Of all UM 0.6% is considered to be familial (Singh et al, 1996). In a retrospective study 0.0017% of the primary UM patients were in the setting of familial atypical mole and melanoma syndrome (FAMM). These pa‐ tients were relatively young with a mean age of 40 years (Singh et al, 1995). Furthermore, an association of neurofibromatosis type 1 and UM has been suggested, since both are of neural crest origin, however this association remains unclear (Honavar et al, 2000). Ocular and ocu‐ lodermal melanocytosis (Nevus of Ota), dysplastic nevi and cutaneous melanoma are corre‐ lated with an increased risk of UM development (Carreno et al, 2012; Gonder et al, 1982; Hammer et al, 1995; Richtig et al, 2004; Singh et al, 1998; Toth-Molnar et al, 2000; van Hees et al, 1994). Additionally, in UM patients ocular and oculodermal melanocytosis are about 35 to 70 times more common (Carreno et al, 2012; Singh et al, 1998).

## **4. Clinical presentation**

Depending on de location and size of the tumour, patients can present with visual com‐ plaints. Most UMs are detected during a routine ophthalmic examination. Approximately 30% of the patients have no symptoms at time of diagnosis, and if there are any complaints these consist mostly of blurred vision, floaters, photopsias and visual field loss (Damato, 2010) (figure 1). Usually patients do not present with severe ocular pain, however, this can occur secondary to inflammation or neovascular glaucoma.

**Figure 1.** A large amelanotic uveal melanoma leads to a visual field defect.

## **5. Diagnosis**

Italy, about 2 per million, whereas registries in France, the Netherlands, Switzerland and Germany has intermediate values around 4 to 5 per million. The United Kingdom registered over 6 per million, and the highest incidence is up to > 8 per million in Norway and Den‐

Men and women with UM are more or less affected equally (Damato & Coupland, 2012; Singh et al, 2011). Iris melanoma is more common in women than in men (Damato & Coup‐ land, 2012). Several phenotypes, like blue or grey eyes and fair skin have been suggested to predispose for UM (Schmidt-Pokrzywniak et al, 2009). This might explain why Caucasians are approximately 150 times more frequently affected than Africans (Margo et al, 1998;

From all the parts of the uvea the iris is most exposed to ultraviolet light, because of filtering effects of the lens and retinal pigment epithelium (RPE), the choroid receives less light (Singh et al, 2004). Although several epidemiologic and case control studies have been per‐ formed to investigate the influence of sunlight exposure on UM, the results are not conclu‐ sive (Guenel et al, 2001; Holly et al, 1990; Pane & Hirst, 2000; Shah et al, 2005; Vajdic et al, 2002). UM may occur as a part of familial syndromes, like xeroderma pigmentosa, Li-Frau‐ meni syndrome and familial breast and ovarian cancer. Of all UM 0.6% is considered to be familial (Singh et al, 1996). In a retrospective study 0.0017% of the primary UM patients were in the setting of familial atypical mole and melanoma syndrome (FAMM). These pa‐ tients were relatively young with a mean age of 40 years (Singh et al, 1995). Furthermore, an association of neurofibromatosis type 1 and UM has been suggested, since both are of neural crest origin, however this association remains unclear (Honavar et al, 2000). Ocular and ocu‐ lodermal melanocytosis (Nevus of Ota), dysplastic nevi and cutaneous melanoma are corre‐ lated with an increased risk of UM development (Carreno et al, 2012; Gonder et al, 1982; Hammer et al, 1995; Richtig et al, 2004; Singh et al, 1998; Toth-Molnar et al, 2000; van Hees et al, 1994). Additionally, in UM patients ocular and oculodermal melanocytosis are about 35

Depending on de location and size of the tumour, patients can present with visual com‐ plaints. Most UMs are detected during a routine ophthalmic examination. Approximately 30% of the patients have no symptoms at time of diagnosis, and if there are any complaints these consist mostly of blurred vision, floaters, photopsias and visual field loss (Damato, 2010) (figure 1). Usually patients do not present with severe ocular pain, however, this can

Singh et al, 2005a). In Asians UM is less common (Biswas et al, 2002).

to 70 times more common (Carreno et al, 2012; Singh et al, 1998).

occur secondary to inflammation or neovascular glaucoma.

mark (Virgili et al, 2007).

138 Melanoma - From Early Detection to Treatment

**3. Predisposing factors**

**4. Clinical presentation**

Diagnosis of UM is based on a combination of clinical examination with slit lamp biomicro‐ scopy, indirect ophthalmoscopy (figure 1, 2a, 3a) and ultrasonography (US) (figure 2b, 3b). Iris melanomas are readily detectable by slit lamp biomicroscopy, whereas ciliary body tu‐ mours are hidden behind the iris and can be visualized by US. Choroidal tumours, depend‐ ing on their location, are diagnosed by dilated indirect ophthalmoscopy and US. In suspect cases of intravenous fluorescein angiography can be helpful in differentiating melanomas from other diagnoses. Also optical coherence tomography (OCT) and autofluorescence can provide additional information (Lavinsky et al, 2007; Shields et al, 2008). In selected cases, when in doubt, an intraocular biopsy is taken of the tumour.

Indirect ophthalmoscopy through a dilated pupil provides a correct diagnosis in more than 95% of the cases (Char et al, 1980). Accuracy of the right diagnosis is established to be over 99% by experienced clinicians with US, ophthalmoscopy, and fluorescein angiography and confirmed by histopathology (Collaborative Ocular Melanoma Study Group, 1990). The abil‐ ity to differentiate melanoma from other lesions has improved over the last decades. When comparing studies of 1964 and 1973, in 19% of the enucleated patients with the clinical diag‐ nosis melanoma no histopathological evidence of a melanoma was found (Ferry, 1964; Shields, 1973). The accuracy in diagnosing medium to small sized tumours is quite challeng‐ ing. Nine percent of presumed melanomas are found to have another diagnosis by fine nee‐ dle aspiration biopsy (Char & Miller, 1995). Most important is to minimise the delay in referring patients with melanoma to a specialised centre. It is reported that in 29% of the pa‐ tients a melanoma is missed during the first visit by an ophthalmologist, and that 31.5% of the patients referred to an oncology centre with the diagnosis of melanoma actually had a mimicking lesion (Eskelin & Kivelä, 2002; Khan & Damato, 2007).

## **5.1. Characteristics**

Melanoma are generally pigmented, but one fourth are relatively non-pigmented or amela‐ notic (figure 1). Melanoma can develop into two different directions: towards the vitreous and outwards, through the underlying sclera. Having broken through Bruch's membrane, into the vitreous, UMs achieve a characteristic shape, even pathognomonic, like a 'collar but‐ ton' or 'mushroom'. Small melanomas can appear flat or dome shaped.

## **5.2. Clinical prognostic factor**

Well-known clinical prognostic factors are age and location of the tumour. Older patients tend to have a worse prognosis (Shields et al, 2012). One study found that UMs were located predominantly posterior and temporal or had a preference for macular zone, while others found a more equal distribution of melanoma (Krohn et al, 2008; Li et al, 2000; Shields et al, 2009b). Patients with larger tumours, tumours that ruptured through Bruch membrane and in patients who have developed metastasis, the tumours were significantly more often locat‐ ed anterior to the equator (Krohn et al, 2008).

**Figure 3.** a: Pigmented uveal melanoma with orange pigment (lipofuscin); 3b: A homogeneous grey scale in the tu‐ mour and choroidal excavation on B-scan ultrasonography; 3c: Optical coherence tomography of the same tumour

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141

The most important clinical prognostic factor is tumour size, and is often used for selection of the treatment. There are several treatment options, which will be discussed later in this chapter. UM are subdivided into different categories depending on the apical size and diam‐ eter, however, many centres use their own definition. Most widely used definition is sug‐ gested by the COMS study. Small melanomas are 1.0 - 2.5 mm in apical height and > 5.0 mm in largest basal dimension (Collaborative Ocular Melanoma Study Group, 1997). Medium tumours are defined as tumours 2.5 to 10 mm in apical height and ≤ 16 mm in largest basal diameter. Large tumours are ≥ 2 mm in apical height and > 16 mm in maximal basal diame‐ ter, or a melanoma > 10 mm in apical height, regardless of the basal diameter (Collaborative Ocular Melanoma Study Group, 2003). One large study described that each increase in milli‐ meter of tumour thickness increased the risk for metastasis by 5% (Shields et al, 2009b). The mortality rate for small (< 2 - 3 mm height), medium (3 - 8 mm height) and large (> 8 mm height) melanoma was 16%, 32% and 53% in 5 years, respectively, and has not changed in recent years (Diener-West et al, 1992). This supports the model of tumour doubling time of melanoma and its' related metastasis. The model suggests that micrometastasis already exist

with subretinal fluid.

**Figure 2.** a: A dark pigmented uveal melanoma with orange pigment; 2b: On B-scan ultrasonography acoustic hol‐ lowing and choroidal excavation is present, 2c: Subretinal fluid and retinal pigment epithelial alterations are visible on optical coherence tomography scan at the top of the tumour.

**5.1. Characteristics**

**5.2. Clinical prognostic factor**

140 Melanoma - From Early Detection to Treatment

ed anterior to the equator (Krohn et al, 2008).

optical coherence tomography scan at the top of the tumour.

