Ocular Tumors

Ocular Tumors NV Laver, K Sitko, J Duker, and NA Farhat, Tufts Medical Center, Boston, MA, USA ã 2014 Elsevier Inc. All rights reserved.

Introduction Malignancies of the eye include metastases and tumors primary to the eye. Of these, metastases are the most common. This article discusses the features of the two most common intraocular tumors that arise de novo in the eye: retinoblastoma and malignant melanoma. Included is a review of the important clinical findings, histopathology, molecular genetics, and animal models.

Background Information: An Overview of the Structure and Function of the Eye The eye consists of three ‘coats’ (Figure 1). The outer coat of the eye, seen clinically as the white of the eye, is called the sclera. The sclera encircles the entire eye except anteriorly where it is continuous with the cornea. The cornea is clear, permitting transmission and refraction of light into the eye. In the posterior eye, the sclera is penetrated by the optic nerve, which carries the visual information to the brain. Behind the cornea is a space called the anterior chamber, which is filled with a small amount of watery fluid called the aqueous humor. Posteriorly to this chamber is the iris, which is a colored tissue, frequently brown or blue. The iris is part of the second coat of the eye called the uveal track. The central hole in the iris is known as the pupil. Behind the pupil is the lens, which, like the cornea, is normally transparent and also serves to refract the light. Another part of the uveal track, the ciliary body is located posterior to the iris at the equator of the lens. It is responsible for making the aqueous humor, holding the lens in place, and changing the shape of the lens by contracting and relaxing a small muscle to facilitate the eye’s ability to focus between distant and close objects. This process is called ‘accommodation.’ The choroid is the third part of the uveal tract. It is a highly vascular layer that lies under the sclera in the posterior part of the eye and is responsible for the blood supply to the outer parts of the retina. The iris, the ciliary body, and the choroid are the three structures that make up the uveal tissue or uvea. Throughout the uvea are many melanocytes that give these tissues their pigmentation, which can be quite variable from person to person. The third innermost ocular coat is the retina. It is the light-sensing portion of the eye and lines the inner wall of the posterior half of the eye just on top of the choroid. Signals from the retina leave the back of the eye through the optic nerve and are carried to the brain for higher-level perception of light and light patterns known as vision. The large cavity of the eye between the lens anteriorly and the retina posteriorly is filled with a viscous substance called the vitreous humor.

Retinoblastoma Epidemiology

Most cases are diagnosed by 3 years of age and 98% are diagnosed before the age of 5. It has a yearly incidence in the United States of 1/15 000–18 000 live births with 250–300 new cases in the United States per year. The mean age at diagnosis overall is 18 months. There is no gender or racial predilection for this tumor. The consistency of incidence measured in many distinct populations of the world suggests no role for environmental factors in the etiology of this disease.

Clinical Presentation Retinoblastoma is most commonly diagnosed when a child is brought in for an evaluation of an ocular abnormality first noticed by a parent or caregiver. This abnormality is most commonly leukocoria: a whitening of the pupil seen as a difference in color when compared to the normal pupil. Leukocoria is most evident in photographs or in the partially dilated state common in dim light (Figure 2). Leukocoria is the presenting sign in about half of retinoblastoma cases. The second most common presenting sign, in about a quarter of cases, is strabismus, or a noticeable misalignment of the eyes. This occurs due to a large or a macular tumor (a tumor involving the central region of the retina) that blocks the central vision of the affected eye. Without a good visual signal reaching the brain, latent muscle imbalance (which is almost universal) overcomes an individual’s ability to align the eyes properly, which is called strabismus. Less common presenting signs are a red, painful, inflamed eye; orbital cellulitis; and exceedingly rarely cataract or a fungating mass on the outside of the eye.

Differential Diagnosis The three diseases most commonly confused for retinoblastoma that should be ruled out are Coats’ disease, persistent fetal vasculature (PFV, formerly called persistent hyperplastic primary vitreous), and toxocariasis. Coats’ disease is usually unilateral, most common in young males, and manifested by telangiectasis of the retinal vasculature resulting in yellow subretinal exudates and exudative retinal detachment. ‘Yellow’ leukocoria can result as opposed to the ‘white’ leukocoria seen in retinoblastoma. PFV results from the failure of the normal regression of the fetal vascular supply to the developing lens that upon contracture can result in a cataract and a funnel retinal detachment resulting in leukocoria. Microphthalmia is typical in the affected eye. PFV is almost exclusively unilateral. Toxocariasis is an ocular infection of the canine ascarid, Toxocara canis. It results in intraocular inflammation and a retinal inflammatory mass or granuloma that can be mistaken for a tumor. The differential diagnosis of leukocoria includes also congenital cataract and congenital corneal opacity.

Diagnosis

Retinoblastoma is the most common ocular malignancy of childhood. It makes up 3–4% of all pediatric malignancies.

The diagnosis of retinoblastoma is most commonly made based on the clinical characteristics of the tumor as visualized

Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms

http://dx.doi.org/10.1016/B978-0-12-386456-7.04711-0

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Retina Lens Sclera

Iris Cornea

Choroid Vitreous body

Optic nerve

Papilla of the optic nerve

Anterior chamber

Ciliary body

Posterior chamber

Figure 1 Histology of the normal eye.

Figure 3 Retinoblastoma. Retinoblastoma (black asterisk) may grow in the macular region of the retina, where the signals for central vision originate. Also seen in the posterior pole are the optic nerve head (black arrow) and the retinal vasculature (white arrows) that may course over the surface of the tumor demonstrating the abnormal tumor protrusion into the vitreous body.

Figure 2 Leukocoria. In children with retinoblastoma, an area of whitening (white arrow) may be seen within the dilated pupil instead of the normal red reflex.

during indirect ophthalmoscopy, a test that allows seeing inside the fundus of the eye (Figure 3). In young children, this is most commonly performed by an ophthalmologist while the patient is under general anesthesia, to facilitate a proper examination. The tumor varies from near translucent to chalk white and can be seen to have a large feeding arteries and draining veins. The growth pattern can be endophytic in which it grows into the vitreous gel of the eye and can seed new smaller tumors, or it can be exophytic in which the bulk of its mass is beneath the retina causing a retinal detachment. The exophytic growth pattern can result in subretinal seeding of tumor. A far less common growth pattern is diffuse infiltrating type that can lead to diagnostic confusion given the lack of a single tumor focus. Ancillary testing assists in making the diagnosis. Most commonly, this involves ultrasonography of the eye, which can better demonstrate the extent and growth pattern of the tumor and calcifications that are a hallmark of retinoblastoma tumors (Figure 4). An MRI of the head and orbits can also demonstrate calcifications and can be particularly useful in the

Figure 4 A-scan and B-scan ultrasound of retinoblastoma. In patients with retinoblastoma, an elevated lesion is present at the level of the retina (white arrow) and can be visualized and measured with ultrasonography. The A-scan is the thin yellow tracing at the bottom of the image. It is a one-dimensional measure of the echogenicity of the tissue. The B-scan is a collection of A-scans that form a two-dimensional image (ultrasonography image).

bilateral form of the disease to help rule out the midline intracranial tumors (discussed in the succeeding text) that can be associated with the heritable form of the disease. Due to the radiation involved, CT scanning should be avoided in young children, especially given the propensity for non-ocular tumors to occur in inherited forms of retinoblastoma. This type of imaging is usually not needed to make the diagnosis, however. Fluorescein angiography (FA) is a testing modality by which a fluorescent dye is injected through a peripheral vein and allowed to circulate through the retinal and choroidal vasculature while photographs are taken to highlight vascular

Opthalmic Pathology | Ocular Tumors

patterns and problems with vascular competency. FA is sometimes utilized to aid in ruling out some conditions that can mimic retinoblastoma.

Genetics There exist both heritable and nonheritable forms of retinoblastoma. The heritable form occurs due to mutational inactivation of both alleles of the retinoblastoma gene (RB1). The mutation of one abnormal allele is transferred as an autosomal dominant condition with 90% penetrance; however the disease only manifests with inactivation of both alleles. This requires a second mutation of the second allele. The result is often bilateral multifocal retinoblastoma tumors. In the nonhereditary form, two separate somatic mutations of the RB1 alleles are required for tumor development. This often results in unifocal, unilateral tumor formation. The RB1 gene is located on the long arm of chromosome 13, involving the chromosome band (13q14), and contains 27 exons and 26 introns. The RB1 gene is an anti-oncogene or growth suppressor gene. The RB1 gene encodes a 110 kDA nuclear phosphoprotein that negatively regulates the progression of the cell cycle. In its unphosphorylated active form, the protein binds to the E2F transcription factor family, needed to initiate the genes that control DNA synthesis and cell proliferation. In the phosphorylated inactive form, the protein releases transcription factors so that DNA synthesis and cell proliferation take place with progression into the G1 phase. Although several genes control cell proliferation, it is believed that the RB1 gene is a master cell cycle regulator and its dysfunction allows unregulated cell-cycle progression. A patient harboring the heritable form of the disease will have a 50% chance of passing the disease on to each offspring. Screening for heritable mutations is possible and requires a combination of molecular tests for high sensitivity. Molecular testing is done both on the tumor cells and in the peripheral blood to determine if there is a germ-line mutation. When a tumor of the pineal gland (pinealoblastoma or more accurately primitive neuroectodermal tumor) is diagnosed in a patient with the bilateral, heritable form of the disease, it is termed ‘trilateral retinoblastoma.’ This is a very poor prognostic sign proving to be almost universally fatal (Figure 5).

