Chapter 142 Pathology of Choroidal Melanoma
The diagnosis of choroidal melanoma is made clinically by fundus examination and ultrasonography. In equivocal cases, a tumor may be biopsied and evaluated by an ophthalmic pathologist. Examination of cytologic features and immunohistochemical stains, provided there is a sufficient amount of tissue, allows for a reliable diagnosis. Gross and microscopic examination of enucleated eyes harboring a uveal melanoma provides relevant prognostic information, e.g., cell type, invasion of vortex veins, and extraocular growth. Implementation of the American Joint Committee on Cancer (AJCC) and the International Union Against Cancer (UICC) TNM classification in the pathology report allows for uniform staging. Assessment of complications due to brachytherapy such as radiation retinopathy and plaque failure with subsequent tumor growth may lead to improvements in clinical care. Increased knowledge of the molecular genetic aspects of choroidal melanoma including tests such as chromosome 3 status and gene expression profiling provides clinically relevant information. All in all, close cooperation between ophthalmic oncologists and pathologists within the framework of scheduled tumor conferences optimizes patient care. This chapter describes the pathologic findings in choroidal melanoma.
For routine pathologic examination, tissue specimens are fixed in paraformaldehyde or formaldehyde (4–10%) immediately after surgery. As formalin diffuses at a rate of approximately 1 mm/hour, an entire globe (about 24 mm in diameter) should be fixed at least 12 hours prior to further processing. If tissue is going to be sent for molecular genetic analysis, an appropriate amount of material should be obtained prior to formalin fixation, though testing of formalin-fixed paraffin-embedded tissue is also possible.
Different types of tissue obtained include cytologic material, a tumor biopsy (incisional or excisional), or an enucleated globe. Clinical information and the type of fixative are noted. Gross examination depends on the submitted specimen:1–4 For cytologic material, the submitted material (e.g., number of slides, quantity and appearance of fluid) is registered. For tumor biopsies, the number of pieces, size (three dimensions if possible), and descriptive features (e.g., color) are noted. A globe should be evaluated according to the guidelines of the Association of Directors of Anatomic and Surgical Pathology.1–4 After orientation of the globe, careful inspection for extraocular growth is followed by transillumination to localize the tumor and to measure its basal diameter. Vortex veins are submitted in different cassettes labeled respective to their localization. A standard section of the globe incorporates the pupil-optic nerve (p-o) section and the tumor. Detailed macroscopic examination and photodocumentation of the entire globe as well as a thorough description (including evidence of previous treatment) and tumor measurements are recommended.
Routine stains for choroidal melanoma comprise hematoxylin and eosin (H&E), periodic acid Schiff (PAS), and bleached H&E stains, to reveal the cytologic features in heavily pigmented tumors (Fig. 142.1). Immunohistochemical stains are not routinely used for the diagnosis of uveal melanoma but may be helpful in selected cases including fine-needle aspiration biopsy (FNAB) samples.
Fig. 142.1 Choroidal melanoma (molecular profile class I) in a 43-year-old woman. (A) On gross examination, a heavily pigmented uveal melanoma is present. (B) The extracellular matrix pattern of the tumor with vascular loops and arches is detected with a PAS stain (PAS, ×4). (C) The uveal melanoma consists of spindle cells and epithelioid cells (mixed-cell type) and exhibits pigmentation in uveal melanoma cells and pigment-laden macrophages (H&E, ×40). (D) Bleached sections enable the evaluation of the cytologic features (bleached H&E, ×40).
The gross examination of intraocular tumors is the basis for clinicopathologic correlations as it allows for a detailed macroscopic evaluation and measurement of the tumor size including the prognostically relevant largest basal diameter.5,6 The largest basal diameter can be reliably assessed on gross examination, although the tumor size may be underestimated when measured after fixation due to tumor shrinkage.7 Choroidal melanomas exhibit an oval or fusiform shape (Fig. 142.2) when confined by Bruch’s membrane or a collar-button/mushroom configuration (Fig. 142.3) when the tumor has broken through Bruch’s membrane.
