Materials and methods

CASE REPORT

In April 1983, a 65-year-old white woman noted a pigmented fleck on the lower nasal quadrant of her iris (Fig 1A). In December, gonioscopy (Fig 1B) and fluorescein angiography revealed a vascularised tumour of the ciliary body that was diagnosed as melanoma. It was circled with cryotherapy and removed a month later by iridocyclectomy. It was found to be a mixed cell type melanoma.

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Figure 1

Clinical and macroscopic findings of retinoinvasive uveal melanoma. (A) The initial pigmented tumour at the base of the iris. (B) The adjacent chamber angle is uninvolved. (C) Amelanotic episcleral nodules (n) are seen 9 years later, the largest is at the site of the iridocyclectomy (between arrows). (D) The optic nerve (on) and space between the pia (arrowheads) and dura (white arrow) are largely replaced by tumour; the episclera is dark from haemorrhage (haem; × 8). (E) Iris and ciliary body are replaced by tumour (arrowheads) that perforates the sclera through the iridocyclectomy flap (arrow) and along aqueous channels (double arrowhead). Note coloboma from iridocyclectomy (ic) and scars from cryocoagulation (cr) (× 5). (F) Scattered intraretinal tumour nodules (arrowheads) and small dark haemorrhages; note a larger nodule in front of the thickened optic nerve (double arrowhead), and blood under the inner limiting membrane (asterisk) (× 6).

The tumour margins were not all unequivocally free. Residual tumour could not be seen, however, and careful follow up only was recommended. Semiannual examinations revealed no recurrence. In 1989, the intraocular pressure rose and dispersed pigment was seen in the chamber angle. The pressure did not respond to medication or cyclocryotherapy. In 2 years the eye lost light perception.

In 1992, several lightly pigmented nodules appeared close to the limbus (Fig 1C). A biopsy was not diagnostic because of crush artefact. The nodules slowly increased in size and recurrent tumour was now seen in the ciliary body. A second biopsy in June 1992 confirmed recurrent melanoma. No tumour was seen in the posterior pole in repeated examinations, but visibility was poor.

The blind eye was enucleated with a wide conjunctival margin in August 1992. Unexpectedly, a large part of the retrobulbar optic nerve was black and tumour was found in the subarachnoid space (Fig 1D). The cut surface of the nerve was free of tumour. Histopathology confirmed the presence of a ring melanoma perforating the limbus. The patient declined suggested radiotherapy.

Computed tomography 1 year later revealed no evidence of orbital recurrence. A clinically suspicious, small pigmented conjunctival spot was excised in October 1993, revealing residual melanoma. In April 1997, the patient is without any clinical or radiological evidence of further local or systemic disease.

METHODS FOR HISTOPATHOLOGY

All excised tissues and the enucleated eye were formalin fixed and paraffin embedded. Routine stains included haematoxylin and eosin and van Gieson. The retinal pigment epithelium (RPE) was used as an internal positive control for cytokeratin (CK) 8 and 18,39vascular endothelial cells for vimentin, and retinal glia for glial fibrillary acidic protein (GFAP) and the HNK-1 carbohydrate epitope.40

Sections, 5 μm thick, were cut on chromium gelatin coated glass slides and immunostained with the avidin-biotinylated-peroxidase complex method (Vectastain ABC Elite Kits, mouse and rabbit IgG, Vector Laboratories, Burlingame, CA, USA) as has been described.3940 To enable evaluation of the positive immunoreaction in pigmented cells, 3,3′-diaminobenzidine tetrahydrochloride was used as chromogen.39 Melanin was then bleached by incubating the sections with 3.0% (v/v) hydrogen peroxide and 1.0% (w/v) disodium hydrogen phosphate for 18 hours at room temperature.41