Melanoma are generally pigmented, but one fourth are relatively non-pigmented or amela‐ notic (figure 1). Melanoma can develop into two different directions: towards the vitreous and outwards, through the underlying sclera. Having broken through Bruch's membrane, into the vitreous, UMs achieve a characteristic shape, even pathognomonic, like a 'collar but‐

Well-known clinical prognostic factors are age and location of the tumour. Older patients tend to have a worse prognosis (Shields et al, 2012). One study found that UMs were located predominantly posterior and temporal or had a preference for macular zone, while others found a more equal distribution of melanoma (Krohn et al, 2008; Li et al, 2000; Shields et al, 2009b). Patients with larger tumours, tumours that ruptured through Bruch membrane and in patients who have developed metastasis, the tumours were significantly more often locat‐

**Figure 2.** a: A dark pigmented uveal melanoma with orange pigment; 2b: On B-scan ultrasonography acoustic hol‐ lowing and choroidal excavation is present, 2c: Subretinal fluid and retinal pigment epithelial alterations are visible on

ton' or 'mushroom'. Small melanomas can appear flat or dome shaped.

**Figure 3.** a: Pigmented uveal melanoma with orange pigment (lipofuscin); 3b: A homogeneous grey scale in the tu‐ mour and choroidal excavation on B-scan ultrasonography; 3c: Optical coherence tomography of the same tumour with subretinal fluid.

The most important clinical prognostic factor is tumour size, and is often used for selection of the treatment. There are several treatment options, which will be discussed later in this chapter. UM are subdivided into different categories depending on the apical size and diam‐ eter, however, many centres use their own definition. Most widely used definition is sug‐ gested by the COMS study. Small melanomas are 1.0 - 2.5 mm in apical height and > 5.0 mm in largest basal dimension (Collaborative Ocular Melanoma Study Group, 1997). Medium tumours are defined as tumours 2.5 to 10 mm in apical height and ≤ 16 mm in largest basal diameter. Large tumours are ≥ 2 mm in apical height and > 16 mm in maximal basal diame‐ ter, or a melanoma > 10 mm in apical height, regardless of the basal diameter (Collaborative Ocular Melanoma Study Group, 2003). One large study described that each increase in milli‐ meter of tumour thickness increased the risk for metastasis by 5% (Shields et al, 2009b). The mortality rate for small (< 2 - 3 mm height), medium (3 - 8 mm height) and large (> 8 mm height) melanoma was 16%, 32% and 53% in 5 years, respectively, and has not changed in recent years (Diener-West et al, 1992). This supports the model of tumour doubling time of melanoma and its' related metastasis. The model suggests that micrometastasis already exist several years before diagnosis of the primary tumour (Eskelin et al, 2000). This emphasizes the importance of identifying small melanoma and reducing the risk of metastases.

### **5.3. Clinical predictive factors of small melanoma**

In general, choroidal nevi have a less than 5 mm basal diameter and are minimal in height (< 2 mm), although several definitions of nevi have been proposed. Due to differ‐ ent examination methods and definitions, the prevalence of nevi is between 0.2% and 30% (Gass, 1977; Wilder, 1946). Overall in a Caucasian population the incidence is 6.5% (Sumich et al, 1998). Whenever, growth of a nevus is measured on US in a short time a transformation into a small melanoma is suspected. On the other hand benign nevi can also grow slowly. Mashayekhi *et al* observed in 31% of nevi a slight growth, without evi‐ dence of development into a melanoma over a mean follow up of 15 years (Mashayekhi et al, 2011). As described by Singh and co-workers, assuming that all melanoma result from nevi, 1 out of 8845 choroidal nevi can undergo malignant transformation into mela‐ noma in the Caucasian population in the USA (Singh et al, 2005b). In Australia this is es‐ timated 1 out of 4300 nevi (Sumich et al, 1998).

**Figure 4.** a: Peripapillary nevus, barely elevated, with margin located < 3 mm to the optic disc in the right eye of a 72

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143

US is a non-invasive tool and helps to establish the diagnosis of UM, despite media opacities or whether the tumour is located far peripherally. UM shows characteristic low to medium internal reflectivity on A-scan. B-scan US is primarily used to plan therapy based on the first measurement, and to periodically measure tumour prominence (height) and basal diameter for follow-up. The B-scan can identify possible extraocular extension as an empty area be‐ hind the sclera. On B-scan US the internal structure of the tumour is typically seen as a rela‐ tive homogeneous grey scale, although this pattern is not specifically diagnostic (figure 3b). At the base of the tumour an acoustically silent zone (called acoustic hollowing) is seen, as well as choroidal excavation and shadowing in the orbit (figure 2b). Eighty-eight percent of the UM show US hollowness or low acoustic reflectivity (Boldt et al, 2008). Choroidal exca‐ vation is not observed in all melanomas and varies from 42% to 70% (Coleman et al, 1974; Sobottka et al, 1998; Verbeek, 1985). US provides accurate measurements with an interob‐

The diagnostic value of fluorescein angiography in UM is limited. Fluorescein angiography does not show pathognomonic patterns and is especially helpful in differentiating lesions, which simulate melanoma. The pigmentation, size and effect on the RPE of the tumour in‐ fluence the fluorescein angiogram. It is of little help in some medium to large melanomas that have an intrinsic tumour circulation. This 'double circulation' (simultaneous visualiza‐ tion of retinal and choroidal circulation) consists of late staining of the lesion and multiple pin-point leaks at the level of the RPE, which is evident in the early phase of the angiogram. Blockage of background fluorescence and late staining, when fluorescein leaks from the ves‐ sels can be seen on an angiogram as well (Atmaca et al, 1999). Characteristic signs are hypo‐

year-old man; 4b: High reflectivity on B-scan ultrasonography.

server variability of 0.5 mm (Char et al, 1990).

*5.4.2. Fluorescein angiography*

**5.4. Ancillary testing**

*5.4.1. Ultrasonography*

It is important to differentiate melanoma form other choroidal pathologies, such as choroi‐ dal nevi, by identifying indicators of potential malignancy which may differentiate nevi from small UM. Shields *et al* constructed a mnemonic "TFSOM", i.e. "to find small ocular melanoma" to assist in identifying small choroidal melanoma at risk for growth (Shields et al, 1995). The letters of the mnemonic indicate: Thickness > 2 mm, subretinal Fluid, Symp‐ toms, Orange pigment and Margin to the optic disc. Tumours with no, one or more than two factors have 4%, 36% or > 45% chance of growth, respectively (Shields et al, 2000). A tumour with a thickness of more than 2 mm is considered suspect of being a melanoma rather than a nevus. Subretinal fluid is the strongest indicator of malignancy. Exudative retinal detach‐ ment, overlying or adjacent to the tumour, is associated with tumour growth (Augsburger et al, 1989). Presence of symptoms, as mentioned earlier or a change in symptoms is a risk fac‐ tor for malignancy. Orange pigment is formed on melanomas of the posterior pole, although this can also be seen on the surface of presumed benign nevi and haemangioma. Orange pigment is an accumulation of lipofuscin within the RPE. In amelanotic tumours it appears brown-black of colour. Besides orange pigment as a risk factor, a tumour margin within 3 mm of the optic disc is also suspect for malignant potential (figure 4a).

Later "Using Helpful Hints Daily" was added to "TFSOM" mnemonic (Shields et al, 2009a). These features indicate a low acoustic profile or Ultrasound Hollowness, absence of a Halo around the tumour and absence of Drusen over the tumour. US hollowness is shown in 25% of nevi that transformed into melanoma, compared to the 4% with growth without US hollowness (Shields et al, 2009a). A halo around a tumour is a pigmented le‐ sion with a surrounding depigmentation, as can also be noticed in dysplastic nevi. Dru‐ sen suggest a chronic lesion and usually indicate that the tumour is benign, however this is not conclusive.

**Figure 4.** a: Peripapillary nevus, barely elevated, with margin located < 3 mm to the optic disc in the right eye of a 72 year-old man; 4b: High reflectivity on B-scan ultrasonography.