Fig. 142.2 (A) Oval-shaped, heavily pigmented choroidal melanoma (molecular profile class I) in a 66-year-old man. (B) Histologically, a mixed-cell type melanoma (asterisk) is present within the uvea (H&E, ×40). Bruch’s membrane (arrow) is intact, the overlying RPE and retina display atrophic changes.
Ocular structures may be invaded by choroidal melanomas. Invasion into the ciliary body may be detected macroscopically, whereas invasion into the sclera, retina, and optic nerve as well as invasion into the vitreous, iris, and anterior chamber is usually detected by microscopic examination. Extraocular extension (Fig. 142.4) usually occurs along vortex veins or emissary canals and, dependent on the extent, may be diagnosed clinically or on gross examination.
Fig. 142.4 An 86-year-old woman presented with (A) extraocular extension of a uveal melanoma (molecular profile class II). (B) On gross examination, extraocular extension (arrow) of a mildly pigmented bilobulated tumor was detected that invades the choroid and the ciliary body. (C) Histologic examination (H&E, ×10) shows the extraocular part of the tumor (asterisk) composed of epithelioid cells. The patient died from liver metastasis less than 1 year after enucleation.
The amount of pigmentation of uveal melanomas varies from tumor to tumor or even within a single tumor and may be related to overall survival.5,8 Most tumors exhibit mild to moderate pigmentation, although heavily or non-pigmented (amelanotic) choroidal melanomas (Fig. 142.5) may be observed.
The prognosis of choroidal melanoma is based on clinical, histologic, and genetic parameters (see Chapter 141, Molecular genetics of choroidal melanoma). Some parameters such as ciliary body invasion, extraocular extension and the largest basal diameter are incorporated into the TNM classification.9 Histopathologic examination allows for further characterization of the tumor including its cytologic features. In FNAB specimens, the cell type is more difficult to determine than in enucleated eyes or completely excised tumors due to the scarce amount of tissue and artifacts (Fig. 142.6).
Choroidal melanomas usually arise de novo but may also arise from a nevus or a melanocytoma (magnocellular nevus). In these cases, remnants of the underlying nevus consisting of spindle A cells (spindle cell nevus (Fig. 142.7)) or, extremely rare magnocellular nevus cells (melanocytoma10,11), may be histologically detected.
Fig. 142.7 (A) Choroidal melanoma arising in a choroidal nevus (H&E, ×4). There is vitreous spread of melanoma cells present (arrow and inset). (B) Higher magnification reveals nevus cells (spindle A cells, arrow) at the base of the choroidal lesion (H&E, ×10). Some of these cells are pigmented. The RPE (arrowhead) shows degenerative changes and underlying drusen and exudates.
First developed by Callender in 1931,12 the so-called “Callender classification” distinguished five types of uveal melanomas depending on the most prominent cell type: spindle cell subtype A; spindle cell subtype B; epithelioid type; fascicular type, and mixed cell type (consisting of mixtures of spindle and epithelioid cells) of uveal melanoma.
Addressing the disagreement about the interpretation and application of Callender’s classification, a “modified Callender classification” was introduced by McLean and associates in 1983, comprising three different types in uveal melanoma based on cell morphology13: spindle cell melanoma (composed of spindle B cells); epithelioid cell melanoma, and melanoma of mixed-cell type. Spindle A cells were no longer regarded as malignant cells and were recognized as benign nevus cells. In addition, a new cell type was introduced, the small epithelioid cell (intermediate cell), that was smaller than the Callender’s epithelioid cell and exhibited features intermediate between spindle B and epithelioid cells.
