PEER REVIEWED

The Role of Biopsy in the Assessment of Uveal Melanoma

Its value can vary according to the methodology used and the diagnostic criteria applied.

Mary E. Aronow, MD • Charles V. Biscotti, MD • Chi-chao Chan, MD • Arun D. Singh, MD

Uveal melanoma is the most common primary intraocular malignancy in adults and has an incidence of 5.1 per million in the United States.¹ Despite successful treatment of the primary tumor, a significant risk of metastasis exists. The cumulative melanoma-related mortality 25 years following treatment is approximately 52% for individuals with medium-sized tumors.²

Most experts agree that micrometastases exist at the time of diagnosis and perhaps even precede clinically recognizable ophthalmic disease.³ Dormancy may persist for years before macrometastatic disease develops, which occurs almost exclusively in the liver.⁴ Unfortunately, metastatic uveal melanoma is largely resistant to currently available therapies.⁵

Significant progress has been made in the understanding of the role of histopathology, cytogenetics, and gene expression patterns in predicting the risk of the metastatic uveal melanoma. For this reason, it has become increasingly common to biopsy for prognostic purposes.

In this article, the role of biopsy and frequently utilized prognostic tests, including fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) array, multiplex ligation-dependent probe amplification (MLPA) and gene expression profiling (GEP), will be reviewed.

INDICATIONS

Intraoperative biopsy may be performed for diagnostic or for prognostic purposes. Typically, uveal melanoma can be diagnosed based upon clinical features alone or in combination with noninvasive ancillary tests, such as ultrasonographic and angiographic studies. In certain situations, such as atypical presentation (Figure 1) or dense media opacity, or for diagnostic confirmation prior to the initiation of therapy, biopsy may be warranted.

Figure 1. Fundus photograph depicting a completely amelanotic tumor (A). B-scan ultrasonography demonstrated a collar-button-shaped tumor (B). A-scan ultrasonography showed low to medium internal reflectivity (C). Fine-needle aspiration biopsy revealed spindle cells lacking melanin, consistent with the clinical impression of amelanotic choroidal melanoma (D). A localized preretinal hemorrhage is seen overlying the needle biopsy site, which generally clears in several weeks (E).

Overall, approximately 1% to 2% of all intraocular tumors, including uveal melanoma, require biopsy to establish diagnosis.⁶ In such instances, the success rate of biopsy when performed via fine-needle aspiration biopsy (FNAB) is 88% to 95%.^6,7

TECHNIQUES

Fine-needle aspiration biopsy is the most frequently used technique for biopsy of uveal tumors, although excisional biopsy and incision biopsy (in combination with vitrectomy) can also be performed in special circumstances. For iris tumors, entry into the anterior chamber can be accomplished using a 26- to 30-gauge needle under direct visualization with an operating room microscope.⁸ The needle is inserted bevel up and is swept gently over the surface of the tumor while approximately 0.5 mL of aqueous fluid is aspirated.⁹ Posterior-segment tumors can be biopsied via a transscleral or a transvitreal approach. The transscleral approach, by far the most common technique employed, is often preferred for tumors located within the ciliary body or the anterior choroid.

The tumor margins are first marked using transillumination. The sclera can be entered directly, though some surgeons prefer the creation of a 3-mm partial-thickness (approximately 80% depth) scleral flap, followed by a small scratch incision in the scleral bed. A short 25-gauge needle attached to short tubing is used to perform aspiration of tumor tissue.⁶

Posteriorly located tumors are often more easily accessible by a transvitreal approach. This technique employs a 25- to 30- gauge needle attached to a 5-mL syringe by short tubing. The needle is inserted into the pars plana 4 mm behind the limbus and generally 180º away from the tumor. The needle is then viewed through the pupil as it is advanced into the mid-vitreous cavity. Under visualization with indirect ophthalmoscopy, the needle is guided into the tumor, and gentle aspiration is performed by pulling the needle plunger to the 2-mL mark. To improve cellular yield, the needle can be rotated along its long axis or carefully moved in and out of the tumor during aspiration. The needle is finally withdrawn, being careful to follow the original path of its insertion.¹⁰

PROGNOSTICATION IN UVEAL MELANOMA Cytologic Features

In additional to confirming the diagnosis of uveal melanoma, tumors in general are comprised of epithelioid cells that are more aggressive than their spindle-cell or mixedcell counterparts.^11,12

There are problematic issues relating to inter-observer variation and lack of consensus as to the number of epithelioid cells required to distinguish mixed from epithelioid cytology. An increased number of mitotic figures has consistently been demonstrated to be correlated with poorer prognoses.¹³ Other cytologic features, such as high numbers of tumor-infiltrating lymphocytes, are also associated with adverse outcomes.^13-16