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Classification There are many different ways of classifying retinoblastoma. Arguably most important is the differentiation between the heritable and the nonheritable forms as described in the previous section. The heritable form of retinoblastoma develops in about 40% of patients with retinoblastoma; of those, about 7% of cases have a known family history of the disease. A rare subclass of heritable retinoblastoma sometimes shows a chromosomal deletion. The remaining 60% of cases are nonheritable forms, in which the mutation is acquired de novo, with no ties to family genetic history. The nonheritable forms develop when a single cell in the developing retina undergoes inactivating changes to both of the RB1 alleles. This differentiation is very important because a patient who harbors the heritable form of the disease is at a high risk of secondary cancers including osteosarcoma, leukemia, melanoma, rhabdomyosarcoma, and pineoblastoma. The risk of secondary malignancies is as high as 26% within the first 30 years after initial diagnosis. A second distinction for retinoblastoma classification is unilateral versus bilateral cases. Almost all cases of bilateral retinoblastoma occur in heritable forms and nearly all cases of unilateral tumors arise sporadically. An exception to this rule is in the case of germ-line mosaicism that occurs when a mutation arises in one cell sporadically during embryonic development leaving a portion of somatic cells with a diseased genotype and the remainder without. One of the more commonly used classification schemes used today is the International Classification of Retinoblastoma (ICRB) that takes into account the size and location of the tumor and the extent of intraocular seeding and extraocular extension (Table 1). This system is useful in that it helps to predict the response to chemoreduction and will be discussed in detail later.

Pathobiology of Retinoblastoma Background information: retinal development and normal histology of the retina A basic understanding of retinal development is necessary to fully understand some of the subsequent discussion of the pathobiology of retinoblastoma. The cells that will make up

Figure 5 Pinealoblastoma. Patients with hereditary bilateral retinoblastoma can develop pineal gland tumors (white arrows) also referred to as ‘trilateral retinoblastoma.’ A well-defined mass growing in the area of the pineal gland may affect adjacent structures and obstruct the ventricular system (axial (left) and sagittal (right) views, magnetic resonance image).

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International classification of retinoblastoma

Table 1 Group

Subgroup

Quick reference

Specific features

A B

A B

Small tumor Large tumor Macula Juxtapapillary

Rb < 3 mm in size Rb > 3 mm in size or Macular Rb (>3 mm foveola) Juxtapapillary Rb (<1.5 mm disk) Clear subretinal fluid

Subretinal fluid Focal seeds

C C1 C2 C3 D

Diffuse seeds D1 D2 D3

E

E

Extensive Rb

Rb with Subretinal seeds <3 mm from Rb Vitreous seeds <3 mm from Rb Both subretinal and vitreous seeds <3 mm from Rb Rb with Subretinal seeds >3 mm from Rb Vitreous seeds >3 mm from Rb Both subretinal and vitreous seeds >3 mm from Rb Rb occupying >50% of globe or Neovascular glaucoma Opaque media from hemorrhage in anterior chamber, vitreous, or subretinal Invasion of postlaminar ON, choroid, sclera, orbit, amacrine cell

Source: Shields, C.L., Shields, J.A., 2006. Basic understanding of current classification and management of retinoblastoma. Curr. Opin. Ophthalmol. 17, 228–234.

the retina come from the neural ectoderm layer of the developing embryo. These cells begin as retinal progenitor cells (RPCs) that are undifferentiated and are capable of division into other cell types and self-renewal. As these cells are dividing during the earliest development of the retina, they take up a position in the cross section of the retina that is dependent upon what stage of the cell cycle they are in. As retinal development progresses, the RPCs give birth to a higher percentage of retinal transitional cells (RTCs), which are cells that are normally no longer capable of mitosis and begin the differentiation process along a certain path that will give rise to fully developed retinal cell types (i.e., photoreceptor, amacrine cell (AC), and bipolar cell (BP)). The retina is the neurosensory part of the eye that lines the inner side of the posterior half of the eye. It is from the retina that the retinoblastoma tumor develops. The retina is responsible for transforming light energy that reaches the retina’s outer layer into a nerve action potential (signal) that is then transmitted through the optic nerve into the brain where it is perceived. The retina itself is composed of many layers of cells and cell parts (Figure 6). The outer part of the retina is made up of the photoreceptors (rods (Rs) and cones (Cs)) that contain the molecules that respond actively to light. These

photoreceptors then interact with other cells called BPs that connect to the ganglion cells on the most inner part of the retina whose long axons join together to become the optic nerve. Also within the middle layers of the retina are other types of cells called horizontal cells (HCs), ACs, and Mu¨ller cells (MCs). HCs connect different photoreceptors to one another, ACs help in signal processing between the outer and inner portions of the retina, and MCs are glial cells that serve as scaffolding among other functions. Rb is the protein product of the RB1 gene, first identified by searching the long arm of chromosome 13, which was found to be missing or altered in the cells of many human retinoblastomas. In mice, a homologous gene was identified, which we will herein also refer to as RB (and protein Rb). Through work with mouse embryonic fibroblast cell lines and later also with chimeric mice that will be described in the succeeding text, it was determined that mice harbor another pair of RB family protein members, p107 and p130 (also known as retinoblastoma-like 1 and retinoblastoma-like 2), that seem to work in parallel with the Rb pathway and can compensate for its loss. It was also shown that Rb has effects on the cell cycle and that these effects are mediated through its interactions with E2F, a gene known to positively influence the progression through the cell cycle.

Animal models Most of our understanding of retinoblastoma and how it develops comes from work on various animal models of the disease that can be divided into three broad groups: (1) spontaneous models or those induced by chemical or viral carcinogens, for example, the introduction of adenovirus; (2) xenograft models in which tumor cells are transplanted into an immunosuppressed host in athymic or nude mice lacking most T cells or mice with severe combined immunodeficiency; and (3) genetically engineered systems in which specific genetic modifications are introduced into the animal genome to produce an altered phenotype. The first attempts at creating an animal model of retinoblastoma involved the injection of carcinogens into rats’ eyes and were mostly failures. The first successful attempts came about through the intraocular injection of adenovirus 12 into mice, rats, and baboons and of JC papovavirus into hamsters. Successful tumor growth was also achieved with intraocular implantation of human retinoblastoma cell lines into rabbits and rodents and of adenovirus-induced rodent retinoblastoma cell lines into rodents. All of these attempts generated tumor growth but did not allow for study of the origins of retinoblastoma tumors. Several models involving xenografts of retinoblastoma cell lines have been developed using orthotopic tumor models or models utilizing human tumor cells. Examples include immunocompromised mice or rats injected with human retinoblastoma cells into the anterior chamber, in the subretinal space of the eye, or intravitreally. A model utilizing the Y79 retinoblastoma cells develop retinal, choroidal, optic nerve, brain, and metastatic growth patterns of disease. The model utilizing WERI-Rb1 cell line does not show extraocular tumor spread or metastasis. A recent study using Y79 and SNUOT-Rb-1 cell lines injected into mice detected the expression of natural killer cell associated antigen in retinoblastoma.

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Internal limiting membrane Nerve fiber layer

GC

Ganglion cell layer Internal plexiform layer

AC MC

Inner nuclear layer

HC Outer plexiform layer

R BC

Outer nuclear layer

C External limiting membrane

Photoreceptors outer segment

Retinal pigment epithelium Choroid

Sclera

Figure 6 Histology of the retina.