The classification of uveal melanomas into either spindle cell (composed of spindle B cells), mixed-cell type (most common), or epithelioid cell still is now widely accepted. The main cell types are depicted as follows:
• Spindle A cells are fusiform, cohesive cells with poorly defined cell borders. The nucleus is also spindle-shaped and its central fold can be seen light microscopically as a dark stripe (Fig. 142.8A). This cell type is regarded as a benign nevus cell, but can be observed in uveal melanomas arising from a nevus.
• Spindle B cells are also fusiform and cohesive with poorly defined cell borders but are plumper than spindle A cells. Prominent nucleoli are detected in the spindle-shaped nucleus (Fig. 142.8B). This is the most common cell type in choroidal melanoma.
Fig. 142.8 (A) Fusiform spindle A cells (representing benign nevus cells) with a central fold are depicted (arrow) (H&E, ×40). (B) Spindle B cells that are plumper than spindle A cells exhibit a prominent nucleolus (arrow) (H&E, ×40). (C) Epithelioid cells characteristically contain a round, large nucleus with a prominent nucleolus (arrow) (H&E, ×40). (D) Small epithelioid cells (intermediate cells) represent a cell type intermediate between spindle B and epithelioid cells (arrow) (H&E, ×40). Mitotic figures are also present (arrowhead).
Although not incorporated into the TNM classification, the cell type of uveal melanomas is also an important prognostic parameter.6 Melanomas solely composed of spindle cells have a more favorable prognosis than mixed-cell type tumors. Epithelioid cell melanomas are associated with monosomy 3 and class 2 molecular profile, a higher metastatic and mortality rate – and thus displays the worst prognosis.6,14,15
Evaluation of the number of mitotic figures per high-power field in the tumor cells (Fig. 142.8D) is part of the routine evaluation of choroidal melanomas. It serves as an indicator for the proliferative activity and is thus a prognostic parameter.6
Morphometry (measurement of the largest nucleoli using a digital filar micrometer) as well as measurements of the nuclear DNA ploidy (DNA content) by image cytometry of Feulgen stained sections, are historical approaches to establish criteria with regard to long-term survival in patients with uveal melanoma.6,16 These tests were replaced by newer approaches such as analysis of the chromosome 3 status15 and gene expression profiling.14
The most important immunohistochemical markers expressed by the tumor cells are HMB45, S100, Melan A, MITF, and tyrosinase.17–21 HMB 45 (Fig. 142.9) and Melan A are melanocytic markers. While Melan A stains melanocytes in general, HMB45 is predominantly expressed in “activated” melanocytes and is therefore more suggestive of malignant melanocytic lesions.21,22 S100 is expressed in different types of cells including melanocytes and very often used in combination with HMB45 as a marker for uveal melanoma.21 Microphthalmia transcription factor (MITF) is essential for the development and survival of melanocytes and therefore expressed in various melanocytic lesions including uveal melanoma.18–20 Tyrosinase is an enzyme that is involved in the metabolism of melanocytes and was recently introduced as a melanoma marker.17 At least two markers (e.g., HMB45 and S100) should be employed for the diagnosis of uveal melanoma if the diagnosis cannot be made on histologic features alone.
Ki67 antigen – a proliferation marker expressed in the nucleus – is suitable to detect the proliferative activity in tumors and has prognostic relevance.23,24 Immunohistochemical stains for Phospho-Histone H3 Ser10 (PHH3) may help to detect mitotic figures in uveal melanomas.25
The different cell types in choroidal melanoma such as spindle A, spindle B, and epithelioid cells can also be differentiated by transmission electron microscopy although the ultrastructural differences between the cell types are not clear-cut.26
Spindle A cells appear elongated and show spindle-shaped nuclei with marked indentations and an unsuspicious nucleolus. In the cytoplasm, there are cell organelles present such as mitochondria, a relatively small number of free ribosomes, rough-surfaced endoplasmic reticulum (RER), largely immature melanin granules (premelanosomes), and numerous cytoplasmic filaments.26
The shape of spindle B cells (Fig. 142.10A) is similar to spindle A cells except they have a plumper cell body. The nucleoli of spindle B cells are also larger and show a reticular pattern. While spindle B cells have more RER and free ribosomes in their cytoplasm, the number of cytoplasmic filaments is decreased compared to spindle A cells.26
Fig. 142.10 (A) Transmission electron microscopy shows a spindle-shaped cell with a lot of rough-surfaced endoplasmic reticulum (RER) in the cytoplasm (×1900). (B) The reticulated and slightly indented nucleus is also spindle-shaped and contains a nucleolus. There are a few melanin granules (empty vacuoles) present. Epithelioid cell with cell organelles within the scanty cytoplasm (×5800). The round nucleolus is reticulated and exhibits a slight indentation.