#### **5.4. Ancillary testing**

several years before diagnosis of the primary tumour (Eskelin et al, 2000). This emphasizes

In general, choroidal nevi have a less than 5 mm basal diameter and are minimal in height (< 2 mm), although several definitions of nevi have been proposed. Due to differ‐ ent examination methods and definitions, the prevalence of nevi is between 0.2% and 30% (Gass, 1977; Wilder, 1946). Overall in a Caucasian population the incidence is 6.5% (Sumich et al, 1998). Whenever, growth of a nevus is measured on US in a short time a transformation into a small melanoma is suspected. On the other hand benign nevi can also grow slowly. Mashayekhi *et al* observed in 31% of nevi a slight growth, without evi‐ dence of development into a melanoma over a mean follow up of 15 years (Mashayekhi et al, 2011). As described by Singh and co-workers, assuming that all melanoma result from nevi, 1 out of 8845 choroidal nevi can undergo malignant transformation into mela‐ noma in the Caucasian population in the USA (Singh et al, 2005b). In Australia this is es‐

It is important to differentiate melanoma form other choroidal pathologies, such as choroi‐ dal nevi, by identifying indicators of potential malignancy which may differentiate nevi from small UM. Shields *et al* constructed a mnemonic "TFSOM", i.e. "to find small ocular melanoma" to assist in identifying small choroidal melanoma at risk for growth (Shields et al, 1995). The letters of the mnemonic indicate: Thickness > 2 mm, subretinal Fluid, Symp‐ toms, Orange pigment and Margin to the optic disc. Tumours with no, one or more than two factors have 4%, 36% or > 45% chance of growth, respectively (Shields et al, 2000). A tumour with a thickness of more than 2 mm is considered suspect of being a melanoma rather than a nevus. Subretinal fluid is the strongest indicator of malignancy. Exudative retinal detach‐ ment, overlying or adjacent to the tumour, is associated with tumour growth (Augsburger et al, 1989). Presence of symptoms, as mentioned earlier or a change in symptoms is a risk fac‐ tor for malignancy. Orange pigment is formed on melanomas of the posterior pole, although this can also be seen on the surface of presumed benign nevi and haemangioma. Orange pigment is an accumulation of lipofuscin within the RPE. In amelanotic tumours it appears brown-black of colour. Besides orange pigment as a risk factor, a tumour margin within 3

Later "Using Helpful Hints Daily" was added to "TFSOM" mnemonic (Shields et al, 2009a). These features indicate a low acoustic profile or Ultrasound Hollowness, absence of a Halo around the tumour and absence of Drusen over the tumour. US hollowness is shown in 25% of nevi that transformed into melanoma, compared to the 4% with growth without US hollowness (Shields et al, 2009a). A halo around a tumour is a pigmented le‐ sion with a surrounding depigmentation, as can also be noticed in dysplastic nevi. Dru‐ sen suggest a chronic lesion and usually indicate that the tumour is benign, however this

mm of the optic disc is also suspect for malignant potential (figure 4a).

the importance of identifying small melanoma and reducing the risk of metastases.

**5.3. Clinical predictive factors of small melanoma**

142 Melanoma - From Early Detection to Treatment

timated 1 out of 4300 nevi (Sumich et al, 1998).

is not conclusive.

#### *5.4.1. Ultrasonography*

US is a non-invasive tool and helps to establish the diagnosis of UM, despite media opacities or whether the tumour is located far peripherally. UM shows characteristic low to medium internal reflectivity on A-scan. B-scan US is primarily used to plan therapy based on the first measurement, and to periodically measure tumour prominence (height) and basal diameter for follow-up. The B-scan can identify possible extraocular extension as an empty area be‐ hind the sclera. On B-scan US the internal structure of the tumour is typically seen as a rela‐ tive homogeneous grey scale, although this pattern is not specifically diagnostic (figure 3b). At the base of the tumour an acoustically silent zone (called acoustic hollowing) is seen, as well as choroidal excavation and shadowing in the orbit (figure 2b). Eighty-eight percent of the UM show US hollowness or low acoustic reflectivity (Boldt et al, 2008). Choroidal exca‐ vation is not observed in all melanomas and varies from 42% to 70% (Coleman et al, 1974; Sobottka et al, 1998; Verbeek, 1985). US provides accurate measurements with an interob‐ server variability of 0.5 mm (Char et al, 1990).

#### *5.4.2. Fluorescein angiography*

The diagnostic value of fluorescein angiography in UM is limited. Fluorescein angiography does not show pathognomonic patterns and is especially helpful in differentiating lesions, which simulate melanoma. The pigmentation, size and effect on the RPE of the tumour in‐ fluence the fluorescein angiogram. It is of little help in some medium to large melanomas that have an intrinsic tumour circulation. This 'double circulation' (simultaneous visualiza‐ tion of retinal and choroidal circulation) consists of late staining of the lesion and multiple pin-point leaks at the level of the RPE, which is evident in the early phase of the angiogram. Blockage of background fluorescence and late staining, when fluorescein leaks from the ves‐ sels can be seen on an angiogram as well (Atmaca et al, 1999). Characteristic signs are hypo‐ fluorescence in the early phase followed by diffuse hyperfluorescence and hyperfluorescent spots (due to changes in RPE). In the late phase the dye accumulates in the tumour tissue and hyperfluorescents (figure 5b). Hypofluorescent spots correspond with deposits of or‐ ange pigment on the surface of the tumour.

teristic or subtle patterns of autofluorescence were observed (Lavinsky et al, 2007; Shields et al, 2008). Choroidal melanoma and related retinal and RPE changes, show different auto‐ fluorescence patterns, and secondary changes, such as subclinical retinal detachments asso‐ ciated with presence of small amounts of subretinal fluid can discriminate small choroidal melanoma and nevi at risk for growth (Muscat et al, 2004). Like some nevi UM show bright‐ er hyperautofluorescence in overlying orange pigment, RPE detachment and subsequently decreased brightness in subretinal fluid and drusen (Shields et al, 2008) (figures 2c and 3c).

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Magnetic resonance imaging (MRI) and computed tomography (CT) can be of additional value in the differential diagnosis of UM. On CT an UM appears as a hyperdense lesion with moderate contrast enhancement. Tumours thinner than 2 mm are not detectable on CT. Besides that, CT is less accurate than US in differentiating melanoma and is more ex‐ pensive (Mafee et al, 1986; Peyster et al, 1985). For extrascleral extension CT is inferior to US (Scott et al, 1998). On the other hand, MRI seems more sensitive and more specific than US for detection of extraocular extension of UM (Hosten et al, 1997). A choroidal melanoma appears hyperintense on a T1 and hypointense on a T2 weighted scan. As this can also be the appearance of a melanocytoma, MRI is not specific for uveal melanoma. Due to the higher expenses of CT and MRI and the superiority of US, both techniques

About 54 different conditions are able to simulate UM. The most frequent diagnosis is cho‐ roidal nevus, accounting for 49% of the approximately 1739 presumed melanoma patients referred to a large tertiary Oncology Department in the USA (Shields et al, 2005b). The dif‐ ferentiation between small melanomas and choroidal nevi remains a clinical challenge. Clin‐ ical features that are more prevalent in *choroidal nevi* than in melanomas are drusen and RPE changes, whereas retinal detachment, choroidal neovascularisation or haemorrhagic retinal detachment can occur in both. On B-scan US, nevi have a high internal reflectivity (figure 4b). Also orange pigment and subretinal fluid, which are features of potential malignancy as mentioned previously, can be present in nevi. Ten percent of the nevi have orange pigment

*Congenital hypertrophy of the retinal pigment epithelium* (CHRPE) has sharper edges than mela‐ noma and usually sharply bordered nonpigmented areas (lacunae), or a depigmentated or pigmented halo within. The lesions might be slightly elevated and are black or grey of col‐ our. CHRPE is a benign lesion and is typically located in the peripheral fundus. On the other hand, adenocarcinomas arising from a CHRPE have been reported (Shields et al, 2009e).

*Optic disc melanocytoma* is a heavily pigmented benign lesion with a fibrillated or feathery margin. Although it can occur anywhere in the uveal tract, the tumour is most often located unilateral and on or nearby the optic disc. Optic disc melanocytoma is a variant of melano‐ cytic nevus. Most patients (75%) have no visual complaints, whereas patients with visual

*5.4.5. Magnetic resonance imaging and computed tomography*

are not routinely used for diagnostic evaluation.

**5.5. Differential diagnosis**

and 18% have subretinal fluid.

**Figure 5.** a: A partly pigmented and non-pigmented uveal melanoma; 5b: Fluorescein angiogram with blockage of the background and fluorescein leaking from the vessels.

#### *5.4.3. Indocyanine green angiography*

Indocyanine green angiography is designed to visualize the choroidal vessels and provides more information than fluorescein angiography. Whether an evident pattern can be seen on an angiogram depends on the pigmentation, thickness, disruption through Bruch's mem‐ brane and vascularisation of the tumour (Atmaca et al, 1999). More fluorescence is seen in less pigmented and larger tumours. The choroidal vasculature can be better visualised with indocyanine green than fluorescein. On indocyanine green late staining is observed, because of the leaking of indocyanine green in the extracellular space of the tumour (Frenkel et al, 2008; Guyer et al, 1993; Stanga et al, 2003).

#### *5.4.4. Optical coherence tomography and fundus autofluorescence*

OCT and fundus autofluorescence imaging have limited use in detecting changes in the cho‐ roid, however, both techniques are non-invasive and of help in identifying subtle changes in the RPE, retina and vitreoretinal interface. By means of an OCT subretinal fluid can be vi‐ sualized and quantified, small tumours can be measured, whereas with fundus autofluores‐ cence orange pigment can be shown. Spectral domain OCT can be useful in the detection of subretinal deposits, vitreous seeding and transretinal tumour extension (Heindl et al, 2009).

Although OCT itself is not useful in diagnosing uveal melanoma, it aids in differentiating other pigmented lesions from melanomas (Schaudig et al, 1998). For example, melanocyto‐ ma tend to have a high reflective signal anteriorly, corresponding with the nerve fibre layer, and an optical shadowing posteriorly (Muscat et al, 2001). In most choroidal nevi no charac‐ teristic or subtle patterns of autofluorescence were observed (Lavinsky et al, 2007; Shields et al, 2008). Choroidal melanoma and related retinal and RPE changes, show different auto‐ fluorescence patterns, and secondary changes, such as subclinical retinal detachments asso‐ ciated with presence of small amounts of subretinal fluid can discriminate small choroidal melanoma and nevi at risk for growth (Muscat et al, 2004). Like some nevi UM show bright‐ er hyperautofluorescence in overlying orange pigment, RPE detachment and subsequently decreased brightness in subretinal fluid and drusen (Shields et al, 2008) (figures 2c and 3c).