Subsequent models utilize transgenic mice or genetically engineered animals with the use of viral oncoproteins. This strategy employs the addition of a foreign piece of DNA (DNA that codes for a tumor-causing protein that comes from tumorassociated viruses) into the animal germ line by genomic recombination that becomes incorporated into the genome of the host and is expressed according to the promoter with which it was formulated. These transgenic mice have been shown to grow tumors consistent with retinoblastoma. However, the study of retinoblastoma in these models is complicated by the fact that the viral oncoproteins utilized have been shown to interact with many different proteins that likely influence their effects on transformation of cells, in addition to their effects on Rb. The human luteinizing hormone (b subunit) (LHb)-Tantigen mouse was the first genetically engineered retinoblastoma model and is still widely used. The LHb promoter is linked to SV40 Tag (a proto-oncogene derived from the polyomavirus SV40). This model shows bilateral and multifocal retinoblastoma tumors within a couple of months, resembling the human tumors; the mice also develop primary subependymal midbrain multifocal CNS tumors at a higher frequency than in humans. Other constructs include SV40 Tat combined with mouse rhodopsin promoter to produce a photoreceptor-selective T-antigen expression with early rod degeneration and CNS tumor development; the alpha-A crystalline SV40 T-antigen mouse model, the human papilloma virus E6/E7 (HPV viral oncoproteins identified in malignant transformation) transgenic mouse model, and the alphacrystallin-E6/E7 transgenic mouse model show development of microphthalmia, cataracts, and retinoblastoma with

metastatic capability. The IRBP (inter-photoreceptor retinoidbinding protein)-E7 and IRBP-E7 þ p53/ transgenic mouse models show inactivation of pRB and the p53 tumor suppressor proteins leading to retinal degeneration in the IRBP-E7 model and to retinoblastoma development in the IRBPE7 þ p53/ model. Knockout mice are genetically engineered by introducing one or both copies of the Rb gene. A targeted insertion of a fragment of DNA that contains a stop codon is introduced into the gene in mice cell lines. These cloned cells are grown and later injected into developing mouse blastocysts, and then interbreeding occurs to create germ-line heterozygotes (RB þ/) and homozygotes (RB /). Unlike in humans, the resulting Rb þ/ mice do not develop retinal tumors but pituitary, thyroid, and neuroendocrine tumors. Rb / mice die in utero and demonstrate problems in neural and erythroid development. These findings support a critical role for pRb in tissue differentiation and development. Since Rb / mice with wild-type extraembryonic tissues survive to birth without these developmental issues, it is felt that they are secondary to Rb/-associated placental developmental anomalies. By generating chimeric mice without the further step of interbreeding, the aforementioned problems were solved. In studies with Rb þ/ cells making up 50% of retinal tissue, there is no resulting retinal phenotype. However, with Rb / cells making up 20% of retinal cells, there is resulting evidence of embryonic inner retinal ectopic proliferation and cell death. The aforementioned Rb / chimeric mice do not form retinal tumors, suggesting the need for further mutations. In order to achieve retinoblastoma growth in knockout mice, conditional knockout models (CKO Rb) were devised

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using a Cre–loxP system utilizing an inducible Cre recombinase to excise a loxP-flanked RB gene in the developing retina. Sixty percent of such derived CKO Rb mice that were also p107 / (p107 is a closely related RB protein that mimics many of its activities) developed retinoblastoma, and these tumors demonstrated only amacrine neuronal markers. Other similar models that resulted in retinal mosaics of conditionally knocked-out Rb cells in mice showed similar results in terms of retinoblastoma growth and AC markers. The findings suggest that there is some functional overlap between pRb and p107 in the developing and adult mouse retina, p107 //RB1 / cells but not RB1 / cells that can give rise to retinoblastoma.

Cell of origin There is good evidence that Rb has its first effects in RTCs as opposed to RPCs and that the former are therefore the cells of origin of retinoblastoma. First, RbKO retinas labeled with BrdU (which gets incorporated into dividing cells) showed ectopic division in the inner retina where there are no RPCs. Markers for ganglion cells, photoreceptors, and amacrine, horizontal, and Mu¨ller cells do not colocalize with BrdU in the wild-type retina but do so in both RbKO and Rb/p107 double knockout (DKO). Secondly, clones that are RbKO or Rb/p107 DKO do not show evidence of an increase in mitotic cells that are located in the normal location of the RPCs in the developing embryonic retina or that label with RPC markers. This suggests that Rb absence does not lead to expansion of RPCs. Lastly, p130 is only present in mouse RTCs and is a closely related RB protein that mimics many of its activities. The deletion of p130 and Rb results in retinoblastoma. Since the deletion of Rb does not affect the expression or activity of p130, the cells of origin must be the RTCs.

Rb and genomic instability In addition to the direct effects that Rb inactivity has on the E2F family of proteins and their subsequent influence over cell cycle progression, Rb loss also influences genome stability. Aneuploidy is the state of a cell with an abnormal number of chromosomes commonly seen in cancer cells. This is often a result of abnormalities in the normal chromosome segregation that occurs during mitosis. Normally interactions between the polar spindle apparatuses and the kinetochores of chromosomes result in each member of a pair of chromosomes being pulled to opposite ends of a dividing cell. In the absence of Rb, chromosomal kinetochores have a propensity to associate with microtubules from both spindles. This occurs through effects on proteins downstream of E2F and through interactions with other molecules such as condensin II components leading to stretched kinetochores. In addition to effects of chromosome mis-segregation, the lack of Rb also plays a role in increasing DNA damage. Rb-deficient cells progress unregulated into Sphase even in the absence of sufficient nucleoside pools leading to an accumulation of DNA damage. Most retinoblastoma tumors reveal many genetic changes, some of which are seen with higher frequencies (such as 1q31–32 gain, 6p22 gain, and 16q loss). Some of these changes are associated with higher rates of other aberrations suggesting a cycle of worsening genome instability through single Rb allele loss.

Cell of origin and number of hits theory Knudson’s two-hit hypothesis proposed that retinoblastoma formation requires two rate-limiting events; it was later shown that both hits target the same gene (RB). However, some studies indicate that RB-mediated tumorigenesis requires additional genetic defects such as the gain of 6p22 and 1q31 in RB-deficient cells in humans. RB loss not only confers infinite proliferative capacity to transition cells or RPCs but also makes them more susceptible to cell death. Therefore, additional mutations in genes that regulate apoptosis must occur for tumorigenesis to occur. The initial events occur at the level of the RTC, and it is most commonly the RTCs that become ACs that seem to be transformed. It is reasoned that the AC is the cell of origin because of its death-resistant character as compared to other retinal cell lineages discussed earlier. When cells, for whatever reason, harbor a natural resistance to apoptosis in the face of unregulated cell growth, there is one less block to overcome on the path to malignant transformation. This does not mean that other cell lines cannot be transformed. Transgenic mice with viral oncoproteins have resulted in the transformation of cells with photoreceptor markers, and most human retinoblastomas show some evidence of photoreceptor differentiation. Cells such as these that are not naturally resistant to apoptosis (at least in mice) would have to acquire mutations that allow for escape from the apoptosis pathway in addition to systems that couple growth arrest to differentiation.

Pathology The International Retinoblastoma Staging Working Group published pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma. Sectioning an eye with retinoblastoma usually shows the vitreous cavity filled with a whitish (Figure 7), necrotic tumor material that is discohesive and can easily dislodge and seed other portions of the globe (Figure 8). It is important to section the optic nerve margin prior to sectioning the globe to avoid tumor contamination since optic nerve involvement is relevant to patient treatment and prognosis. The tumor may show different growth patterns (Figure 9 and Virtual Microscopy Slide 1 eSlide: VM00277); endophytic growth occurs when the tumor breaks through the internal limiting membrane of the retina and is usually associated with vitreous tumor seeding. Exophytic growth occurs in the subretinal space and is often associated with subretinal fluid accumulation and retinal detachment (Figures 10 and 11). The tumor cells may infiltrate through Bruch’s membrane into the choroid and then invade either blood vessels or ciliary nerves or vessels. A rare subtype comprising 1.5% of all retinoblastomas is characterized by a relatively flat infiltration of the retina by tumor cells but without a discrete tumor mass. This variant grows slowly compared with the other patterns of growth. The growth pattern does not influence prognosis but can have implications for patient management. One of the most important histopathologic prognostic features is optic nerve invasion (Figure 12 and Virtual Microscopy Slide 2 eSlide: VM00278 and Figure 13). Other poor prognostic features include extraocular extension and massive choroidal invasion (larger than 3 mm in diameter) (Figure 14).

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Figure 7 Large retinoblastoma. Retinoblastomas (black arrows) can occupy the entire vitreous cavity, displace the lens anteriorly, and lead to a lens cataract (white arrow). The retina commonly detaches (asterisk) (Gross picture, original magnification 1).

Figure 8 Retinoblastoma with calcifications. In retinoblastoma, the tumor may grow as much as to occupy the majority of the vitreous cavity. Foci of microcalcifications may also be seen (black arrow) and proteinaceous exudate may accumulate adjacent to the tumor (white arrow) (Gross picture, original magnification 1).