Chromosomal/DNA Features

The first uveal melanoma cytogenetic studies were conducted in the early 1990s using standard karyotyping.^17-19 Tumor cells with minimal chromosomal aberration were found to be similar to early cutaneous melanomas and were not associated with an increased risk of mortality when followed for extended intervals.²⁰

The first reported genetic abnormalities in uveal melanoma involved chromosomes 1, 3, 6 and 8.^18,19 In 1992, Horsthemke and Prescher proposed that aberrations in chromosomes 3 and 8 accounted for survival differences in patients with uveal melanoma.^17,21 In 1996, Prescher confirmed her results in a series of 54 uveal melanomas treated by enucleation, in which monosomy 3 was detected in 30 (56%). At three years of follow-up, 50% of patients with monosomy 3 had developed metastases, while in comparison, not a single patient with disomy 3 had developed metastatic disease.²²

In 1997, Sisley reported similar results in a series of 42 tumors, in which monosomy 3 was detected in 50% and duplication of chromosome 8q was detected in 54% using standard karyotyping.²³ While several karyotype abnormalities, including the loss of chromosome 1p, monosomy 3, loss of 6q, and gain of chromosome 8q, have a statistically significant association with an increased risk of mortality,^18,24-26 monosomy 3 is by far the most significant prognostic indicator.²²

Several of the more commonly used techniques for prognostication of uveal melanoma using DNA include FISH, SNP array, and MLPA.²⁷

Fluorescence in Situ Hybridization

Fluorescence in situ hybridization takes advantage of fluorochromes linked to DNA probes, a technique that allows for enumeration and approximate spatial localization of targeted DNA sequences. FISH provides an economical and rapid assay that can be directly visualized in the laboratory under a fluorescence microscope (Figure 2).

Figure 2. Fluorescence in situ hybridization of choroidal melanoma demonstrates monosomy 3 (one orange alphacentromeric signal) and aneusomy of chromosome 8 (CEP8 Aqua) with an approximately equal number of 8q24 MYC signals (green; no net gain compared to the reference CEP8 probe; for MYC amplification to be present, the MYC-to-CEP8 ratio must be >2.0). Figure courtesy of Raymond R. Tubbs, DO, Department of Molecular Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic.

In general, three basic types of DNA probes are used: centromeric (also known as chromosome enumeration probes or CEPs), whole chromosome probes (or whole chromosome paints), and locus-specific probes.²⁸

Single Nucleotide Polymorphism Array

Single nucleotide polymorphism array, a type of DNA microarray, provides a powerful means by which to evaluate changes in chromosome copy numbers and loss of heterozygosity (LOH) in uveal melanoma. Microarray allows a large number of tumor cells to be examined in a single array and may therefore reduce bias introduced by sampling error.

Single nucleotide polymorphism array is able to detect a DNA sequence variation occurring when a single nucleotide in the genome differs between an individual and a population, which can be missed using standard karyotyping and FISH techniques.²⁹ Importantly, SNP array can identify tumors with isodisomy 3, the scenario that exists when a single copy of chromosome 3 is lost, and the remaining damaged copy is then duplicated. This condition is present in 5% to 10% of cases of uveal melanoma and, from a prognostic standpoint, is equivalent to monosomy 3 status.³⁰

There have been very few studies comparing FISH and SNP array findings in uveal melanoma. In 2007, Onken et al. reported that loss of chromosome 3 heterozygosity detected by SNP array was superior to FISH in predicting metastatic outcomes in a series of 53 patients with uveal melanoma.³⁰ It is possible that the relatively high threshold of 30%, used to define monosomy 3 status, may have accounted for the lower detection rate by FISH observed in this study.

In a second study by Young et al., monosomy 3 was detected in 64% of choroidal melanomas by FISH, compared to a 73% detection rate using SNP array.³¹ In the authors' experience, SNP array has higher sensitivity in detecting uveal melanoma that will metastasize (0.89) compared to FISH, using centromeric probes for chromosome 3 (0.78). FISH, using locus-specific probes for 3p26, had intermediate sensitivity (0.83).³²

Multiplex Ligation-dependent Probe Amplification

Multiplex ligation-dependent probe amplification is a PCR-based technique that permits amplification of multiple targets, thereby allowing for relative quantification of up to 45 loci in a single experiment. In contrast to MLPA, FISH can only evaluate a limited number of loci (generally four or fewer) in a standard assay, and the technique is difficult to automate.

One drawback of MLPA is that it only allows for an overall estimation of chromosomal aberrations, which can be difficult to detect if present in a limited number of cells. In comparison to MLPA, FISH is a visual technique, which allows for examination of individual cells and is readily applicable for simultaneous cytologic confirmation of diagnosis.