#### *5.4.5. Magnetic resonance imaging and computed tomography*

Magnetic resonance imaging (MRI) and computed tomography (CT) can be of additional value in the differential diagnosis of UM. On CT an UM appears as a hyperdense lesion with moderate contrast enhancement. Tumours thinner than 2 mm are not detectable on CT. Besides that, CT is less accurate than US in differentiating melanoma and is more ex‐ pensive (Mafee et al, 1986; Peyster et al, 1985). For extrascleral extension CT is inferior to US (Scott et al, 1998). On the other hand, MRI seems more sensitive and more specific than US for detection of extraocular extension of UM (Hosten et al, 1997). A choroidal melanoma appears hyperintense on a T1 and hypointense on a T2 weighted scan. As this can also be the appearance of a melanocytoma, MRI is not specific for uveal melanoma. Due to the higher expenses of CT and MRI and the superiority of US, both techniques are not routinely used for diagnostic evaluation.

#### **5.5. Differential diagnosis**

fluorescence in the early phase followed by diffuse hyperfluorescence and hyperfluorescent spots (due to changes in RPE). In the late phase the dye accumulates in the tumour tissue and hyperfluorescents (figure 5b). Hypofluorescent spots correspond with deposits of or‐

**Figure 5.** a: A partly pigmented and non-pigmented uveal melanoma; 5b: Fluorescein angiogram with blockage of

Indocyanine green angiography is designed to visualize the choroidal vessels and provides more information than fluorescein angiography. Whether an evident pattern can be seen on an angiogram depends on the pigmentation, thickness, disruption through Bruch's mem‐ brane and vascularisation of the tumour (Atmaca et al, 1999). More fluorescence is seen in less pigmented and larger tumours. The choroidal vasculature can be better visualised with indocyanine green than fluorescein. On indocyanine green late staining is observed, because of the leaking of indocyanine green in the extracellular space of the tumour (Frenkel et al,

OCT and fundus autofluorescence imaging have limited use in detecting changes in the cho‐ roid, however, both techniques are non-invasive and of help in identifying subtle changes in the RPE, retina and vitreoretinal interface. By means of an OCT subretinal fluid can be vi‐ sualized and quantified, small tumours can be measured, whereas with fundus autofluores‐ cence orange pigment can be shown. Spectral domain OCT can be useful in the detection of subretinal deposits, vitreous seeding and transretinal tumour extension (Heindl et al, 2009).

Although OCT itself is not useful in diagnosing uveal melanoma, it aids in differentiating other pigmented lesions from melanomas (Schaudig et al, 1998). For example, melanocyto‐ ma tend to have a high reflective signal anteriorly, corresponding with the nerve fibre layer, and an optical shadowing posteriorly (Muscat et al, 2001). In most choroidal nevi no charac‐

ange pigment on the surface of the tumour.

144 Melanoma - From Early Detection to Treatment

the background and fluorescein leaking from the vessels.

2008; Guyer et al, 1993; Stanga et al, 2003).

*5.4.4. Optical coherence tomography and fundus autofluorescence*

*5.4.3. Indocyanine green angiography*

About 54 different conditions are able to simulate UM. The most frequent diagnosis is cho‐ roidal nevus, accounting for 49% of the approximately 1739 presumed melanoma patients referred to a large tertiary Oncology Department in the USA (Shields et al, 2005b). The dif‐ ferentiation between small melanomas and choroidal nevi remains a clinical challenge. Clin‐ ical features that are more prevalent in *choroidal nevi* than in melanomas are drusen and RPE changes, whereas retinal detachment, choroidal neovascularisation or haemorrhagic retinal detachment can occur in both. On B-scan US, nevi have a high internal reflectivity (figure 4b). Also orange pigment and subretinal fluid, which are features of potential malignancy as mentioned previously, can be present in nevi. Ten percent of the nevi have orange pigment and 18% have subretinal fluid.

*Congenital hypertrophy of the retinal pigment epithelium* (CHRPE) has sharper edges than mela‐ noma and usually sharply bordered nonpigmented areas (lacunae), or a depigmentated or pigmented halo within. The lesions might be slightly elevated and are black or grey of col‐ our. CHRPE is a benign lesion and is typically located in the peripheral fundus. On the other hand, adenocarcinomas arising from a CHRPE have been reported (Shields et al, 2009e).

*Optic disc melanocytoma* is a heavily pigmented benign lesion with a fibrillated or feathery margin. Although it can occur anywhere in the uveal tract, the tumour is most often located unilateral and on or nearby the optic disc. Optic disc melanocytoma is a variant of melano‐ cytic nevus. Most patients (75%) have no visual complaints, whereas patients with visual loss were related to neuroretinitis from tumour necrosis and secondary subretinal fluid of the fovea (Shields et al, 2006; Shields et al, 2004). In addition, visual field defects have been described (Meyer et al, 1999; Shields et al, 2006). Ocular melanocytosis is associated with melanocytoma in 8% of cases, and melanocytoma enlargement is noticed in 57% within 8 years (Lee et al, 2010) and 32% within 10 years (Shields et al, 2004). Although malignant transformation is extremely rare, it has been reported (Meyer et al, 1999; Shields et al, 2004).

*Peripheral exudative hemorrhagic chorioretinopathy* (PEHC) lesions, unilateral and often bilater‐ al, have peripheral (> 3 mm outside the fovea) subretinal or sub-RPE haemorrhage that aris‐ es from choroidal neovascularisation. In the periphery signs of macular degeneration, such as lipid exudation, subretinal fluid and fibrosis can be observed (Mantel et al, 2009; Shields et al, 2009c). Also in the macula drusen, RPE alterations or choroidal neovascularisation can be present, which is then consistent with macular degeneration (Shields et al, 2009c). On Bscan internal lesion characteristics show a solid or hollow acoustic quality and no choroidal excavation (Mantel et al, 2009; Shields et al, 2009d). The majority of the peripheral lesions

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*Choroidal haemorrhage* may be distinguished from UM by partially or totally resorption of the haemorrhage over a few weeks, and on US an after-movement can be noticed by kinetic evaluation. Key features are elevated eye pressure, forward movement of diaphragm com‐

*Posterior nodular scleritis* is rare, but often underdiagnosed. It is twice as common in women as in men, and in 35% of the patients it occurs in both eyes. The most common symptoms are periocular pain, pain with eye movement and decreased vision. The differentiation be‐ tween scleritis and melanoma can be made by US. On B-scan echogenic scleral nodules, flu‐ id in Tenon's capsule, swelling of the optic disc and serous retinal detachment are found

*Intraocular leiomyoma* is a rare benign amelanotic tumour of the uvea and mimics an UM. It presents as a dome-shaped lesion, showing light translucency and often contains dilated episcleral vessels, with a predilection in young females (Shields et al, 1994). Sometimes the diagnosis cannot be made by non-invasive examination and intraocular biopsy is necessary

*Adenoma of the RPE* is infrequently diagnosed before enucleation. RPE adenoma is domeshaped and has in contrast to melanoma a higher internal reflectivity on A-scan US (Naka‐ mura et al, 2012). Compared to UM, RPE adenoma has more frequently retinal feeder vessels, retinal or subretinal exudates and exudative retinal detachment (Wei et al, 2010).

UMs develop from melanocytes of the uvea that are derived from neural crest cells. Initially Callender and colleagues described several melanoma cell types, (Callender, 1931) currently three histopathological uveal melanoma categories are being recognised: spindle, epithelioid and mixed cell type (Campbell et al, 1998). Haematoxylin and eosin (H&E) staining is used to differentiate between cell types. Spindle cells exhibit elongated nuclei that may contain eosinophilic nucleoli. In general, Ums containing spindle cells grow slowly and might be as‐ sociated with better prognosis. On the other hand, UMs consisting of faster growing epithe‐ lioid cells, have a more aggressive behaviour, and are therefore associated with poor clinical

resolve spontaneously over time, leaving a scar.

bined with severe pain (Yang et al, 2003).

**6. Classification and histopathologic features**

(McCluskey et al, 1999).

(Richter et al, 2003).

*Hemorrhagic detachment of the retina and RPE* may also simulate melanoma.

*Hyperplasia of the RPE* is a common ocular finding, which is idiopathic or develops in re‐ sponse to trauma, inflammation, haemorrhage and retinal detachment. It is characterised as a black irregular usually small retinal lesion consisting of proliferated RPE cells. Intraretinal pigmented spicules can be seen, and when it manifests as a subretinal localized mass, a mel‐ anoma can be suspected.

*Choroidal haemangioma* is a benign tumour consisting of blood vessels with a typical red to orange colour. Some areas of increased pigmentation can be observed, which makes it diffi‐ cult to differentiate from melanoma. On angiography typical early hyperfluorescence is shown and on US a characteristic high internal reflectivity is present.