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Figure 9 Histopathology of retinoblastoma (black thick arrow). The sclera, the outer coat of the eye that is in continuity with the cornea (square) anteriorly, shows an indentation due to an artifact of tissue preparation (thin black arrow). The lens (asterisk) is dark pink and cataractous due to the tumor growing in the vicinity and pushing the lens forward. Note the retina near the optic nerve head (circle) is seen in continuity with the tumor. The tumor does not show optic nerve or choroidal invasion. The choroid is located between the retina and the sclera and is best seen in the digitally scanned slide (blue arrow). The tumor shows the typical sleeves of necrosis and debris (red arrow). Use this figure to aid you in the use of (Virtual Microscopy Slide 2 eSlide: VM00277) (H&E stain, original magnification 10 ).

Figure 10 Growth patterns of retinoblastoma. Retinoblastomas may exhibit mixed exophytic and endophytic growth patterns (black arrow), fill the posterior pole of the eye, and lead to retinal detachment with subretinal exudate accumulation (white arrow). The tumor can be found growing in continuity with the retina (black asterisk) (H&E stain, original magnification 10 ).

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Figure 11 Histopathology of large size retinoblastoma. Retinoblastomas may reach large sizes filling the entire globe (thick black arrow). Tumor growth into the optic nerve head may be seen in large tumors (thin black arrow). A lens cataract and displacement (asterisk) may develop due to tumor growth posteriorly (H&E stain, original magnification 10 ).

Figure 12 Retinoblastoma with optic nerve invasion. There is a large area of calcification within necrotic retinoblastoma (enclosed in black circle). There is subretinal tumor growth and subretinal fluid accumulation (white asterisk). The tumor invades the optic nerve (thin black arrow). The lens shows an artifactual fold of tissue preparation (black asterisk). Use this figure to aid you in the use of (Virtual Microscopy Slide 2 eSlide: VM00278) of this article (H&E stain, original magnification 10).

Figure 13 Comparison of a normal optic nerve and nerve with retinoblastoma tumor invasion. In a cross section of a normal histology of an optic nerve, nerve fibers are divided into bundles by fibrovascular septa (black asterisk) ((a) H&E stain, original magnification 10). In invasive retinoblastoma, tumor cells may extend from the retina into the optic nerve past the lamina cribrosa leaving a peripheral rim of uninvolved optic nerve (black arrow) ((b) H&E stain, original magnification 10).

On histopathology, most tumor cells resemble the cells of an undifferentiated retina of the embryo called retinoblasts. These consist of small- to medium-sized round cells with large nuclei and scant cytoplasm. Undifferentiated tumors show sleeves of tumor cells with large areas of necrosis and microcalcifications (Figure 15). Well-differentiated tumors may have Flexner–Wintersteiner rosettes (tumor cells in a wreath-like pattern with a central lumen formation recapitulating the retina), Homer Wright rosettes (tumor cells in a wreath-like pattern with central pink material – no lumen formation), and

fleurettes, which probably represent photoreceptor differentiation into lumens (Figure 16). Some tumors may have a combination of both undifferentiated and differentiated areas (Figure 17). As previously mentioned, the differential diagnosis includes medulloepithelioma, a childhood tumor arising from the ciliary epithelium and presenting usually by age 5. The tumor is arranged in cords and sheets (Figure 18) that may resemble embryonic retina. Cystic spaces lined by the tumor cells and containing hyaluronic acid are a common diagnostic

Figure 14 Retinoblastoma with choroidal invasion. Retinoblastoma tumor cells (white arrows) can invade the choroid (black hash lines) (H&E stain, original magnification 100).

Figure 15 Undifferentiated retinoblastoma. In undifferentiated retinoblastoma, cells are medium-sized round cells with large nuclei and scant cytoplasm (H&E stain, original magnification 100 ). Inset: In large size retinoblastoma, the tumor can replace the majority of the vitreous and show large areas of necrosis seen as pink material within the tumor (H&E stain, original magnification 10).

(a)

Flexner–Wintersteiner rosettes

(b)

Figure 17 Mixed histological patterns of retinoblastoma. Retinoblastoma may show a mixture of undifferentiated and differentiated tumor cells. Undifferentiated cells grow in a sheetlike pattern (white arrows) with necrosis (shape). Inset: Differentiated areas (black arrow) show rosettes (H&E stain, original magnification 200).

Figure 18 Medulloepithelioma, benign variant. The tumor cells are arranged in cords and sheets. Cystic spaces are lined by cells that have elongated hyperchromatic nuclei (H&E stain, original magnification 400). Inset: Tumor (black arrow) arises from the ciliary body epithelium (H&E stain, original magnification 10).

Homer Wright rosettes

(c)

Fleurettes

Figure 16 Differentiation patterns in retinoblastoma. Flexner–Wintersteiner rosettes are formed by cells shaped in a wreath-like pattern with a central lumen limited by a basement membrane ((a) H&E stain, original magnification 600). Homer Wright rosettes are pseudorosettes that consist of wreath-like patterns of multitiered cells that enclose eosinophilic material ((b) H&E stain, original magnification 600). Fleurettes probably represent photoreceptor differentiation ((c) H&E stain, original magnification 600).

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feature. Although toxocara endophthalmitis, PFV, and Coats’ disease may be clinically mistaken for retinoblastoma, these are easily diagnosed on histopathology. The nematode Toxocara endophthalmitis is a sclerosing, chronic vitreous body inflammation caused by the toxocara larva. PFV shows a retrolental fibrovascular mass, with ruptured posterior lens capsule (Figure 19). Coats’ disease shows telangiectatic vessels with accumulations of eosinophilic exudates in the outer retinal layers and subretinal space (Figure 20).

Treatment and Prognosis Advances in treatment of retinoblastoma in the last 20 years have markedly decreased the mortality of this disease in developed countries like the United States and those of Western Europe. In fact, enucleation (the surgical removal of the eye and a portion of the optic nerve) is no longer the only mortality-reducing modality, and globe-sparing treatment options are now commonly employed when appropriate. Also, external beam radiotherapy has been mostly phased

due to undesirable secondary tumor complication rates in hereditable cases. In recent years, 5-year survival rates approach 100% in the United States and England compared to higher mortality rates in Latin America (20%), Asia excluding Japan (39%), and Africa (70%). Using the ICRB classification discussed earlier, the factors that influence staging for treatment purposes include laterality, tumor size, tumor location, presence and extent of vitreous or subretinal seeds, and evidence of invasion of certain ocular and periocular tissues. Small unilateral tumors are now mostly treated with ablative modalities such as cryotherapy, thermotherapy, laser photocoagulation, and plaque radiotherapy, sometimes with the addition of adjuvant chemotherapy (Figure 21). Larger, more involved tumors often require pretreatments with chemoreduction followed by one of the ablative procedures discussed earlier. The most utilized chemoreduction regimen involves 6–9 cycles of the combination of vincristine, etoposide, and carboplatin. Some unilateral tumors can be so advanced (ICRB group E) at diagnosis that enucleation is still the recommended treatment. This involves the careful surgical removal of the

Figure 19 Persistent fetal vasculature (PFV). PFV is commonly seen in trisomy 13 and is a differential diagnosis of retinoblastoma. In PFV, a persistent fetal vessel (black arrow) is seen originating from the optic nerve head area and surrounding the lens posteriorly. In this example, the persistent vessel becomes a fibrovascular plaque (white asterisk) attached to the posterior surface of a cataractous lens (black asterisk) and shows cartilage differentiation (ciliary body, small black arrow) ((a) H&E stain, original magnification 20). Persistent fetal vessel ((b) H&E stain, original magnification 200 ).

Figure 20 Coat’s disease. Abnormal dilatation and leakage of retinal vessels leads to the accumulation of subretinal and vitreous exudate (black asterisk) and retinal detachment (black arrow) ((a) H&E stain, original magnification 10). Telangiectatic retinal vessels (black asterisk) are seen with the accumulation of proteinaceous debris (black arrow) in the vitreous ((b) H&E stain, original magnification 20).

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however, including the eye. Melanocytes are the pigmentproducing cells that give skin and many ocular tissues their color. Melanocytes are found in the normal eyelid skin, conjunctiva, and uveal tissue, as well as in the orbit. Each of these tissues can be the primary site of an ocular melanoma. Melanomas of the conjunctiva, eyelid skin, and orbit are relatively rare tumors and will not be discussed in this work. Intraocular melanoma can involve the iris, ciliary body, and choroid, all components of the uveal tract or the vascular intraocular coat. The incidence of uveal melanoma in the United States is 6 per 1 million people; choroidal melanomas make up more than 90% of the tumors. Iris and ciliary body melanomas are less common in frequency, about 5% each. The different types of uveal melanoma demonstrate differences in epidemiology, pathology, treatment strategies, and prognosis.