The largest nucleolus is found in the round to polygonal epithelioid cells (Fig. 142.10B) and exhibits a reticular pattern. The nucleolus of the epithelioid cells can have indentations but they are less prominent than in spindle cells. Many cell organelles, in particular mitochondriae – some of them exhibiting a bizarre form and structure – and free ribosomes, but only a few filaments, are present in the cytoplasm.26
In summary, the ultrastructural features are consistent with the present consensus that the cellular malignancy increases from spindle A (nevus cells) through spindle B to epithelioid types of melanoma cells in conjunction with an increasing number of cell organelles (e.g., mitochondriae, free ribosomes) and increasing size of nucleoli that are regarded as signs of activity.26
As choroidal melanomas metastasize hematogenously (Fig. 142.11), much attention has been paid to “intratumoral vessels and vascular-like structures” – nine different morphologic patterns can be distinguished: normal, silent, straight, parallel, parallel with crosslinking, arcs, arcs with branching, closed vascular loops (large “vessel” occluded by tumor cells), vascular networks (at least three back-to-back closed vascular loops) (Fig. 142.12).27 These vascular-like structures have been later shown to reflect fibrovascular septae rather than microvasculature leading to the term “vasculogenic mimicry”.28,29 The presence of these extravascular matrix patterns, in particular loops, that are identified by a PAS stain is associated with death from metastatic melanoma.27,30 Microvascular density (MVD) itself has been identified as prognostically relevant,27,31 and the presence of tumor cells in intratumoral blood vessels is also regarded as a factor for unfavorable outcome.32
Fig. 142.11 (A) Liver metastasis of a choroidal melanoma with basophilic melanoma cells (arrow) next to hepatocytes (arrowhead) (H&E, ×40). (B) The primary tumor consists predominantly of epithelioid cells (arrow) (H&E, ×40) and exhibits extraocular extension (not shown).
Fig. 142.12 (A) Choroidal melanoma with extracellular matrix patterns such as arches (arrow) (PAS, ×4) and (B) loops (arrow) (PAS, ×4). (C) Vascular channels within the tumor are also present (arrow) (PAS, ×4).
A high number of tumor-infiltrating/associated lymphocytes (TILs/TALs) (Fig. 142.13) is associated with a more aggressive behavior and a higher risk of metastasis than a low number of TILs33 – and so are tumor-infiltrating/associated macrophages (TIMs/TAMs).6,34 In particular, M2 macrophages that exhibit proangiogenic and anti-inflammatory characteristics (in contrast to M1 macrophages with antibacterial and antiangiogenic features) are linked to increased microvascular density, ciliary body involvement, monosomy 3 and thus, a worse prognosis for survival.35,36
Fig. 142.13 (A) Choroidal melanoma infiltrated by pigmented macrophages (arrow) (H&E, ×4). (B) Higher magnification shows pigment-laden macrophages (arrow) (H&E, ×40). (C) An immunohistochemical double-stain for CD68 and CD163 reveals M2-macrophages (inset; CD68–CD163 double staining, ×40). Lymphocytes are also present within the tumor (arrow) intermixed with epithelioid cells (arrowhead) (H&E, ×40).
