The biggest challenge in treating choroidal melanoma is the potential risk of metastatic spread. There is no cure for choroidal melanoma metastasis, which may affect 50% of those diagnosed with this uncommon but deadly cancer. Molecular testing of a primary tumor biopsy at the time of treatment can reveal whether there is a lower or higher risk of metastasis. Determining metastatic risk is valuable to both the patient and to the health care team. Studies assessing the desire to know molecular prognostic test results have shown that patients want as much information about their cancer as possible^1,2 and the oncologist will oversee a screening program for systemic surveillance or may direct a high-risk patient toward a clinical trial.

Controversy regarding the safety of fine-needle aspiration biopsy still exists despite most ocular oncology centers having adopted biopsy for molecular prognostication. Fears of seeding cancer in the orbit and increasing the risk of metastatic spread are serious concerns, despite there being no evidence for these complications in large published series.^3-6

Fortunately for patients, ophthalmologists treating choroidal melanoma are continuing to adopt fine-needle biopsy and to improve their experience and techniques. Additionally, an increasing number of ophthalmic oncologists are also vitreoretinal surgeons, which allows for utilization of expanding biopsy and treatment options that include vitrectomy. Providing prognostic information in addition to treating the primary ocular melanoma has now become the standard of care in treating choroidal melanoma patients.⁷

This article will review 3 questions about fine-needle biopsy in uveal melanoma: (1) What information does biopsy provide? (2) What are some methods for performing biopsy? and (3) What tips and tricks are helpful to maximize sample yield and to minimize complications?

INFORMATION PROVIDED BY BIOPSY

Cytopathology

Although in most cases, the diagnosis of choroidal melanoma is made on clinical examination, obtaining tissue for pathology helps to confirm that the tumor is indeed a primary choroidal melanoma. This is important because the most common type of intraocular tumor of adults is a metastatic tumor to the choroid. Patients with choroidal melanoma may have a past medical history of cancer, such as breast, lung, colon, or prostate. It may be helpful to the patient’s general oncologist to confirm that the cancer in the eye does not represent metastatic recurrence of another primary malignancy. Furthermore, some metastatic tumors may have a similar clinical appearance to a primary choroidal melanoma, sharing features of pigmentation, a dome-shaped appearance, and serous retinal detachment.

For cytopathology, the pathologist usually recommends a dry smear of the aspirate on a slide. If tissue is visible on the slide with the naked eye there will likely be adequate sample for analysis. The best sampling method for obtaining an undiluted smear is via a transscleral biopsy or following fluid-air exchange after complete vitrectomy using a long 25 g or 27 g needle (details below). A cell block may also be created with the aspirate.⁸

It is important to remember that the smaller the tumor, the less likely there may be sufficient material to make a cytopathologic diagnosis. When pathologic confirmation is essential to the systemic management of the patient, and the tumor has a height less than 2 mm, we find that vitrectomy with retinochoroidal biopsy for tissue acquisition offers higher tissue yield to establish a diagnosis.

Molecular Prognosis

Biologic markers can be identified that help predict a patient’s risk for developing choroidal melanoma metastasis. The molecular risk factor most strongly associated with metastasis is monosomy 3, the loss of one whole copy of chromosome 3 in the tumor tissue.⁹ Additional chromosomal aberrations in uveal melanoma occur on chromosomes 6 and 8. Tests that assess chromosomal aberrations use DNA-based platforms. Both DNA and RNA can be analyzed from the tumor to determine prognosis.

In addition to institution-based DNA-based protocols, such as fluorescent in-situ hybridization (FISH), single nucleotide polymorphism (SNP) analysis, or next-generation sequencing (NGS), most ophthalmic oncology centers include commercially available testing platforms to provide prognostic information to patients. At this time, there are 2 available commercial tests, a multiplex-ligation probe amplification (MLPA) test, which is a DNA-based test by Impact Genetics, and a gene expression profile (GEP) test, an RNA-based test by Castle Biosciences.