Risk factors Figure 21 Posttreatment retinoblastoma. Retinoblastoma tumors that have been treated with brachytherapy can show features of regression in the form of a macular scar (black arrow).

globe with the longest piece of connected optic nerve that can be resected, followed by insertion of an orbital implant that is sutured to the extraocular muscles to improve mobility of the ocular prosthesis that is later placed in the conjunctival fornices. The implant helps to ensure proper facial bone growth and improves cosmesis. When high-risk pathological features such as choroidal invasion, optic nerve invasion beyond the lamina cribrosa, scleral or orbital involvement, or seeding into the anterior chamber are noted, adjuvant chemotherapy has been shown to decrease the metastasis rate from 24% to 4%. Bilateral tumors are all treated with chemoreduction in addition to other modalities listed earlier because there is some evidence that suggests that this strategy lowers the risk of secondary tumors, particularly the pinealoblastomas discussed previously. The use of locally directed chemotherapy is currently under investigation and has shown some promise as a treatment modality. This method employs selective cannulation of the ophthalmic artery and local administration of tiny doses of chemotherapeutics over multiple cycles. Subconjunctival injection of carboplatin may also prove beneficial as an adjuvant to ablative strategies on small tumors or combined with other chemotherapeutic regimens for more advanced tumors. Overcoming the mortality differential between developed and developing countries is one of the most important current obstacles in the treatment of retinoblastoma. Education programs in countries such as Honduras aim to help parents and caregivers recognize the signs of the disease and the importance of early presentation to medical care. One study showed a reduction of extraocular tumor extension from 73% to 35%. Much work still needs to be done to try to decrease the mortality rates in developing countries.

Melanoma Epidemiology Melanoma is a well known potentially fatal cancer of the skin. Melanoma can arise in any tissue that has melanocytes,

Sunlight exposure is a known risk factor for cutaneous melanoma. The relationship is not as clear with uveal melanomas. Some evidence, such as the rarity of the disease in non-white races supports an association, while other evidence such as the lack of variance in incidence with latitude argues against it. Xeroderma pigmentosum (XP) is a rare inherited disorder of DNA repair that is a risk factor for uveal melanoma, which is 23 times more likely to develop in a person with XP than in the general population. Ocular melanocytosis (or oculodermal melanocytosis when involving the periocular skin) is a condition of increased pigmentation caused by higher numbers of melanocytes in the ocular and periocular tissues on one side. Persons with this condition have about a 30-fold increased risk of developing uveal melanoma. The presence of a nevus, a benign melanocytic tumor of the uvea, is also a recognized risk factor, with a risk of transformation of a nevus into melanoma estimated at between 1 in 4800 and 1 in 8845 in white populations. A meta-analysis also identified fair skin, light eyes, and an inability to tan as host-specific risk factors for the development of uveal melanoma.

Clinical Presentation Most iris melanomas are noted by the patient as a darkly colored spot inferiorly on the colored part of the eye that has recently changed in size (Figure 22). Rarely, the lesion or the inflammatory cells associated with it can clog the trabecular meshwork (or drainage system of the eye) and result in increased intraocular pressure and subsequent glaucomatous field loss. A diffuse growth pattern can present as difference in iris color of the two eyes. Ciliary body melanomas can grow to be quite large at the time of diagnosis secondary to their secluded location behind the iris. When small, they are challenging to diagnose even to the well-trained ophthalmologist and would be unapparent to the patient. They can manifest as a changing astigmatic refractive error secondary to their effect on the morphology and position of the adjacent lens. When advanced, they can be seen on the episcleral ocular surface after infiltrating through the underlying sclera. They can also cause glaucoma upon infiltration of the nearby angle structures (Figure 23). Choroidal melanomas are often asymptomatic and mostly discovered on routine eye examination (Figure 24). Their

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posterior location hides them from a patient’s or family member’s view, though occasionally symptoms can prompt a patient to seek an ophthalmologist (Figure 25). Such symptoms include visual loss, flashes, floaters, and visual field defect. Many of these symptoms are related to subretinal fluid that can accumulate in the area around the tumor (Figure 26).

Differential diagnosis The diseases that might be confused for malignant melanoma and need to be ruled out are nevi and metastatic tumors. Benign choroidal nevi are common intraocular tumors seen in approximately 6% of the population; only 1 in 500 will evolve into choroidal melanoma. They are managed by observation; they are usually not greater than 2 mm in thickness; they rarely show detachment of the pigment epithelium or choroidal neovascularization (Figure 27). Melanocytomas (Figure 28) are a heavily pigmented variant of nevi that rarely occur in the choroid. Metastatic cancer (Figure 29), a common form of intraocular malignancy, usually presents to the ophthalmologist in advanced cancers of known primary

Figure 22 Iris melanoma. Iris melanomas (black arrow) are most commonly found on the inferior aspect of the iris. Features that support the diagnosis include increased thickness, evidence of growth, and prominent vasculature (white arrow). Courtesy of Helen K. Wu, MD.

Figure 24 Peripheral choroidal melanoma. Choroidal melanomas demonstrate variable amounts of pigmentation (white arrow). A greater thickness and evidence of growth help distinguish a melanoma from a nevus.

Figure 23 Ciliary body melanoma. A malignant melanoma (black arrow) may involve the angle, root of the iris (black asterisk), and ciliary body (small black arrow) ((a) H&E stain, original magnification 40 ). A layer of tumor cells can infiltrate the surface of the iris (black arrows) ((b) H&E stain, original magnification 100 ).

Figure 25 Choroidal melanoma of the posterior pole. The macula, which is the central vision part of the retina, may be affected by choroidal melanomas (shape) (fundoscope image).

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sites. The great majority are carcinomas, breast cancer in women (Figure 30) and lung cancer in males. The iris, ciliary body, or choroidal metastases usually present as single or multiple yellow lesions. There is a long list of nonneoplastic lesions that can simulate a uveal melanoma including vascular and hemorrhagic lesions, inflammatory and infectious lesions, and miscellaneous conditions including medulloepithelioma (Figures 18 and 31) and bilateral diffuse uveal melanocytic proliferation.

Diagnosis Careful eye examination by an experienced clinician is the primary tool used in diagnosing a malignant melanoma. Small choroidal lesions are often observed initially. Definitive growth on subsequent examinations typically means transformation to a melanoma. A choroidal melanoma usually

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presents as a dome-shaped mass located under the retina. Clinical findings that increase the likelihood that a suspicious nevus will undergo growth while being observed include tumor thickness greater than 2 mm, presence of subretinal fluid (Figure 32), visual symptoms, orange pigment on the surface of the tumor (Figure 33), and tumor margin touching the optic disk. None of these findings are diagnostic for malignancy, however, and even if all exist initially in a specific lesion, that lesion could still be benign. FA (Figure 26) and ultrasonography are valuable in the initial evaluation of suspicious lesions. Ultrasonography helps in substantiating the diagnosis and can accurately determine tumor size and subsequent response to therapy. Serial fundus color photographs and FA can aid in determining tumor diameter and subsequent lateral growth. The A-scan ultrasonography provides accurate information about internal reflectivity and the B-scan provides two-dimensional topographic information (Figure 34).

Pathobiology of Malignant Melanoma Background information

Figure 26 Fluorescein angiography (FA) of choroidal melanoma. Fluorescein angiography is an additional ancillary test that can demonstrate areas of leakage within the tumor (white asterisk) (photograph of fluorescein angiography).

The uveal tract is a vascular intraocular coat composed of the iris, the ciliary body, and the choroid. The iris and ciliary body are located anterior to the ora serrata, the site that marks the beginning of the retina. The ciliary body is contiguous with the iris anteriorly and the choroid posteriorly. It can be divided into an anterior ring, or the pars plicata, and a posterior ring, or the pars plana. The vascular layer of the ciliary body is located between its muscle layer and the two layers of ciliary epithelium. The choroid is the largest and most posterior portion of the uvea, located between the retinal pigment epithelium and the sclera and extending from the ora serrata to the optic nerve. It consists mainly of blood vessels, nerve fibers, and pigmented melanocytic cells in a loose connective tissue matrix (Figure 35). The choroid’s prime function is to nourish the outer half of the retina. It is supplied by branches of the posterior and anterior ciliary vessels derived from the

Figure 27 Comparison of malignant melanoma to a nevus. There is overlap in the clinical appearance of a choroidal nevus (white arrow) and malignant melanoma. A feature that favors a nevus rather than melanoma is thickness of two millimeters. The optic disk is a well-defined, yellow, pale, off-centered area (black arrow) ((a) color photograph). Choroidal nevi cells are spindle-shaped, small to medium in size, with homogenous nuclei (white arrow), inconspicuous nucleoli, and low mitotic rates ((b) H&E stain, original magnification 400 ).

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ophthalmic artery and drained by tributaries of the four or more vortex veins into the orbital ophthalmic veins and through the superior orbital fissure into the cavernous sinus.