*Choroidal metastases* are the most common intraocular malignancies. The prevalence of uveal metastases from all forms of carcinoma is between 2% and 9%, with a mean of 7% for breast cancer and 5% for lung cancer (Kanthan et al, 2007). The origin of choroidal metastases is predominantly breast cancer in woman and lung cancer in man. Less fre‐ quently patients are diagnosed with other primary tumours, such as gastrointestinal tract, kidney, skin and prostate carcinoma (Shields et al, 1997). Choroidal metastases typi‐ cally develop after the diagnosis of breast cancer and in some cases systemic metastases have already been detected. In 66% to 97% of lung cancer patients, choroidal metastases are detected after the primary tumour has been diagnosed (Kanthan et al, 2007). In con‐ clusion, uveal metastases can also be observed before the diagnosis of breast or lung can‐ cer (Demirci et al, 2003; Singh et al, 2012). The median interval between diagnosis of the primary tumour and uveal metastasis is 1 - 4.5 years (Amer et al, 2004; Ratanatharathorn et al, 1991; Rosset et al, 1998; Rottinger et al, 1976; Tsina et al, 2005). Choroidal metasta‐ ses are creamy yellow, flat or elevated and often multilobulated lesions that can occur bi‐ lateral. More than half of the patients may develop subretinal fluid (Demirci et al, 2003). The lesion can show clumps of brown pigmentation, known as leopard spots and RPE al‐ terations. Metastases grow in a different fashion than primary UMs, they infiltrate and replace the normal choroidal architecture more diffusely. On US metastases from breast carcinoma show a higher internal reflectivity than UM (Sobottka et al, 1998).

*Choroidal osteoma* is a rare ossifying benign lesion of the choroid that appears as a yellowish to orange well-defined, juxtapapillary or macular choroidal tumour. These lesions mostly occur in young women with a mean age of 26 years; usually it occurs unilateral, although in 20-30% of cases it appears to be bilateral. Over time an osteoma may enlarge and decalcify partially or totally (Ross & Kemp, 2009; Shields et al, 2005a). There is a 31% chance of devel‐ oping choroidal neovascularisation after 10 years (Shields et al, 2005a). On B-scan US a high‐ ly reflective lesion that shadows the orbit can be seen.

*Peripheral exudative hemorrhagic chorioretinopathy* (PEHC) lesions, unilateral and often bilater‐ al, have peripheral (> 3 mm outside the fovea) subretinal or sub-RPE haemorrhage that aris‐ es from choroidal neovascularisation. In the periphery signs of macular degeneration, such as lipid exudation, subretinal fluid and fibrosis can be observed (Mantel et al, 2009; Shields et al, 2009c). Also in the macula drusen, RPE alterations or choroidal neovascularisation can be present, which is then consistent with macular degeneration (Shields et al, 2009c). On Bscan internal lesion characteristics show a solid or hollow acoustic quality and no choroidal excavation (Mantel et al, 2009; Shields et al, 2009d). The majority of the peripheral lesions resolve spontaneously over time, leaving a scar.

*Hemorrhagic detachment of the retina and RPE* may also simulate melanoma.

loss were related to neuroretinitis from tumour necrosis and secondary subretinal fluid of the fovea (Shields et al, 2006; Shields et al, 2004). In addition, visual field defects have been described (Meyer et al, 1999; Shields et al, 2006). Ocular melanocytosis is associated with melanocytoma in 8% of cases, and melanocytoma enlargement is noticed in 57% within 8 years (Lee et al, 2010) and 32% within 10 years (Shields et al, 2004). Although malignant transformation is extremely rare, it has been reported (Meyer et al, 1999; Shields et al, 2004).

*Hyperplasia of the RPE* is a common ocular finding, which is idiopathic or develops in re‐ sponse to trauma, inflammation, haemorrhage and retinal detachment. It is characterised as a black irregular usually small retinal lesion consisting of proliferated RPE cells. Intraretinal pigmented spicules can be seen, and when it manifests as a subretinal localized mass, a mel‐

*Choroidal haemangioma* is a benign tumour consisting of blood vessels with a typical red to orange colour. Some areas of increased pigmentation can be observed, which makes it diffi‐ cult to differentiate from melanoma. On angiography typical early hyperfluorescence is

*Choroidal metastases* are the most common intraocular malignancies. The prevalence of uveal metastases from all forms of carcinoma is between 2% and 9%, with a mean of 7% for breast cancer and 5% for lung cancer (Kanthan et al, 2007). The origin of choroidal metastases is predominantly breast cancer in woman and lung cancer in man. Less fre‐ quently patients are diagnosed with other primary tumours, such as gastrointestinal tract, kidney, skin and prostate carcinoma (Shields et al, 1997). Choroidal metastases typi‐ cally develop after the diagnosis of breast cancer and in some cases systemic metastases have already been detected. In 66% to 97% of lung cancer patients, choroidal metastases are detected after the primary tumour has been diagnosed (Kanthan et al, 2007). In con‐ clusion, uveal metastases can also be observed before the diagnosis of breast or lung can‐ cer (Demirci et al, 2003; Singh et al, 2012). The median interval between diagnosis of the primary tumour and uveal metastasis is 1 - 4.5 years (Amer et al, 2004; Ratanatharathorn et al, 1991; Rosset et al, 1998; Rottinger et al, 1976; Tsina et al, 2005). Choroidal metasta‐ ses are creamy yellow, flat or elevated and often multilobulated lesions that can occur bi‐ lateral. More than half of the patients may develop subretinal fluid (Demirci et al, 2003). The lesion can show clumps of brown pigmentation, known as leopard spots and RPE al‐ terations. Metastases grow in a different fashion than primary UMs, they infiltrate and replace the normal choroidal architecture more diffusely. On US metastases from breast

shown and on US a characteristic high internal reflectivity is present.

carcinoma show a higher internal reflectivity than UM (Sobottka et al, 1998).

ly reflective lesion that shadows the orbit can be seen.

*Choroidal osteoma* is a rare ossifying benign lesion of the choroid that appears as a yellowish to orange well-defined, juxtapapillary or macular choroidal tumour. These lesions mostly occur in young women with a mean age of 26 years; usually it occurs unilateral, although in 20-30% of cases it appears to be bilateral. Over time an osteoma may enlarge and decalcify partially or totally (Ross & Kemp, 2009; Shields et al, 2005a). There is a 31% chance of devel‐ oping choroidal neovascularisation after 10 years (Shields et al, 2005a). On B-scan US a high‐

anoma can be suspected.

146 Melanoma - From Early Detection to Treatment

*Choroidal haemorrhage* may be distinguished from UM by partially or totally resorption of the haemorrhage over a few weeks, and on US an after-movement can be noticed by kinetic evaluation. Key features are elevated eye pressure, forward movement of diaphragm com‐ bined with severe pain (Yang et al, 2003).

*Posterior nodular scleritis* is rare, but often underdiagnosed. It is twice as common in women as in men, and in 35% of the patients it occurs in both eyes. The most common symptoms are periocular pain, pain with eye movement and decreased vision. The differentiation be‐ tween scleritis and melanoma can be made by US. On B-scan echogenic scleral nodules, flu‐ id in Tenon's capsule, swelling of the optic disc and serous retinal detachment are found (McCluskey et al, 1999).

*Intraocular leiomyoma* is a rare benign amelanotic tumour of the uvea and mimics an UM. It presents as a dome-shaped lesion, showing light translucency and often contains dilated episcleral vessels, with a predilection in young females (Shields et al, 1994). Sometimes the diagnosis cannot be made by non-invasive examination and intraocular biopsy is necessary (Richter et al, 2003).

*Adenoma of the RPE* is infrequently diagnosed before enucleation. RPE adenoma is domeshaped and has in contrast to melanoma a higher internal reflectivity on A-scan US (Naka‐ mura et al, 2012). Compared to UM, RPE adenoma has more frequently retinal feeder vessels, retinal or subretinal exudates and exudative retinal detachment (Wei et al, 2010).

## **6. Classification and histopathologic features**

UMs develop from melanocytes of the uvea that are derived from neural crest cells. Initially Callender and colleagues described several melanoma cell types, (Callender, 1931) currently three histopathological uveal melanoma categories are being recognised: spindle, epithelioid and mixed cell type (Campbell et al, 1998). Haematoxylin and eosin (H&E) staining is used to differentiate between cell types. Spindle cells exhibit elongated nuclei that may contain eosinophilic nucleoli. In general, Ums containing spindle cells grow slowly and might be as‐ sociated with better prognosis. On the other hand, UMs consisting of faster growing epithe‐ lioid cells, have a more aggressive behaviour, and are therefore associated with poor clinical outcome. Epithelioid cells have more polygonal cytoplasm and contain eccentric placed large pleomorphic nuclei and prominent eosinophilic nucleoli (figure 6). The mixed-cell type melanoma has variable proportion of spindle and epithelioid cells with a minimum of 10% of any one type (Edge & American Joint Committee on Cancer, 2010). Other inter-tumour factors, like the presence of certain extracellular matrix patterns (three closed loops located back to back identified by Periodic-acid Schiff (PAS) staining) and increased mitotic figures (number of mitoses per 50 high-power fields equal to 8mm2) can both provide additional adverse prognostic information (Folberg et al, 1993; Mooy et al, 1995). Other histological fea‐ tures associated with mortality and metastases are mean diameter of ten largest nucleoli, de‐ gree of pigmentation, presence of inflammation and tumour necrosis (Gill & Char, 2012). Extrascleral extension by perineural, perivascular, intravascular or direct scleral invasion is correlated with a worse prognosis, especially when the orbital fat resection margin is posi‐ tive (Collaborative Ocular Melanoma Study Group, 1998).