The degree of pigmentation varies between different tumors and also within one tumor. Tumors cells as well as pigment-laden macrophages (that are typically larger than tumor cells) contribute to the degree of pigmentation that is roughly classified into amelanotic, mildly, moderately, and heavily pigmented (Figs 142.1, 142.5). In heavily and moderately pigmented tumors, bleaching is mandatory in order to analyze the cytologic features. The degree of pigmentation in comparison with other features only has weak prognostic value.5,6
“Melanoma-associated spongiform scleropathy (MASS)” is a degenerative, noninflammatory process in the sclera underlying the tumor that occurs in 38% of enucleated eyes harboring uveal melanoma.37 MASS is associated with age and basal tumor diameter (extent of direct contact between tumor and sclera) but not with long-term survival.38 A high incidence of MASS is found in eyes with scleral invasion and extrascleral extension as altered sclera collagen may allow for tumor invasion. On gross examination, whitish spindle-shaped areas within the sclera adjacent to the tumor may be detected.37 Degraded collagen fibers and glycosaminoglycan accumulation leading to the typical picture of spongiotic areas surrounded by feathery fragmented collagen fibers are microscopically observed (Fig. 142.14).37
Choroidal melanomas may invade into several ocular tissues: Most uveal melanomas invade into the sclera (Fig. 142.15). Continuous horizontal growth may lead to invasion of the adjacent ciliary body and is associated with a worse prognosis. Choroidal melanomas invading into the ciliary body do not necessarily also invade into the iris and tumor growth is usually restricted by the reticulum of Müller. Once the tumor has broken through Bruch’s membrane it may invade into the retina but rarely gains access to the vitreous cavity with vitreous spread (Fig. 142.16).39 Melanoma cells in the anterior chamber and trabecular meshwork are rarely observed in choroidal tumors that do not continuously invade into the ciliary body and iris (Fig. 142.16). Peripapillary uveal melanomas may invade into the optic nerve head, but rarely extend far retrolaminary (Fig. 142.17).6
Fig. 142.15 (A) Overview of a 48-year-old man’s eye harboring a mushroom-shaped choroidal melanoma (H&E). (B) Scleral invasion of the mixed-cell type tumor (arrow) is present and associated with MASS.
Fig. 142.16 (A) Partially necrotic choroidal melanoma (s/p brachytherapy) in an 80-year-old female patient (H&E, ×4). (B) Tumor cells are present adjacent to the zonules (arrow) (H&E, ×10). (C,E) Superficial retinal invasion can be observed (arrows) (C, H&E, ×10; E, H&E, ×40). (D) The chamber angle and the iris are also non-continuously infiltrated by tumors cells (H&E, ×4). Higher magnification of the cells in the chamber angle exhibits choroidal melanoma cells and macrophages (inset).
Fig. 142.17 (A) Peripapillary choroidal melanoma compressing the optic nerve head (asterisk) (H&E, ×4). (B) There are islands of tumor cells in an emissary canal (arrow) adjacent to the optic nerve (asterisk) (H&E, ×10). (C) The tumor is composed of spindle cells and epithelioid cells (H&E, ×40).
Extraocular extension occurs most frequently along emissary canals which include ciliary nerves and vascular channels, in particular vortex veins, as well as aqueous channels (Fig. 142.18).40 Extension via the optic nerve or through iatrogenic wounds has also been described.40–42 Extensive orbital extension is rarely observed (Fig. 142.19). Histologic examination of step sections of the globe and vortex veins enables pathologists to issue reliable reports that include these prognostically important parameters. Extraocular extension of uveal melanoma is associated with a significant risk for liver metastases and an increased local recurrence rate.43
Fig. 142.18 (A) Gross appearance of a vortex vein invaded by a mixed-cell type choroidal melanoma (molecular profile class II). (B) Tumor cells (asterisk) are detected in the lumen of a vortex vein (arrow) (H&E, ×10).