Primary mouse monoclonal antibodies (Mab) CAM 5.2 to CK 8 (IgG2a, Lot P0522, Becton Dickinson, Mountain View, CA, USA, diluted 1:2),39 CY-90 to CK 18 (IgG1, Lot 70H-4866, Sigma, St Louis, MO, USA, 1:3000),39 Vim 3B4 to vimentin (IgG2a, Lot 11454324-01, Boehringer Mannheim, Mannheim, Germany; 1:50), and HNK-1 to the HNK-1 carbohydrate epitope (Anti-Leu-7, IgM, Lot 30015, Becton Dickinson, 1:25) were obtained commercially. Rabbit antisera to GFAP (Lot 119, 1:1000) and S-100 protein (Lot 026, 1:1000) were bought from Dakopatts (Glostrup, Denmark).

The sections were pretreated with 0.4% (w/v) pepsin to reduce background and to enhance the intensity of specific staining when antibodies to intermediate filaments were used.3940Omitting the primary or secondary antibody or the ABC complex resulted in loss of all immunoreaction. Sections stained with different antibodies of the same isotype were carefully compared to exclude non-specific binding of antibodies representing that isotype, and normal rabbit serum was used as a negative control with the polyclonal antisera.

LITERATURE SURVEY

A Medline search using the keywords melanoma, retina, vitreous, and optic nerve was conducted for the years 1962–96. Hand searching of older reports was done using the papers found, textbooks, and several indices as starting points. Pertinent original papers were reviewed for data on retinal and optic nerve invasion.

Results

HISTOPATHOLOGY

The iridocyclectomy specimen was a 6 × 8 mm deeply pigmented tumour with adjacent sclera. Pigmented, non-cohesive epithelioid cells and large and plump spindle cells were present (Fig 2A). Where the tumour infiltrated the base of the iris, the cells were smaller and more uniform in shape. Areas of necrosis and haemorrhage from preceding cryotherapy were found (Fig 2A). The tumour reacted for S-100 protein and for vimentin with Mab Vim 3B4, but not with Mab CAM 5.2 and CY-90 to CK 8 and 18.

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Figure 2

Evolution of the ciliary body melanoma (A, C–H, haematoxylin and eosin; B, I, immunoperoxidase). (A) The initial tumour contains pigmented, pleomorphic epithelioid cells (× 340). (B) The limbal nodule consists of small epithelioid cells that react with MAb Vim 3B4 to vimentin (× 360). (C) The iris and anterior ciliary body are replaced by spindle cell melanoma (× 380). (D) The tumour (arrowhead) grows as a sheet on the ciliary epithelium (ce), but the ciliary muscle (cm) and stroma are uninvolved (× 250). (E) The tumour (arrowheads) grows in the anterior vitreous (vit) (× 210). (F) It extends along the hyaloid membrane (arrowhead) to the vitreous base (vb) and invades the peripheral retina (ret) (× 140). (G) It fills the spaces between the septa (s) of the optic nerve normally occupied by nerve fibre bundles (× 280). (H) The residual orbital tumour consists of epithelioid cells with eccentric, often semilunar nuclei (× 450). (I) Some are labelled with MAb CY-90 to CK 18 (× 480).

The second biopsy of a limbal nodule, 3 mm in size, contained smaller and more cohesive, irregularly polygonal cells that largely lacked pigment, and reacted for S-100 protein and for vimentin (Fig 2B), but not for CK 8 and 18.

The enucleated eye measured 23 × 24 mm. Variably pigmented limbal nodules were present, the largest were 4.5 and 6 mm in size. The optic nerve stump was 13 mm long and abnormally dark and thick, measuring 5 mm in diameter (Fig 1D). On sectioning the eye, a ring melanoma of the ciliary body and iris (Fig 1E), multiple retinal haemorrhages, and pale tumour nodules over and within the retina were noted (Fig 1F). The ring melanoma consisted of non-pigmented spindle cells (Fig 2C) unlike its iridocyclectomised predecessor (Fig 2A). They reacted for S-100 protein and for vimentin, but no unequivocal labelling for CK 8 and 18 was present.