anomalies on either the short arm (p) and or long arm (q) of chromosomes 1, 3, 6 and 8,

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To examine chromosomal changes in UM tissue several cytogenetic and molecular techni‐ ques are available. UMs are quite suitable for cytogenetic analysis because of their relatively simply karyotype. Large chromosomal gains, deletions and translocations can be visualized with conventional karyotyping and spectral karyotyping (SKY) (figure 7a). However, for the detection of smaller abnormalities other techniques are necessary, such as FISH (figure 7b), comparative genomic hybridization (CGH) or quantitative polymerase chain reaction (qPCR) based techniques. An approach is the multiplex ligation probe amplification (MLPA) which allows the relative quantification of multiple loci in one single reaction. MLPA can detect patients at risk for metastatic disease using the results for chromosome 3 and 8 with similar accuracy as FISH *(*Damato et al, 2009*;* Vaarwater et al, 2012*).* MLPA and other qPCRbased techniques as Multiplex Amplicon Quantification (MAQ) fill the gap between more expensive genome-wide screening assays and cheaper methods that only provide informa‐ tion on a single locus (Kumps et al, 2010). A different technique is microsatellite analysis (MSA). Microsatellites are tandem repeats of polymorphic sequences located in the non-cod‐ ing regions of DNA. An extreme form of microsatellite instability was first described in he‐ reditary nonpolyposis colorectal cancer syndrome (Thibodeau et al, 1993). This technique is used to study loss of heterozygosity (LOH) as an indicator of chromosomal loss. A draw‐ back of MSA is that only a limited number of markers can be analyzed in one experiment.

**Figure 7.** a: Example of a karyogram showing monosomy 3 and trisomy of chromosome 8; 7b: FISH analysis of a tu‐ mour demonstrates 1 signal for the probe on centromere 3 (green signals) and 3 to 4 signals of the probe on centro‐

After completion of the human genome project, genome-wide DNA assays became availa‐ ble. Micro-assay based CGH, single nucleotide polymorphism (SNP) analysis and gene ex‐ pression profiling (GEP) analysis are the frequently applied techniques. With the

which can serve as prognostic markers.

mere 8 (red signals).

**7.1. Cytogenetic and molecular techniques in UM research**

Immunohistochemistry may be of diagnostic value. S-100 is expressed by cells of neuroecto‐ dermal origin. HMB-45 binds to gp100, an antigen expressed by melanocytes that can be useful in differentiating UM from nonmelanocytic tumours (Burnier et al, 1991).

**Figure 6.** a: Haematoxylin and eosin staining of formalin fixed and paraffin embedded eye sample with a typical mushroom shaped melanoma.; 6b: Uveal melanoma tissue with spindle cell type characterised by elongated nuclei; 6c: Uveal melanoma tissue with epithelioid cells containing large pleomorphic nuclei and prominent eosinophilic nu‐ cleoli.

## **7. Genetic classification**

Cytogenetic studies in solid tumours have been a greater challenge than in haematological malignancies since metaphase chromosome spreads of good quality are more difficult to ob‐ tain. Solid tumours frequently have highly complex chromosome alterations and are more heterogeneous. Despite this, UM has been well studied since the late eighties with different techniques, such as cytogenetic and fluorescent in situ hybridization (FISH) analysis. Over the years, we have learned that the majority of UMs contain non-random chromosomal anomalies on either the short arm (p) and or long arm (q) of chromosomes 1, 3, 6 and 8, which can serve as prognostic markers.

#### **7.1. Cytogenetic and molecular techniques in UM research**

outcome. Epithelioid cells have more polygonal cytoplasm and contain eccentric placed large pleomorphic nuclei and prominent eosinophilic nucleoli (figure 6). The mixed-cell type melanoma has variable proportion of spindle and epithelioid cells with a minimum of 10% of any one type (Edge & American Joint Committee on Cancer, 2010). Other inter-tumour factors, like the presence of certain extracellular matrix patterns (three closed loops located back to back identified by Periodic-acid Schiff (PAS) staining) and increased mitotic figures (number of mitoses per 50 high-power fields equal to 8mm2) can both provide additional adverse prognostic information (Folberg et al, 1993; Mooy et al, 1995). Other histological fea‐ tures associated with mortality and metastases are mean diameter of ten largest nucleoli, de‐ gree of pigmentation, presence of inflammation and tumour necrosis (Gill & Char, 2012). Extrascleral extension by perineural, perivascular, intravascular or direct scleral invasion is correlated with a worse prognosis, especially when the orbital fat resection margin is posi‐

Immunohistochemistry may be of diagnostic value. S-100 is expressed by cells of neuroecto‐ dermal origin. HMB-45 binds to gp100, an antigen expressed by melanocytes that can be

**Figure 6.** a: Haematoxylin and eosin staining of formalin fixed and paraffin embedded eye sample with a typical mushroom shaped melanoma.; 6b: Uveal melanoma tissue with spindle cell type characterised by elongated nuclei; 6c: Uveal melanoma tissue with epithelioid cells containing large pleomorphic nuclei and prominent eosinophilic nu‐

Cytogenetic studies in solid tumours have been a greater challenge than in haematological malignancies since metaphase chromosome spreads of good quality are more difficult to ob‐ tain. Solid tumours frequently have highly complex chromosome alterations and are more heterogeneous. Despite this, UM has been well studied since the late eighties with different techniques, such as cytogenetic and fluorescent in situ hybridization (FISH) analysis. Over the years, we have learned that the majority of UMs contain non-random chromosomal

useful in differentiating UM from nonmelanocytic tumours (Burnier et al, 1991).

tive (Collaborative Ocular Melanoma Study Group, 1998).

148 Melanoma - From Early Detection to Treatment

cleoli.

**7. Genetic classification**

To examine chromosomal changes in UM tissue several cytogenetic and molecular techni‐ ques are available. UMs are quite suitable for cytogenetic analysis because of their relatively simply karyotype. Large chromosomal gains, deletions and translocations can be visualized with conventional karyotyping and spectral karyotyping (SKY) (figure 7a). However, for the detection of smaller abnormalities other techniques are necessary, such as FISH (figure 7b), comparative genomic hybridization (CGH) or quantitative polymerase chain reaction (qPCR) based techniques. An approach is the multiplex ligation probe amplification (MLPA) which allows the relative quantification of multiple loci in one single reaction. MLPA can detect patients at risk for metastatic disease using the results for chromosome 3 and 8 with similar accuracy as FISH *(*Damato et al, 2009*;* Vaarwater et al, 2012*).* MLPA and other qPCRbased techniques as Multiplex Amplicon Quantification (MAQ) fill the gap between more expensive genome-wide screening assays and cheaper methods that only provide informa‐ tion on a single locus (Kumps et al, 2010). A different technique is microsatellite analysis (MSA). Microsatellites are tandem repeats of polymorphic sequences located in the non-cod‐ ing regions of DNA. An extreme form of microsatellite instability was first described in he‐ reditary nonpolyposis colorectal cancer syndrome (Thibodeau et al, 1993). This technique is used to study loss of heterozygosity (LOH) as an indicator of chromosomal loss. A draw‐ back of MSA is that only a limited number of markers can be analyzed in one experiment.

**Figure 7.** a: Example of a karyogram showing monosomy 3 and trisomy of chromosome 8; 7b: FISH analysis of a tu‐ mour demonstrates 1 signal for the probe on centromere 3 (green signals) and 3 to 4 signals of the probe on centro‐ mere 8 (red signals).

After completion of the human genome project, genome-wide DNA assays became availa‐ ble. Micro-assay based CGH, single nucleotide polymorphism (SNP) analysis and gene ex‐ pression profiling (GEP) analysis are the frequently applied techniques. With the development of Next Generation Sequencing (NGS) technologies, the genome can be ana‐ lyzed at base pair level. Genome-wide mutation analysis of tumour samples led to the dis‐ covery of a subset of genes in UM such as *GNAQ* and *BAP1*.

6p is frequently found in tumours with disomy 3 (Ehlers et al, 2008; Hoglund et al, 2004; Sisley et al, 1997). However, this combination of gain of 6p with disomy 3 could not be con‐ firmed by others (Mensink et al, 2009). Aberrations resulting in a relative increase of 6p have been found to be related with both a longer survival (White et al, 1998) or a decreased sur‐ vival (Aalto et al, 2001). The effect of chromosome 6 aberrations on patient outcome is not

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**Figure 8.** Single nucleotide polymorphism (SNP) array of an uveal melanoma. The upper panel (LogR ratio) shows loss of chromosome 3, partial loss of chromosome 8p and gain of chromosome 8q. The lower panel depicts the B-allele

In cutaneous melanoma rearrangements on the short arm of chromosome 1 are a common abnormality, occurring in about 80% of all cases (Fountain et al, 1990; Zhang et al, 1999). In UM this region on 1p is also frequently affected, giving rise to a deletion of 1p. However, these anomalies on chromosome 1 are less common than those in skin melanomas with a frequency of approximately 30% (Horsman & White, 1993; Parrella et al, 1999; Prescher et al,

Aberrations on other chromosomes have been explored, such as chromosome 9p21 (Scholes et al, 2001), chromosome 11q23 (Sisley et al, 2000), chromosome 18q22 (Mensink et al, 2008;

frequency representing allelic imbalance at these chromosomes.