The MLPA Test

Copy number testing using the MLPA technique is performed on chromosomes 1p, 3, 6, and 8 from a biopsy sample. Monosomy 3 portends a high risk for metastasis. Chromosome 8q gain combined with monosomy 3 portends a worse prognosis than monosomy 3 alone.¹⁰ In samples where no monosomy 3 is detected (ie, disomy 3), additional sequencing for a GNAQ or GNA11 mutation, SF3B1 mutation, and EIF1AX mutation are performed. These GNA mutations are related to tumor development and are found in more than 90% of all uveal melanoma.^11,12 They also confirm that actual melanoma tumor tissue was submitted for analysis. A mutation in SF3B1 without monosomy 3 indicates risk for late-onset metastasis, and an EIF1AX mutation is associated with good prognosis.¹³

The molecular test results are then entered into a validated uveal melanoma prognostic online calculator,¹⁴ which incorporates a patient’s gender, age, and tumor size to determine an individualized 3-, 5- and 10-year survival estimate for that patient alongside a similar control individual without uveal melanoma.

The GEP Test

The expression of a panel of genes is analyzed in the tumor tissue submitted. The pattern of over- and/or underexpressed genes is matched to either a class 1 (good prognosis) or a class 2 (poor prognosis) profile. Class 1 is further divided into a class 1A and class 1B.¹⁵ PRAME, a biomarker for metastatic risk, is analyzed in class 1A tumors to help determine a risk for late-onset metastasis.¹⁶

Unlike the MLPA test, the GEP test does not provide additional sequencing to confirm that the biopsy sent was actually uveal melanoma tissue. It is recommended that a biopsy for cytopathology be performed in addition to the biopsy for GEP.

Are the results from MLPA testing and GEP testing equivalent? This is a difficult question to answer, as both tests examine different parameters and there has been no published study with enough follow-up to determine the actual metastatic outcome associated with prognostic test result. Because the reporting from the MLPA test includes clinical parameters to provide a more individualized estimate of metastatic risk, the information may be more useful for patients. Furthermore, several reports have found that at least 1 in 5 class 1A tumors contain monosomy 3, generally considered a marker of high risk of metastasis.^17,18

BAP1 Mutation Testing

A mutation in the BRCA-associated protein-1 (BAP1) gene, located on chromosome 3, results in loss of expression of this tumor suppressor gene in choroidal melanoma tissue. BAP1 mutations are highly correlated with a poor prognosis for metastasis in choroidal melanoma. In the author’s experience, BAP1 testing can be performed by immunohistochemical staining for BAP1 protein from a fine needle biopsy sample on a slide. Also, we have noted correlation with BAP1 mutation, presence of monosomy 3, and class 2 in choroidal melanoma, although there are no published clinical outcomes to date regarding this association.

When fine-needle aspiration biopsy is performed, the sample may be submitted for any or all of cytopathology, MLPA, GEP, and BAP1 testing. There may be instances where one of the prognostic test results does not yield sufficient tissue, and in this case, one of the other samples may inform the patient regarding risk of metastasis. When test results are discordant (for example, biopsy results in class 1A and monosomy 3 by MLPA), we generally feel that the patient must be counseled from a high-risk for metastasis perspective. Until we have longer follow-up regarding metastatic outcome, many ophthalmic oncologists feel that it may be premature to tailor metastatic surveillance to prognostic test result.

Multiple prognostic tests may offer a more complete picture for the patient. A class 1A may underestimate metastatic risk in 20% of patients.¹⁷ A patient may want to know both his or her class and monosomy 3 status, as both provide more risk information.

METHODS FOR PERFORMING BIOPSY

There are several techniques for obtaining fine-needle biopsy, and ophthalmic oncologists should become familiar with multiple techniques so that fine-needle biopsy can be performed on every uveal melanoma. If the ophthalmologist is comfortable performing biopsy on only medium- to large-sized melanomas, those patients with the smallest tumors (who may have the greatest chance at altering metastatic prognosis with future systemic therapies) will not benefit.

Transscleral Biopsy

Transscleral biopsy is a straightforward approach to obtain tissue, and when possible we recommend that this approach be the first consideration when a fine-needle aspiration biopsy is planned. After the tumor is identified on the sclera with transillumination, the biopsy can be performed.