Tumour cells extended as a sheet along the endothelium of the peripheral cornea and adhered as aggregates to the central endothelium. They perforated the sclera at multiple sites through the iridocyclectomy flap and via aqueous outflow channels (Fig 1E). The tumour replaced the pars plicata and the anterior pars plana, but did not involve the posterior pars plana or choroid (Fig 2D). Instead, it grew along the ciliary epithelium (Fig 2D) and detached to the vitreous (Fig 2E) that had guided cells to peripheral retina (Fig 2F). The vitreous base was uninvolved.

Most of the optic nerve was replaced by melanoma (Fig 2G). Only scattered myelinated axons remained. The tumour extended to the perineural space. It did not react for CK 8 and 18. The later orbital biopsy contained a small focus of melanoma (Fig 2H). The cells were epithelioid in type with abundant eosinophilic cytoplasm and often semilunar nuclei that were pushed to one side (Fig 2H). Little evidence of infiltration of surrounding tissues and no mitoses were found. Many cells reacted for CK 8 and 18 (Fig 2I), respectively, in addition to S-100 protein and vimentin.

Mostly spindle-shaped tumour cells extensively invaded the neuroretina. They grew as variably thick epiretinal membranes (Fig 3A), under the inner limiting membrane (Fig 3B), along the posterior hyaloid face (Fig3C), formed nodules within the retina (Fig 3D,E), and eroded up to Bruch’s membrane (Fig 3F). They invaded the retina preferably close to blood vessels and formed perivascular collars (Fig 3E) and single files on either side of inner limiting membrane (Fig 3B). Many intraretinal tumour cells reacted for CK 8 and 18 (Fig 3G) in addition to vimentin and S-100 protein. The retina showed secondary gliosis, cystic oedema, intraretinal haemorrhages (Fig 3C,D), and a haemorrhagic detachment of the inner limiting membrane (Fig 1F). Antibodies to GFAP and the HNK-1 carbohydrate epitope (Fig 3H) revealed that little if any mixing of neoplastic cells and retinal glia had occurred.

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Figure 3

Invasion of the retina by uveal melanoma (nf, nerve fibre; ip, inner plexiform; and pr, photoreceptor layer; A–F, haematoxylin and eosin; G, H, immunoperoxidase). (A) Epiretinal melanoma (arrowhead) and intraretinal haemorrhages (haem) are seen (× 140). (B) Invading melanoma cells (double arrowhead) align under the inner limiting membrane (arrowheads) and grow as nodules (arrow) among intraretinal haemorrhages (haem) (× 230). (C) The tumour also grows along the posterior hyaloid membrane (× 280). (D) Nodular and diffuse infiltration of the neuroretina (× 230). (E) Tumour grows around blood vessels (v) within the retina (× 280). (F) It replaces the RPE (double arrowhead) but spares Bruch’s membrane (arrowheads); dr, drusen (× 340). (G) Melanoma cells (mm) react with MAb CY-90 to CK 18 (× 420). (H) They are not labelled for the HNK-1 epitope that highlights displaced retinal glia (ret) instead (× 230).

Discussion

Invasion through the vitreous and retina into the retrobulbar optic nerve of a ciliary body melanoma is unique. Nevertheless, we were able to identify three almost identical tumours, the first one reported by Terry in 1940, with a systematic literature survey (Table 1). A ring melanoma of the ciliary body, uninvolved choroid,232529 invasion of anterior sclera232529 and secondary glaucoma,2529 retrocorneal tumour growth,25vitreous seeding,25 invasion of retina preferably adjacent to vessels,2325 intraretinal haemorrhages,232529 growth on the optic disc2325 with invasion of the retrobulbar nerve232529 and perineural space,25 and eventual loss of light perception2529 were common to them. This phenotype, characterised in particular by widespread invasion of non-adjacent retina and optic nerve, could aptly be designated retinoinvasive.