Animal models Uveal melanoma may spontaneously occur in some animals, including dogs, cats, horses, rats, mice, birds, and fish. However, these are rare and are therefore not useful as models of disease. Several methods have been developed to induce intraocular melanoma chemically or by radiation in laboratory animals. Some of these induced tumors resemble human uveal melanoma, although the majority originate from the retinal pigment epithelium. Uveal proliferations have been biologically induced by feline leukemia/sarcoma virus and simian virus 40, although the presence of virus in tumor cells and extraocular tumors resulting from shed virus detracts from the utility of this model. Animal models may be injected subcutaneously with tumor cells or may be inoculated with tumor cells into the eye of the host animal to induce tumor formation. Inoculation of human melanoma cells into animal

Figure 28 Melanocytoma. Melanocytomas show cells with ample cytoplasm filled with melanin pigment (H&E stain, original magnification 400 ).

eyes has the advantage that the inoculation site and size of inoculum can be controlled. Disadvantages include the immune suppression necessary for tumor growth in some models and the fact that many of the melanoma cell lines are of cutaneous origin. Orthotopic tumor models use human melanoma cell lines, while heterotopic tumor models use cutaneous melanoma cell lines injected into the eyes of animals. Orthotopic tumor models better resemble human tumor growth and metastasis. Tumors that do not metastasize from heterotrophic sites in nude mice will form metastases after orthotopic transplantation, suggestive of local organ-specific factors influencing metastatic disease. Animal models of uveal melanoma have been developed using melanoma cells derived from different origins: mouse (B16, Greene, and B16LS9), hamster, and human (i.e., OCM, MKI-BR, KP6.5, 92.1, Mel290, and Mel270). The mouse B16 melanoma cell line was used to study metastasis and the immunologic response to the disease. The hamster Greene melanoma cell line was broadly used to conduct preclinical research in rabbits. Both the Greene and the B16 melanoma cell lines present the disadvantages of being not only of animal origin but also of cutaneous origin; therefore, the biological behavior of the tumor cells probably differs from that observed in human uveal melanoma cells. Human primary OCM-1 uveal melanoma cells injected into the immunosuppressed SCID mouse eye (CB 17 strain of SCID athymic nude mice) showed aggressive tumor growth and internal vascular architecture as seen in human tumors but did not show metastatic disease. Human melanoma cell lines injected into immunosuppressed rabbits led to a rabbit model established in 1989 by Kan-Mitchell and coworkers. In this model, uveal melanoma cell line 92.1 injected into the suprachoroidal ocular spaces grew in the choroid of rabbits and showed a mixed cell tumor type. Circulating malignant tumor cells and primarily lung metastasis were also observed in this model. The main limitations of this model are minimal liver metastasis and the need for continuous immunosuppression to allow for tumor development. Unlike cutaneous melanoma, uveal melanoma lacks mutations in BRAF, NRAS, or KIT. GNAQ, a G-protein alpha subunit and an early oncogenic mutation, has been identified mutated in ocular melanomas in approximately half of the tumors. This mutation leads to melanocyte proliferation in mice and also

Figure 29 Metastatic adenocarcinoma of prostatic origin to the eye. A 74-year-old man presented with episodic blurred vision in the right eye of 2 months duration. Funduscopic examination showed an inferotemporal amelanotic choroidal tumor (black arrow) with associated subretinal fluid ((a) color image). B-scan showed a vascular dome-shaped lesion of medium to high internal reflectivity (white arrow) ((b) B-scan image). Enucleation of the eye showed a choroidal amelanotic mass (black arrow) with an overlying retinal detachment (white arrow) ((c) Gross image). Microscopic evaluation revealed a metastatic adenocarcinoma with prominent nucleoli (black arrow) and mitotic figures (white arrow). Ancillary testing proved the tumor cells to be of prostatic origin ((d) H&E stain, original magnification 200 ). Courtesy of Seymour Brownstein, MD.

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Figure 30 Metastatic breast cancer to the eye. A 53-year-old female with the history of stage II breast cancer presented with a cystic iris lesion that developed into a conjunctival mass 2 weeks later. Her symptoms included severe pain, light sensitivity, headache, and foreign body sensation. The pupil is irregular in shape (white arrow). The edge of a tan white mass is seen in the iris (black arrow) ((a) external examination photograph). Slit-lamp examination shows a protruding lesion (white arrows) with prominent vascularity ((b) color photograph). Ultrasound biomicroscopy of the eye shows a normal portion of the iris (white arrow) and an abnormal thickened area involving the iris and ciliary body corresponding to the tumor growth (white asterisk) ((c) ultrasound image). Fundoscope examination reveals a choroidal lesion (white arrows) ((d) fundoscope image). Tumor cells are round to oval in shape with abundant cytoplasm arranged in nests ((e) H&E stain, original magnification 200). Immunohistochemical staining with estrogen receptor shows positivity viewed as brown staining in the cytoplasm of the tumor cells (black arrow). These findings are consistent with a metastatic tumor of breast origin ((f) IHC, original magnification 200).

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Figure 31 Malignant medulloepithelioma. In a malignant variant of medulloepithelioma, invasion through the sclera is seen (white arrows); notice the tumor growing in the ciliary body (white asterisk) with invasion into adjacent tissues (black arrows) ((a) H&E stain, original magnification 200 ). This tumor (black arrow) shows elements resembling the immature medullary epithelium of the embryonic optic cup and can be seen growing besides the pigmented layer of the ciliary epithelium (white arrow) ((b) H&E stain, original magnification 400). The tumor cells resemble medullary epithelium with elongated blue cells (white arrows) arranged in a tubular architecture with spaces filled with aqueous-like material (black asterisk) ((c) H&E stain, original magnification 1000).

Figure 32 Choroidal melanoma with adjacent exudative retinal detachment. Fluid beneath the retina (black asterisk) adjacent to or overlying a pigmented lesion (white asterisk) is an additional feature that supports the diagnosis of malignant melanoma.

interacts with other oncogenes to transform melanocytes. GNAQ mutations affect the glutamine residue at amino acid position 209 and are present in 46% of uveal melanomas. This mutation occurs in the RAF/MEK/ERK pathway, which has been identified as initiating mutations in other cancers and leads to activation of cell cycle genes, helping explain the overexpression of cyclin D1 present in uveal melanoma. Withaferin A, a natural withanolide steroidal lactone, has efficacy against multiple tumor types and was studied using uveal melanoma cell lines 92.1, MEL290, and OMM2.3. These cell lines carry a WTBRAF genotype; only the 92.1 cell line harbors a GNAQ mutation. Transgenic murine models have been developed using the promoter region of the tyrosinase gene to target expression of oncogenes in melanin-producing cells. Spontaneous intraocular pigmented tumors and distant metastases may

Figure 33 Choroidal melanoma with orange pigment. Orange pigment (white arrow) overlying a lesion is an additional feature that supports the diagnosis of malignant melanoma.

occur, although many, if not all, of the intraocular tumors arise in the retinal pigment epithelium.

Cell of origin It is hypothesized that the earliest event in the pathogenesis of melanoma may be the underactivity of the Rb pathway, the overexpression of cyclin D, or the underexpression of CDKN2A. These features could determine the formation of a choroidal nevus. The switch to melanoma could be determined by the inhibition of the p53 pathway. Monosomy of the chromosome 3 determines the transformation in a more aggressive type of uveal melanoma with features of neoangiogenesis and local infiltration. Additionally, 8q triplication, with the consequent overexpression of several protooncogenes, among others, C-MYC, NBS1, and DDEF1, leads to metastasis formation.

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Pathology Gross examination of an enucleated eye is important to determine if there are signs of extrascleral or anterior chamber invasion, the growth pattern (dome-shaped, mushroomshaped, or diffuse), the extent of secondary retinal detachment, pigmentation, and other important features (eye specimen

Figure 34 A-scan and B-scan ultrasound of a choroidal melanoma. Ultrasonography is an important ancillary test that is helpful in establishing the diagnosis of choroidal melanoma. It is useful in measuring tumor size, demonstrating growth pattern (dome-shaped lesion, white arrow), and determining the lesion echogenicity. It can also demonstrate the presence of adjacent subretinal fluid accumulation (white asterisk). The A-scan is the thin yellow tracing superimposed on the image. In malignant melanoma, it shows a low echogenicity of the substance of the tumor compared to surrounding structures (ultrasound image).

Figure 35 Normal histology of the choroid. The choroid is composed of vessels, melanocytes, and fibroblasts. The choroid (black hashed lines) feeds the outer retina including the outer segments of the photoreceptors (black asterisk) and the retinal pigment epithelium (white asterisk). Bruch’s membrane (white arrow) is the basement membrane of the retinal pigment epithelium (H&E stain, original magnification 600 ).