After marking the anterior tumor border using transpupillary transillumination, we prefer to use a 30-g short needle attached to Eagle tubing attached to a 10-cc syringe. The surgeon holds the base of the needle and directs it at an angle toward the posterior pole. A tangential approach to the sclera with the needle bevel up is better than entering perpendicularly; tumors usually have a greater basal diameter than height. After the tip of the needle passes through the sclera, a slight “give” sensation can be felt when the needle is within the tumor itself (Figure 1).

At this point, with the needle tip held within the tumor, an assistant (who does not need to be scrubbed) pulls back on the 10-cc syringe to create suction at the tip of the needle, which is in the tumor. Once maximum suction is achieved, the surgeon can move the needle back and forth with micromovements like the movements a sewing machine may make. Then, the assistant returns the plunger back down the syringe, which stops the suction. Once there is no more suction, the surgeon withdraws the needle tip from the eye and the assistant carries the entire setup with sample in the syringe off the field. If a sufficient sample yield results from the first pass, the surgeon can use the same site (or be in the near vicinity) for subsequent needle passes.

Although some centers that perform this technique may use a scleral flap that can be sutured after the biopsy, we do not feel this additional step is necessary. Additional scleral surgery may compromise the overall scleral integrity. The sclera receives the highest dose of radiation when brachytherapy is performed, and creating a flap may contribute to scleral necrosis in large tumors. Some centers also perform cryotherapy after passing the needle through the sclera. We also do not feel this additional maneuver is necessary, as any malignant cell left at the scleral surface can be covered with the plaque.

Transvitreal Biopsy

The transvitreal technique incorporates direct visualization of the needle entering the tumor. Transvitreal biopsy methods can be categorized as nonvitrectomy transvitreal biopsy or vitrectomy transvitreal biopsy, where in the latter, vitreoretinal surgical techniques are used (Figure 2).

Nonvitrectomy Approach

The nonvitrectomy transvitreal biopsy may be utilized if the ophthalmic oncologist does not have access to vitrectomy or is not skilled in vitreoretinal surgery. This technique involves the use of binocular indirect ophthalmoscopy to visualize the tumor, and the passing of a needle from a trans-pars plana location opposite to the tumor. The biopsy involves movements that are reversed and upside-down to one’s actual view.

Vitrectomy Approach

Most ophthalmic oncologists who are vitreoretinal surgeons may be more comfortable with the incorporation of widefield imaging from an operating room microscope and standard small-gauge vitrectomy set-up when considering a transvitreal biopsy. Although this review is written from the bias of a vitreoretinal surgeon, we still recommend using a vitrectomy setup when possible. Furthermore, there are more variations that can be introduced in vitrectomy transvitreal biopsy when obtaining tissue is challenging.

Vitrectomy biopsy may be considered as a primary approach when fine-needle biopsy is being performed on tumors less than 2.0 mm in thickness. Although a transscleral technique may work well, the chance of successful tissue yield via a transscleral approach can be less than 50%.³ Furthermore, smaller tumors sometimes are closer to the posterior pole, making a transscleral approach more technically challenging. Finally, needle track injury to nontumor tissues carries the greatest risk to central vision in posterior tumors. Tumors greater than 2.0 mm in thickness can also be biopsied this way, however, the additional cost and time of vitrectomy setup solely for this purpose may not be necessary if the surgeon is comfortable with a transscleral approach.

Both 25-g and 27-g vitrectomy techniques have been reported with good success. Our approach¹⁹ involves a 2-trocar transscleral beveled setup (light pipe and cutter) using a 27 g 2-dimensional cutter (TDC) system (EVA, Dutch Ophthalmic). The cutter is introduced directly into the tumor with consistent force until the port is buried within the tumor. Often a tangential angle is required for this to occur particularly in very small tumors. The cut rate is reduced to 150-200 cpm, and a vacuum setting (rather than flow setting) is used. The pedal is fully depressed with maximal aspiration for 1-2 seconds. The cutter is immediately removed from the eye, and the cutter tip is placed into an empty sterile white receptacle such as vitrectomy packaging or BSS bottle cap. Back-flush is used to expel the sample into the cap. Visualization of actual tissue dots or fragments (with the naked eye or microscope) ensures that tissue is present, and usually ensures that molecular analysis is feasible (Figure 3).