Many uveal melanomas that invade the optic nerve, including most retinoinvasive ones (Table 1), share a set of features postulated by Spencer.29 They contain non-cohesive epithelioid or necrotic cells.310182325293538 They often spread to the optic nerve through the vitreous rather than by direct continuity.102529 However, direct optic nerve invasion by tumours originating adjacent to the optic disc is also rather common.35182736 Finally, secondary angle closure glaucoma31025293538 diverts aqueous with neoplastic cells posteriorly and weakens the optic nerve by causing ischaemia and oedema.29 Loss of light perception alerts the clinician to optic nerve invasion.31025293538 These factors do not, however, explain invasion of non-adjacent retina.

Spencer mentioned that tumour cells invading the optic nerve might specifically adhere to it.29 Today one would give even more emphasis to properties of tumour cells and of tissues they invade rather than to mechanical factors.42-45 As in metastatic cascade, retinoinvasive tumour cells must detach from their primary tumour by altering their interaction with the extracellular matrix.4243 They must adhere to the inner limiting membrane, digest it, migrate into the retina, and proliferate within it.42-44

Metastases probably start when tumour cells bind to the vascular endothelium of susceptible organs.4445 This is how occasional cutaneous melanomas will metastasise to the neuroretina.46-50 Further growth depends on interaction with the local microenvironment and on natural immunity of the host tissue.43-45 In the present case, single file formation along the inner limiting membrane is indicative of binding to the retina. In contrast, the choroid seems to form a barrier which many ring1 and all retinoinvasive melanomas so far reported have been unable to trespass.232529 Intriguingly, electron microscopy and immunohistochemistry have recently revealed uveal compartments not visible by routine light microscopy4051 that might influence tumour invasion.

Interestingly, whereas Mab CAM 5.2 and CY-90 to CK 8 and 18 did not label the initial ciliary body tumour and the ring melanoma, many intraretinal cells were immunoreactive. Perhaps retinal microenvironment had induced cytokeratins, or a tumour cell clone that expressed them was especially capable of invading the retina. On balance, every other primary uveal melanoma contains some cells that react for CK 8 and 18.52

Because random mutations are responsible for tumour progression,4243 uveal melanomas should be able to acquire either some or all of the above properties. If so, some should adhere to and migrate on the retina, but fail to invade into it. Tumours that have negotiated these two steps are indeed on record.1812-17 Like retinoinvasive melanomas,2325 they have often grown adjacent to retinal vessels1415 because nutrition here is better or resistance to invasion is weaker. Only limited infiltration into the superficial retina has been noted,1131416 unlike in retinoinvasive melanomas.

Retinoinvasive melanomas spread widely within the eye, which suggests a relation with diffuse uveal melanomas: ring melanomas of the ciliary body and flat choroidal tumours involving at least one fourth of the uvea.111226 However, diffuse choroidal melanomas that have shed cells to the vitreous cavity and onto the inner limiting membrane apparently did not invade the retina512 and thus did not exhibit retinoinvasive properties. The histopathology in our case suggests an origin from a spindle cell ring melanoma that spawned a rapidly growing epithelioid tumour and then took a retinoinvasive phenotype. Much less likely, residual cells from a circumscribed epithelioid cell tumour developed into spindle cell retinoinvasive ring melanoma. The other retinoinvasive melanomas apparently also evolved from a ring melanoma (Table 1).

Time is probably important for the development of the retinoinvasive phenotype. Analogous to many diffuse melanomas,1112262837 a long delay in diagnosis and, apparently, slow growth are typical.232529 The tumour has time to spread locally, to cause secondary glaucoma that facilitates posterior dissemination, and to develop unusual clones of neoplastic cells.813 Two retinoinvasive tumours proved fatal, one from intracranial growth of an orbital recurrence 7 years after enucleation indicating slow growth,23 and the other from metastases 1 year after enucleation.2529 Thirteen years after iridocyclectomy and 5 years after enucleation our patient is alive without evidence of recurrence.