1990; Prescher et al, 1995; Sisley et al, 2000).

*7.2.4. Chromosome 1*

conclusive.

## **7.2. Chromosomal anomalies**

## *7.2.1. Monosomy 3*

Monosomy of chromosome 3 is observed in approximately 50% of the cases of UM and is strongly associated with clinical and histopathological prognostic factors and with metastat‐ ic death (Horsman et al, 1990; Prescher et al, 1990; Sisley et al, 1990). Prescher and associates were the first to find a strong correlation between loss of chromosome 3 and a poor progno‐ sis of the patient (Prescher et al, 1996). Since then several groups have confirmed the prog‐ nostic value of monosomy 3 (Kilic et al, 2006; Sisley et al, 2000; Sisley et al, 1997; White et al, 1998). It is assumed that loss of chromosome 3 is a primary event, as it often occurs with other chromosomal aberrations in UM such as 1p loss, and gain of 6p and 8q (Prescher et al, 1995). Kiliç and colleagues established that tumours with concurrent loss of chromosome 1p and 3 are at higher risk of metastasizing than the tumours with other aberrations (Kilic et al, 2005). Mostly one entire copy of chromosome 3 is lost, although in some cases, isodisomy of chromosome 3 is acquired (Aalto et al, 2001; Scholes et al, 2001; White et al, 1998). Partial deletions or translocations have rarely been described on this chromosome making it diffi‐ cult to map putative tumour suppressor genes. However, recently a mutation in the *BAP1* gene, located on chromosome 3, has been identified in UMs and this gene seems to play an important role in the tumour progression (Harbour et al, 2010). This gene will be discussed in more detail later in this chapter.

#### *7.2.2. Chromosome 8*

Abnormalities in chromosome 8, and in particular gain of 8q or an isochromosome 8q, are thought to be a secondary event in UM as variable copy numbers can be present in one mel‐ anoma (Horsman & White, 1993; Prescher et al, 1994). Gain of chromosome 8q is frequently found in tumours that also have loss of chromosome 3, and this is associated with a poor patient outcome (Aalto et al, 2001; Prescher et al, 1995; White et al, 1998). A SNP array analy‐ sis with this chromosome status is depicted in figure 8. The relationship between the percen‐ tages of aberrant copy numbers within UM cells and patient outcome has been investigated. A higher percentage of monosomy 3 and chromosome 8q gain in primary UM cells shows a strong relation with poor disease-free survival compared to low percentage aberrations (van den Bosch et al, 2012).

#### *7.2.3. Chromosome 6*

Rearrangements on chromosome 6 affect both arms of the chromosome, resulting in dele‐ tions of 6q and gains of 6p. The relative gain of chromosome 6p can occur either through an isochromosome of 6p or a deletion of 6q. Tumours with gain of 6p are thought to be a sepa‐ rate group within UM with an alternative genetic pathway in carcinogenesis, since gain of 6p is frequently found in tumours with disomy 3 (Ehlers et al, 2008; Hoglund et al, 2004; Sisley et al, 1997). However, this combination of gain of 6p with disomy 3 could not be con‐ firmed by others (Mensink et al, 2009). Aberrations resulting in a relative increase of 6p have been found to be related with both a longer survival (White et al, 1998) or a decreased sur‐ vival (Aalto et al, 2001). The effect of chromosome 6 aberrations on patient outcome is not conclusive.

**Figure 8.** Single nucleotide polymorphism (SNP) array of an uveal melanoma. The upper panel (LogR ratio) shows loss of chromosome 3, partial loss of chromosome 8p and gain of chromosome 8q. The lower panel depicts the B-allele frequency representing allelic imbalance at these chromosomes.

#### *7.2.4. Chromosome 1*

development of Next Generation Sequencing (NGS) technologies, the genome can be ana‐ lyzed at base pair level. Genome-wide mutation analysis of tumour samples led to the dis‐

Monosomy of chromosome 3 is observed in approximately 50% of the cases of UM and is strongly associated with clinical and histopathological prognostic factors and with metastat‐ ic death (Horsman et al, 1990; Prescher et al, 1990; Sisley et al, 1990). Prescher and associates were the first to find a strong correlation between loss of chromosome 3 and a poor progno‐ sis of the patient (Prescher et al, 1996). Since then several groups have confirmed the prog‐ nostic value of monosomy 3 (Kilic et al, 2006; Sisley et al, 2000; Sisley et al, 1997; White et al, 1998). It is assumed that loss of chromosome 3 is a primary event, as it often occurs with other chromosomal aberrations in UM such as 1p loss, and gain of 6p and 8q (Prescher et al, 1995). Kiliç and colleagues established that tumours with concurrent loss of chromosome 1p and 3 are at higher risk of metastasizing than the tumours with other aberrations (Kilic et al, 2005). Mostly one entire copy of chromosome 3 is lost, although in some cases, isodisomy of chromosome 3 is acquired (Aalto et al, 2001; Scholes et al, 2001; White et al, 1998). Partial deletions or translocations have rarely been described on this chromosome making it diffi‐ cult to map putative tumour suppressor genes. However, recently a mutation in the *BAP1* gene, located on chromosome 3, has been identified in UMs and this gene seems to play an important role in the tumour progression (Harbour et al, 2010). This gene will be discussed

Abnormalities in chromosome 8, and in particular gain of 8q or an isochromosome 8q, are thought to be a secondary event in UM as variable copy numbers can be present in one mel‐ anoma (Horsman & White, 1993; Prescher et al, 1994). Gain of chromosome 8q is frequently found in tumours that also have loss of chromosome 3, and this is associated with a poor patient outcome (Aalto et al, 2001; Prescher et al, 1995; White et al, 1998). A SNP array analy‐ sis with this chromosome status is depicted in figure 8. The relationship between the percen‐ tages of aberrant copy numbers within UM cells and patient outcome has been investigated. A higher percentage of monosomy 3 and chromosome 8q gain in primary UM cells shows a strong relation with poor disease-free survival compared to low percentage aberrations (van

Rearrangements on chromosome 6 affect both arms of the chromosome, resulting in dele‐ tions of 6q and gains of 6p. The relative gain of chromosome 6p can occur either through an isochromosome of 6p or a deletion of 6q. Tumours with gain of 6p are thought to be a sepa‐ rate group within UM with an alternative genetic pathway in carcinogenesis, since gain of

covery of a subset of genes in UM such as *GNAQ* and *BAP1*.

**7.2. Chromosomal anomalies**

150 Melanoma - From Early Detection to Treatment

in more detail later in this chapter.

*7.2.2. Chromosome 8*

den Bosch et al, 2012).

*7.2.3. Chromosome 6*

*7.2.1. Monosomy 3*

In cutaneous melanoma rearrangements on the short arm of chromosome 1 are a common abnormality, occurring in about 80% of all cases (Fountain et al, 1990; Zhang et al, 1999). In UM this region on 1p is also frequently affected, giving rise to a deletion of 1p. However, these anomalies on chromosome 1 are less common than those in skin melanomas with a frequency of approximately 30% (Horsman & White, 1993; Parrella et al, 1999; Prescher et al, 1990; Prescher et al, 1995; Sisley et al, 2000).

Aberrations on other chromosomes have been explored, such as chromosome 9p21 (Scholes et al, 2001), chromosome 11q23 (Sisley et al, 2000), chromosome 18q22 (Mensink et al, 2008; White et al, 2006), and chromosome 16q (Kilic et al, 2006; Vajdic et al, 2003). The impact on the prognosis, however, remains unclear due to contradictory findings.

overall these mutations are rare (Cohen et al, 2003; Kilic et al, 2004; Mooy et al, 1991; Sal‐

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With the recent discovery of activating *GNAQ* and *GNA11* mutations new light has been shed on the MAPK pathway. Van Raamsdonk and co-workers demonstrated an alterna‐ tive route to MAPK activation through G-protein signalling in melanocytic neoplasia in‐ cluding UMs. They reported a *GNAQ* mutation in 83% of blue naevi and in 46% of UM cases (Van Raamsdonk et al, 2009). Other studies confirmed these results, *GNAQ* muta‐ tions were found in half of the UM cases (Bauer et al, 2009; Onken et al, 2008). *GNAQ* and its paralog *GNA11* encode the heterotrimeric guanine nucleotide-binding protein G subunit alpha q and 11, respectively. Through mutations these subunits become activated and abrogate their intrinsic GTPase activity, which is required to return them to an inac‐ tive state. This oncogenic conversion is suggested to be the cause of constitutive MAPK pathway activation. A subsequent study reported that 83% of UM samples harboured Gα-protein mutations (*GNAQ* or *GNA11* mutations) affecting specific regions on either exon 4 or 5 (codon R183 or Q209, respectively) in a mutually exclusive pattern (Van Raamsdonk et al, 2010). There is no relation between *GNAQ* mutations and prognosis (Bauer et al, 2009). Hence, the presence of Gα-protein mutations in tumours at all stages of malignant progression and in melanocytic lesions of the choroid, suggests that they

are early events in UM (Onken et al, 2008; Van Raamsdonk et al, 2009).