In some cases, vitreous hemorrhage may occur at the biopsy site. Rather than using the vitreous cutter to remove the blood, we recommend only digital pressure on the globe with a cotton tip upon the cutter exiting the tumor. Localized and minimal vitreous hemorrhage will usually resolve spontaneously with observation alone. It has been rare that vitrectomy for nonclearing hemorrhage is necessary, in our experience. In general, we feel that complete vitrectomy is not required or necessary in these cases, unless 1,000-centistoke silicone oil will be used for radiation attenuation.²⁰ Incomplete vitrectomy may contribute to increased risk of postoperative retinal detachment.

In cases where a complete vitrectomy is performed for 1,000-centistoke silicone oil placement for radiation attenuation, there is also the option of performing a biopsy using a 1.5 inch 27-g needle inserted through a trocar, connected with the same tubing and 10-cc syringe for creating suction. This can be performed if material obtained from a transscleral approach was not adequate. Performing this needle biopsy after fluid-air exchange results in the greatest likelihood of a concentrated dry sample. As in transscleral biopsy, 27-g needle biopsy can be performed multiple times to obtain sufficient material for cytopathology and molecular prognostication.

When neither transscleral 30-g needle biopsy nor 27-g needle transvitreal biopsy results in adequate sample, we recommend introducing the vitreous cutter into the tumor (also under air) and backflushing the sample into a collection receptacle. The use of any of the small-gauge vitrectomy cutters is adequate for this maneuver. A vitreous cutter, like a needle, may also be introduced multiple times into the tumor depending upon the surface area of tumor available. We recommend choosing a different entry site for multiple cutter passes, as there may be hemorrhage within the tumor at the site of the cutter entry point.

TIPS AND TRICKS FOR FINE-NEEDLE BIOPSY

The most important consideration when performing biopsy is sample yield. The biopsy must be approached with the intention to obtain an adequate amount of tissue. A single pass where no tissue is visualized will not likely provide accurate information.

Small Tumors

The smaller the tumor, the less likely a diagnostically sufficient specimen will be obtained. However, the decision to treat a small uveal melanoma is based on clinical features that have caused the surgeon to make the diagnosis. Therefore, biopsy for cytopathology may be of lower priority than molecular prognostication, which cannot be determined without biopsy. Tumors less than 1.0 mm may or may not provide a test result, and with limited tissue, one must decide how best to distribute the material for testing. In cases of small tumors (~1.0 mm), we do not usually send tissue for cytopathology unless there is tissue to spare. It is also less feasible to perform even multiple vitrectomy biopsies on very small tumors.

Large Tumors

Some large-sized choroidal melanomas may pose a challenge for obtaining tissue for biopsy. Often, a large ciliary body melanoma may have areas of tumor necrosis, and the biopsy aspirate does not contain viable cells. In situations like this where transscleral biopsy is performed, multiple needle passes may be necessary with feedback each time from a biopsy assistant to ensure that sample was obtained.

Biopsy Assistant

Having an unscrubbed assistant is very helpful for performing fine-needle aspiration biopsy. It is very cumbersome and time consuming for the surgeon to both manage the specimen collections and perform the surgery. The assistant, who may smear a slide and/or expel the biopsy into a collection tube, plays a critical role. He or she will let the surgeon know whether an additional needle pass is needed or to consider a different part of the tumor for needle pass, based on the specimen yield. We recommend that the biopsy assistant be familiar with the biopsy supplies, the differences with all testing platforms and types of samples collected, and have an established verbal communication protocol with the surgeon as the biopsy is performed.

Multiple Needle Passes

There continues to be concern regarding the safety of passing a biopsy needle or instrument more than one time through the choroidal melanoma itself. There is no single center series to date that has demonstrated an increased risk of orbital dissemination or increased risk of systemic metastasis from fine-needle aspiration. At our institution, multiple needle passes per biopsy has been standard protocol to obtain adequate material for test results and research purposes.

SUMMARY

Fine-needle aspiration biopsy provides important information for patients regarding the metastatic potential of their choroidal melanoma. The most important reason for becoming skilled in performing tumor biopsy is to inform patients of their prognosis. Evolving vitrectomy techniques allow for biopsy in even very small tumors. We recommend that ophthalmic oncologists become familiar with various tumor biopsy techniques to improve the chances of obtaining sufficient material for prognostic testing. RP

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