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gross examination (Movie 1)). Standard histological evaluation of uveal melanoma (Figures 36 and 37) include measurement of the largest basal tumor diameter, tumor height, cell type, presence of scleral invasion, ciliary body involvement, mitotic figures, and presence of inflammation and pigmentation (Figures 38–40). Histopathologically, these tumors have a spectrum of cell types, ranging from thin and plump spindle cells to epithelioid cells. In 1931, Callender developed a cytological classification of uveal melanomas dividing them into spindle A, spindle B, fascicular, mixed, epithelioid, and necrotic cell types. McLean et al. modified the classification and proposed three main tumor types: spindle cell, mixed cell, and epithelioid cell types. Spindle cell tumors tend to grow in a compact cohesive fashion surrounded by a dense reticulin framework (Figures 41 and 42), epithelioid cells grow less cohesively and are not surrounded by a network of reticulin (Figures 43 and 44 and Virtual Microscopy Slide 4 eSlide: VM00280), and mixed cell-type tumors are composed of a mixture of the two cell types (Figure 45). The tumor cell type is related to prognosis; patients with tumors composed of pure spindle cells have a more favorable prognosis, and those with a component of epithelioid cells (mixed or epithelioid cell types) have a worse prognosis (Figure 46). Melanomas with a low mitotic activity show a better prognosis; tumor infiltration by lymphocytes has been associated with decreased survival. Extraocular tumor extension can occur through different routes, mainly the vortex veins, aqueous channels, the ciliary arteries, and ciliary nerves; all are associated with a poor prognosis (Figure 47).

Cytogenetic and molecular diagnostic studies The most common cytogenetic changes in uveal melanoma include loss of DNA on chromosome 3, gain on 6p, loss on 6q, and gain on 8q. Loss of an entire chromosome 3 (or monosomy 3), which is an early event in tumorigenesis, is

Figure 36 Predominantly nonpigmented choroidal melanoma. In a predominantly nonpigmented choroidal melanoma (white arrow), the tumor shows a white appearance with focal areas of melanin deposition (black arrow) (Gross picture, original magnification 1).

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Figure 37 A large choroidal and ciliary body melanoma showing the classic mushroom tumor growth (white arrow) due to breakage of Bruch’s membrane. The retina is displaced by the tumor (black thin arrows) and the lens is cataractous (black asterisk) (Gross picture, original magnification 1).

Figure 38 Small size malignant melanoma. Small malignant melanoma of the choroid (4 mm in greatest dimension) can arise in any location of the posterior pole (thick arrow). In some instances, the retina artifactually detaches (thin arrow) (H&E stain, original magnification 10).

Figure 39 Medium size malignant melanoma of the choroid. In medium-sized malignant melanoma of the choroid (6 mm in greatest dimension) (thick arrow), features of a nodular growth pattern can be appreciated. Accompanying subretinal exudate (asterisk) can be seen (H&E stain, original magnification 10).

Figure 40 A large malignant melanoma of the choroid. In large malignant melanomas of the choroid (15 mm in greatest dimension) (thick arrow), distortion of the shape of the globe can occur. Total retinal detachment (thin arrow) and accumulation of subretinal exudate (asterisk) can be seen (H&E stain, original magnification 10).

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Figure 41 Spindle cell-type uveal melanoma. In uveal melanoma of the spindle cell type, spindle-shaped cells with inconspicuous nucleoli (black arrow) are present (H&E stain, original magnification 600 ).

Figure 42 Spindle cell-type uveal melanoma. Uveal melanoma of the spindle cell type shows spindle-shaped cells with pink cytoplasm. Melanin pigment may be seen as fine granular deposits in the cytoplasm of many cells (black arrow) (H&E stain, original magnification 600).

detected in approximately 50% of tumors. The presence of monosomy 3 may be determined by karyotyping using fluorescence in situ hybridization (FISH) on cultured cells or by FISH analysis of tissue sections or cells obtained by fine-needle aspiration biopsy (Figure 48). Long-term studies have shown that almost 70% of patients with monosomy 3 in the primary tumor die of metastases within 4 years after the initial diagnosis, while tumors with normal chromosome 3 status rarely give rise to metastatic disease. This finding led to the development of a new tumor classification scheme, class 1 (disomy 3) and class 2 (monosomy 3). Similar cytogenetic changes are found in hepatic metastatic lesions. Two regions of deletion overlap on chromosome 3, UVM1 and UVM2, providing information

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Figure 43 Uveal melanoma of epithelioid cell type. The epithelioid cells are discohesive with large, round nuclei, prominent nucleoli (white arrow) and abundant eosinophilic cytoplasm (black arrow) (H&E stain, original magnification 600).

Figure 44 Malignant melanoma, mushroom growth pattern. Large malignant melanomas may rupture through Bruch’s membrane creating a mushroom-like growth pattern appearance (black arrows). In general, large tumors are predominantly of epithelioid cell type and can show melanin pigmentation, prominent mitotic figures, vascular loops, and lymphocytic infiltration, all poor prognostic indicators. Use this still figure to aid you in the use of (Virtual Microscopy Slide 4 eSlide: VM00280) of this article (H&E stain, original magnification 10).

on the location of the suppressor genes in uveal melanoma. Trisomy of 6p appears to be mutually exclusive with chromosome 3 monosomy and links with a better prognosis. Conversely, 6q deletion occurs more commonly in metastasizing tumors. A further poor prognostic factor, strongly associated with metastatic death, is 8q trisomy (also in form of 8q isochromosome), which usually appears later in the natural

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history of uveal melanoma. Other less frequent chromosome abnormalities reported in uveal melanoma include 1p and 13q monosomy and trisomy 21.

Treatment and Prognosis The two most commonly used treatments of uveal malignant melanoma are radiotherapy and enucleation (removal of the eye). The most frequently used system for delivering radiation is brachytherapy (plaque therapy), which involves the temporary attachment of a shielded metallic plaque of radioactive material (usually iodine-125) to the sclera beneath the tumor for a period of a few days. Other methods include proton-beam charged particles and gamma knife. Radiation plaque therapy offers good tumor control, can often preserve

Figure 45 Malignant melanoma, mixed cell type. Mixed uveal melanoma is composed of two cell types: spindle and epithelioid cells. Spindle cells contain nuclei that are elongated. The nucleoli are usually inconspicuous (black arrow). Epithelioid cells are larger than spindle cells and contain abundant cytoplasm and prominent round central nucleoli (white arrow) (H&E stain, original magnification 600).

useful vision, and demonstrates a systemic prognosis that is comparable to that of enucleation. Enucleation remains the standard of care for most large melanomas of the choroid and ciliary body if there is no hope of preserving useful vision. Despite earlier diagnosis and treatment modalities available in the treatment of intraocular melanomas, the prognosis has remained mostly stable. The prognosis of iris melanoma is generally good with incidence rates of metastasis reported to be 2.4–5.0%. The treatment is most commonly local resection although plaque radiotherapy (brachytherapy) and enucleation are occasionally utilized especially among cases that exhibit a diffuse growth pattern and when associated with ipsilateral glaucoma. The Collaborative Ocular Melanoma Study (COMS) has helped define most of what is known about the prognosis and treatment of intraocular melanomas. In this study, choroidal melanomas were categorized according to their size as small, medium, or large. Small choroidal melanomas are defined as those tumors that are between 1 and 3 mm in thickness and between 5 and 16 mm in maximum basal tumor diameter (Figures 49 and 50 and Virtual Microscopy Slide 1 eSlide: VM00277 and Virtual Microscopy Slide 3 eSlide: VM00279). Medium tumors are defined as those with a thickness of between 2.5 and 10 mm with an MBTD of less than 16 mm (Figure 51). Finally, large tumors are those with a thickness greater than 2 mm when the MBTD is at least 16 mm or those of any MBTD whose thickness is at least 10 mm or any tumor within 2 mm of the optic disk with a thickness greater than 8 mm (Figure 40). Among definitive small choroidal melanomas (not suspicious nevi), the COMS reported an all-cause 5-year mortality of 6% and a melanoma-associated mortality of 1%. Among these small tumors, 11% will grow sufficiently to qualify them for a new size category by 1 year, 21% by 2 years, and 31% by 5 years. Increase in thickness greater than 2 mm, the presence of orange pigment, subretinal fluid, any symptoms associated

Figure 46 Comparison of various cell types encountered in malignant melanomas. The epithelioid cell type is composed of round cells that have distinct borders (black arrow) and centrally located round nuclei and prominent nucleoli (white arrow) ((a) H&E stain, original magnification 600 ). The spindle cell type is composed of oval- to spindle-shaped cells that contain small, flat nuclei and inconspicuous nucleoli (white arrow). Melanin pigment may be seen within the cytoplasm of some cells (black arrow) ((b) H&E stain, original magnification 600). The mixed type is made of a mixed population of epithelioid (white arrow) and spindle cells (black arrow) ((c) H&E stain, original magnification 600 ).

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Figure 47 Invasive choroidal melanoma. In choroidal malignant melanoma, tumor cells (black arrows) invade through the sclera (white arrow) into the orbit (H&E stain, original magnification 100).