Exome genome sequencing led to the discovery of the BRCA1 associated protein 1 (*BAP1*) gene in UM (Harbour et al, 2010). *BAP1,* a nuclearly localized enzyme, was origi‐ nally identified as an ubiquitin hydrolase that binds to the RING finger domain of BRCA1 (Farmer et al, 2005; Jensen et al, 1998). It has de-ubiquitinating activity and is in‐ volved in several biological processes, including regulation of cell cycle and cell growth, chromatin dynamics and DNA damage response (Farmer et al, 2005). *BAP1* is located on chromosome 3p21.1 and is thought to be a tumour suppressor gene (Ventii et al, 2008). Mutations in this gene first have been reported in a small number of breast and lung cancer cell lines (Jensen et al, 1998). Recently, inactivating somatic mutations were found in 84% of the metastasizing UMs. These mutations were only found in 1 out of 26 inves‐ tigated class 1 tumours against 26 out of 31 class 2 tumours, implicating that *BAP1* muta‐ tions occur late in the UM progression (Harbour et al, 2010). In addition, co-segregating germline *BAP1* mutations have been described in several families with different range of diseases, such as cutaneous melanomas (Wiesner et al, 2011), malignant pleural mesothe‐ liomas (Testa et al, 2011), and other cancers such as meningioma (Abdel-Rahman et al, 2011). Given the functional complexity of *BAP1*, different germline mutations in *BAP1* may predispose to divergent tumour types. To understand more about the impact of *BAP1* mutations on UM and other types of cancers, more extensive clinical, molecular ge‐

danha et al, 2004).

*7.3.3. BAP1 gene*

netic, and functional studies are ongoing.

*7.3.2. GNAQ and GNA11 gene*

#### *7.2.5. Gene expression profiling*

Using GEP UMs can be classified into two classes of tumours that correspond remarka‐ bly well with the ability of the tumour to metastasize. In a study of 25 UMs, class 1 tu‐ mours had a low risk of metastasizing and class 2 tumours had a high risk of developing metastasis (Onken et al, 2004). This molecular classification strongly predicts metastatic death and outperforms other clinical, histopathological and cytogenetic prognostic indica‐ tors (Petrausch et al, 2008; van Gils et al, 2008; Worley et al, 2007). Class 1 tumours pre‐ dominantly show disomy of chromosome 3, whereas class 2 tumours consist mostly of monosomy 3 (Worley et al, 2007).

#### **7.3. Candidate genes**

After identifying the non-random chromosomal alterations in UM, the search for potential oncogenes and tumour suppressor genes followed. By narrowing down altered regions on chromosomes, researchers have tried to identify genes involved in tumourigenesis or pro‐ gression towards metastasis. This way, studies have been conducted on chromosome 8q re‐ vealing potential oncogenes such as *MYC*, which is amplified in about 30% of the UMs (Parrella et al, 2001). Other oncogenes on chromosome 8q have been described, such as *DDEF1* and *NBS1* (now referred to as *ASAP1* and *NBN*, respectively) (Ehlers & Harbour, 2005; Ehlers et al, 2005). Yet, no specific oncogenic mutations on this region have been re‐ ported thus far. Other candidate genes were proposed, such as *HDM2*, *BCL-2* and *CCND1*. However, the pathogenic significance for any of these genes has not been established.

Mutations in certain genes have been well described for cutaneous melanoma. Examples of such genes are the oncogenes *NRAS*, *BRAF* and *AKT3*, and the tumour suppressors *CDKN2A*, *PTEN* and *TP53*. In contrast to skin melanomas, *PTEN* mutations were not ob‐ served in a study of nine cell lines (Naus et al, 2000). Nevertheless, in 15% of the UM cases mutations in *PTEN* were found resulting in activation of *AKT* and overexpression of the PI3K-PTEN-AKT pathway preventing apoptosis (Abdel-Rahman et al, 2006; Ehlers et al, 2008; Ibrahim & Haluska, 2009).

#### *7.3.1. The RAS-RAF-MEK-ERK pathway*

In a large proportion of the UMs the RAS-RAF-MEK-ERK pathway or mitogen-activated protein kinase (MAPK) pathway is constitutionally activated, leading to excessive cell proliferation and suggesting the presence of activating mutations upstream in the path‐ way (Weber et al, 2003; Zuidervaart et al, 2005). Mutation analysis on potential mutation sites in the *BRAF* gene were performed, since a single substitution (p.V600E) in *BRAF* oc‐ curs frequently in benign and premalignant cutaneous nevi (Davies et al, 2002; Pollock et al, 2003). However, *NRAS* and *BRAF* mutations have been reported in a few UMs but overall these mutations are rare (Cohen et al, 2003; Kilic et al, 2004; Mooy et al, 1991; Sal‐ danha et al, 2004).

#### *7.3.2. GNAQ and GNA11 gene*

White et al, 2006), and chromosome 16q (Kilic et al, 2006; Vajdic et al, 2003). The impact on

Using GEP UMs can be classified into two classes of tumours that correspond remarka‐ bly well with the ability of the tumour to metastasize. In a study of 25 UMs, class 1 tu‐ mours had a low risk of metastasizing and class 2 tumours had a high risk of developing metastasis (Onken et al, 2004). This molecular classification strongly predicts metastatic death and outperforms other clinical, histopathological and cytogenetic prognostic indica‐ tors (Petrausch et al, 2008; van Gils et al, 2008; Worley et al, 2007). Class 1 tumours pre‐ dominantly show disomy of chromosome 3, whereas class 2 tumours consist mostly of

After identifying the non-random chromosomal alterations in UM, the search for potential oncogenes and tumour suppressor genes followed. By narrowing down altered regions on chromosomes, researchers have tried to identify genes involved in tumourigenesis or pro‐ gression towards metastasis. This way, studies have been conducted on chromosome 8q re‐ vealing potential oncogenes such as *MYC*, which is amplified in about 30% of the UMs (Parrella et al, 2001). Other oncogenes on chromosome 8q have been described, such as *DDEF1* and *NBS1* (now referred to as *ASAP1* and *NBN*, respectively) (Ehlers & Harbour, 2005; Ehlers et al, 2005). Yet, no specific oncogenic mutations on this region have been re‐ ported thus far. Other candidate genes were proposed, such as *HDM2*, *BCL-2* and *CCND1*.

However, the pathogenic significance for any of these genes has not been established.

Mutations in certain genes have been well described for cutaneous melanoma. Examples of such genes are the oncogenes *NRAS*, *BRAF* and *AKT3*, and the tumour suppressors *CDKN2A*, *PTEN* and *TP53*. In contrast to skin melanomas, *PTEN* mutations were not ob‐ served in a study of nine cell lines (Naus et al, 2000). Nevertheless, in 15% of the UM cases mutations in *PTEN* were found resulting in activation of *AKT* and overexpression of the PI3K-PTEN-AKT pathway preventing apoptosis (Abdel-Rahman et al, 2006; Ehlers et al,

In a large proportion of the UMs the RAS-RAF-MEK-ERK pathway or mitogen-activated protein kinase (MAPK) pathway is constitutionally activated, leading to excessive cell proliferation and suggesting the presence of activating mutations upstream in the path‐ way (Weber et al, 2003; Zuidervaart et al, 2005). Mutation analysis on potential mutation sites in the *BRAF* gene were performed, since a single substitution (p.V600E) in *BRAF* oc‐ curs frequently in benign and premalignant cutaneous nevi (Davies et al, 2002; Pollock et al, 2003). However, *NRAS* and *BRAF* mutations have been reported in a few UMs but

the prognosis, however, remains unclear due to contradictory findings.

*7.2.5. Gene expression profiling*

152 Melanoma - From Early Detection to Treatment

monosomy 3 (Worley et al, 2007).

2008; Ibrahim & Haluska, 2009).

*7.3.1. The RAS-RAF-MEK-ERK pathway*

**7.3. Candidate genes**

With the recent discovery of activating *GNAQ* and *GNA11* mutations new light has been shed on the MAPK pathway. Van Raamsdonk and co-workers demonstrated an alterna‐ tive route to MAPK activation through G-protein signalling in melanocytic neoplasia in‐ cluding UMs. They reported a *GNAQ* mutation in 83% of blue naevi and in 46% of UM cases (Van Raamsdonk et al, 2009). Other studies confirmed these results, *GNAQ* muta‐ tions were found in half of the UM cases (Bauer et al, 2009; Onken et al, 2008). *GNAQ* and its paralog *GNA11* encode the heterotrimeric guanine nucleotide-binding protein G subunit alpha q and 11, respectively. Through mutations these subunits become activated and abrogate their intrinsic GTPase activity, which is required to return them to an inac‐ tive state. This oncogenic conversion is suggested to be the cause of constitutive MAPK pathway activation. A subsequent study reported that 83% of UM samples harboured Gα-protein mutations (*GNAQ* or *GNA11* mutations) affecting specific regions on either exon 4 or 5 (codon R183 or Q209, respectively) in a mutually exclusive pattern (Van Raamsdonk et al, 2010). There is no relation between *GNAQ* mutations and prognosis (Bauer et al, 2009). Hence, the presence of Gα-protein mutations in tumours at all stages of malignant progression and in melanocytic lesions of the choroid, suggests that they are early events in UM (Onken et al, 2008; Van Raamsdonk et al, 2009).

#### *7.3.3. BAP1 gene*