Both MLPA and FISH have been shown to be similar in their ability to detect high-risk metastasizing uveal melanoma.³³

Gene Expression Profiling

Gene expression profiling has the capability of simultaneously measuring messenger ribonucleic acid (mRNA) expression of multiple genes.^34,35 A modified, commercially available platform for GEP assay has been used to classify tumors into two major subtypes, referred to as class 1 and class 2. In this scheme, class 1 tumors have a relatively low risk of metastasis, whereas class 2 tumors have a very high risk of developing metastatic disease.²⁹

In enucleated specimens, the high-risk class 2 gene expression profile also correlates well with other prognostic indicators, including the largest tumor basal diameter, epithelioid cytology, the presence of extravascular matrix patterns, and monosomy 3.^36-38 The prognostic accuracy of the class 2 gene expression profile may be greater than for any of these other features, alone or in combination.

The superiority of GEP over monosomy 3 status to predict metastatic behavior has been independently verified by several oncology groups.^39,40 Recent advances in GEP have used computational analysis to eliminate redundant genes, thereby requiring only 12 genes to accurately classify tumors into class 1 and class 2 subgroups.^41,42 This refined form of GEP is now a commercially available as a polymerase chain reaction–based platform (DecisionDx-UM).^29,41

However, at least 10% of tumors identified as class 1 may harbor monosomy 3 (according to the manufacturer of DecisionDX-UM, Castle Biosciences) and go on to metastasize, and no long-term studies exist that validate the accurate use of the class 1/class 2 GEP platform.

LIMITATIONS AND COMPLICATIONS

While FNAB of uveal melanoma may be necessary for establishing diagnosis and prognosis, the techniques do have some limitations. Limited cellularity can comprise the diagnostic yield and the ability to perform prognostication studies. In the authors' own experience, 5% to 15% of cases had insufficient tissue for diagnosis.

Several practical considerations reducing the likelihood of limited cellularity include avoiding biopsy of tumors less than 2.5 mm in thickness and using a needle thickness less than 30-gauge. Additionally, surgical experience with FNAB techniques and communication with a cytopathologist who is familiar with uveal melanoma are critical.

In a recent publication on long-term follow-up of FNAB, the yield of FNAB was correlated with tumor height: For tumors with heights of <3.0 mm,="" 3.0="" to="" 5.0="" mm,="" and="">5.0 mm, sufficient biopsy material for FISH was obtained in 53%, 68% and 91%, respectively.⁵¹

Multiple factors specific to FNAB technique, including the surgical approach, needle size, and number of passes taken through the tumor for sampling, can influence outcome of prognostication studies. Furthermore, variability in the techniques utilized impacts prognostication.

Another limitation of FNAB prognostication is the issue of intratumoral heterogeneity. As FNAB allows for only a small sample to be removed from the tumor, it is possible to introduce sampling error. In fact, intratumoral heterogeneity for monosomy 3 has been estimated to occur in at least 14% to 18% of uveal melanomas.^43-45

In general, the complications of FNAB of uveal melanoma are relatively rare. They include vitreous hemorrhage, retinal detachment, and endophthalmitis. Localized hemorrhages overlying the transvitreal biopsy site are normal and can be controlled with gentle pressure on the globe following withdraw of the needle. Vitreous hemorrhages generally clear within a few weeks.

In a report of 170 cases of FNAB for uveal melanoma with up to six years of follow-up, there were no cases of endophthalmitis, local treatment failure, or orbital dissemination. Retinal detachment in three cases was a result of posterior vitreous detachment associated with tumor response to radiation rather than FNAB directly.⁵¹

In the authors' experience, in a series of 130 FNABs of uveal melanoma, only a single case required pars plana vitrectomy for a nonclearing vitreous hemorrhage. Retinal detachment likewise is unusual, as the retinal break is sealed by the blood clot at the biopsy site. Again, in the authors' experience of 130 FNABs, only a single case resulted in rhegmatogenous retinal detachment requiring repair.

There have been only two published case reports of endophthalmitis following FNAB.^46,47 To our knowledge, no documented reports have been published of local tumor extension caused by FNAB using small-diameter needles (23-gauge or smaller).

FUTURE DIRECTIONS

Fine-needle aspiration biopsy is critical to obtaining material for research purposes,⁵² to generating cell lines for study and for drug testing, and for ultimately understanding the biology of metastasizing uveal melanoma so that a cure for metastasis can be developed.

The issue of tumor heterogeneity and the importance of obtaining a representative tumor sample are persistent sources of uncertainty.⁴⁴ The goal is that efforts aimed at improving molecular prognostication will not only identify patients at risk but also implicate specific pathogenic pathways than can be targeted to treat micrometastatic or subclinical systemic disease.⁴⁸ Alternative approaches include identification of serum biomarkers that can not only detect class 2 tumors but that may also be useful in monitoring response to treatment.^49,50 RP

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