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Figure 49 Small size choroidal melanoma. Melanomas may be associated with prominent vascularity (black arrow). Commonly, the retina is artifactually detached due histological preparation (thin black arrow) (H&E stain, original magnification 10 ).

Figure 48 Fine-needle aspiration (FNA) biopsy of a choroidal melanoma. Malignant melanoma cells from an FNA biopsy of a choroidal melanoma (H&E stain, original magnification 600 ). Inset: Fluorescence in situ hybridization of a single cell with a cocktail of probes for the telomeric regions of chromosome 3 and the telomere of 22q. The colors represent the following chromosome arms: 3p-green, 3q-red, and 22q-yellow. The image shows 1 red, 1 green, and 2 yellow signals, consistent with monosomy for chromosome 3 (hybridization probe, original magnification 100).

Figure 50 Small-sized melanoma. Melanomas can present with various sizes (thick arrow). The optic nerve (asterisk) head is usually not involved by the tumor. The retina is located on the inner surface of the choroid and may be artifactually detached during tissue preparation for evaluation (thin black arrow). Use this still figure to aid you in the use of (Virtual Microscopy Slide 1 eSlide: VM00277) of this article (H&E stain, original magnification 10).

with the tumor, and tumor location adjacent to the optic disk have been shown to be factors that can predict subsequent tumor growth. The COMS also compared the mortality rates of persons with medium tumors who had undergone either enucleation or plaque radiotherapy. There was no difference in all-cause or melanoma-associated mortality at 12 years of follow-up. Among this group, 45% were alive and

cancer-free at 12 years. The importance of this report was support for a move toward globe-sparing therapies for appropriate tumors. Among large tumors, the COMS showed that there is no benefit to using external beam radiotherapy prior to enucleation. Large choroidal melanomas have the worst prognosis. At 10 years, only about 1/3 of patients treated appropriately for large choroidal melanoma are alive and cancer-free.

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Opthalmic Pathology | Ocular Tumors

DiCiommo, D., Gallie, B.L., Bremner, R., 2000. Retinoblastoma: the disease, gene and protein provide critical leads to understand cancer. Semin. Cancer Biol. 10, 255–269. Madhavan, J., Ganesh, A., Kumaramanickavel, G., 2007. Retinoblastoma: from disease to discovery. Ophthalmic Res. 40, 221–226. Pacal, M., Bremner, R., 2006. Insights from animal models on the origins and progression of retinoblastoma. Curr. Mol. Med. 6, 759–781. Conway, M.R., Wheeler, S.M., Murray, T.G., Jockovich, M.E., O’Brien, J.M., 2005. Retinoblastoma: animal models. Ophthalmol. Clin. North Am. 18 (1), 1–15. Classon, M., Harlow, E., 2002. The retinoblastoma tumour suppressor in development and cancer. Nat. Rev. Cancer 2, 910–917. Zhang, J., Schweers, B., Dyer, M.A., 2004. The first knockout mouse model of retinoblastoma. Cell Cycle 3, 952–959. Sastre, J., Chantada, G.L., Doz, R., Wilson, M.W., de Davila, M.T.G, Rodrı´guez-Galindo, C., et al., 2009. Proceedings of the consensus meetings from the International Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma. Arch. Pathol. Lab. Med. 133 (8), 1199–1202. Shields, C.L., Shields, J.A., 2010. Retinoblastoma management: advances in enucleation, intravenous chemoreduction, and intra-arterial chemotherapy. Curr. Opin. Ophthalmol. 21, 23–212. Dimaras, H., Kimeni, K., Dimba, E.A.O, Gronsdahl, P., White, A., Chan, H.S.L, et al., 2012. Retinoblastoma. Lancet 379, 1436–1446.

Figure 51 Choroidal melanoma. Melanomas of medium size (thick black arrow) can be associated with subretinal fluid accumulation (thin black arrow) and true retinal detachment. The lens (asterisk) may or may not be cataractous (H&E stain, original magnification 10).

In addition to the therapies described in the preceding text, laser photocoagulation, transpupillary thermotherapy, other forms of radiotherapy such as gamma knife, local resection, orbital exenteration, and systemic therapies have all been reported or are in development. If metastatic disease develops, they are most common to the liver. For this reason, yearly monitoring with serum-based liver function tests is an important part of the care of choroidal melanoma patients. There are no effective therapies for metastatic disease. The new frontier of choroidal melanoma treatment that may finally reduce mortality rates will likely be chemotherapeutic systemic therapies that exploit our evergrowing knowledge of the pathobiology of the disease. Clinical trials of new therapeutic agents have been initiated in patients with metastatic uveal melanoma as well as in the adjuvant setting after primary therapy. New systemic therapies using molecularly targeted agents are being tested in preclinical studies including inhibitors of Bcl-2, ubiquitin–proteasome, histone deacetylase, mitogen-activated protein kinase and phosphatidylinositol-3-kinase–AKT pathways, and receptor tyrosine kinases. Modifiers of adhesion molecules, matrix metalloproteinase, and angiogenic factors also have demonstrated potential benefit.

Melanoma Laver, N.V., McLaughlin, M.E., Duker, J.S., 2010. Ocular melanoma. Arch. Pathol. Lab. Med. 134, 1778–1784. Shields, J.A., Shields, C.L., 2008. Posterior uveal melanoma: clinical features. In: Shields, J.A., Shields, C.L. (Eds.), Intraocular Tumors. An Atlas and Textbook, second ed. Wolters Kluwer/Lippincott Williams and Wilkins, Philadelphia, pp. 86–115. Blanco, P., Marshall, J.C.A, Antecka, E., et al., 2005. Characterization of ocular and metastatic uveal melanoma in an animal model. Invest. Ophthalmol. Vis. Sci. 46 (12), 4376–4382. Van Raamsdonk, C.D., Klauss, G.G., Crosby, M.B., et al., 2010. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363, 2191–2199. Grossniklaus, H.E., Dithmar, S., Albert, D.M., 2000. Animal models of uveal melanoma. Melanoma Res. 10, 195–211. Yang, H., Fang, G., Huang, X., Yu, J., Hsieh, C.L., Grossniklauss, H.E., 2008. In-vivo xenograft murine human uveal melanoma model develops hepatic micrometastasis. Melanoma Res. 18 (2), 95–103. Font, R.L., Croxatto, J.O., Rao, N.A., 2006. Tumors of the eye and ocular adnexa. In: Silverberg, S.G. (Ed.), AFIP Atlas of Tumor Pathology, fourth ed. American Registry of Pathology Press, Washington, DC, pp. 155–214. Onken, M., Worley, L.A., Ehlers, J.P., Harbour, J.W., 2004. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 64 (20), 7205–7209. Van Raamsdonk, C.D., Griewank, K.G., Crosby, M.B., Garrido, M.C., Vemula, S., Weinsner, T., et al., 2012. Mutations in GNA11 in uveal melanoma. N. Engl. J. Med. 363 (23), 2191–2199. Triozzi, P.L., Eng, C., Singh, A.D., 2008. Targeted therapy for uveal melanoma. Cancer Treat. Rev. 34 (3), 247–258. The Collaborative Ocular Melanoma Study Group, 2006. COMS randomized trial of iodine 125 brachytherapy for choroidal melanoma. V. Twelve-year mortality rate and prognostic factors: COMS report n.28. Arch. Ophthalmol. 124 (12), 1684–1693. Patel, M., Smyth, E., Chapman, P.B., Wolchok, J.D., Schwartz, G.K., Abramson, D.H., et al., 2011. Therapeutic implications of the emerging molecular biology of uveal melanoma. Clin. Cancer Res. 17 (8), 2087–2100.

Further Reading

Relevant Websites

Retinoblastoma Font, R.L., Croxatto, J.O., Rao, N.A., 2006. Tumors of the eye and ocular adnexa. In: Silverberg, S.G. (Ed.), AFIP Atlas of Tumor Pathology. American Registry of Pathology Press, Washington, DC, pp. 48–62, 85–104. Shields, J.A., Shields, C.L., 2008. Intraocular Tumors: An Atlas and Textbook, second ed. Lippincott Williams & Wilkins, Philadelphia. Shields, C.L., Shields, J.A., 2006. Basic understanding of current classification and management of retinoblastoma. Curr. Opin. Ophthalmol. 17, 228–234.

http://atlasgeneticsoncology.org/Tumors/UvealmelanomID5047.html. http://www.cancer.gov/cancertopics/pdq/treatment/intraocularmelanoma/ HealthProfessional/page1. http://www.cancer.gov/cancertopics/types/retinoblastoma. http://www.cancer.net/all-about-cancer/genetics/genetics-melanoma. http://emedicine.medscape.com/article/1190564-overview. http://emedicine.medscape.com/article/1222849-overview. http://ghr.nlm.nih.gov/condition/retinoblastoma.