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Retinal Physician

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Diagnostic Biopsy of Intraocular Tumors

Techniques, specimen handling, and complications.

Retinal Physician July 1, 2019

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Diagnosis of intraocular tumors is based on clinical and ancillary imaging studies, including ultrasonographic evaluation. The rate of clinical misdiagnosis of uveal melanoma is estimated to be less than 1%.1 Hence, biopsies are reserved for diagnosis of atypical cases, confirmation of a known diagnosis prior to definitive treatment, such as plaque radiotherapy or enucleation, and prognostic studies. Common indications for diagnostic biopsies include differentiation between melanocytic lesions and amelanocytic lesions, such as amelanotic melanoma, metastasis without a known primary, and uveal or vitreoretinal lymphoma. Depending on the tumor location, size, suspected diagnosis, and media opacities, different biopsy techniques can be performed.

Juan Valenzuela, MD, is with the Department of Ophthalmic Oncology, Consultores Oftalmológicos in Buenos Aires, Argentina. Paola De la Iglesia Niveyro, MD, is with the Service of Anatomic Pathology, Hospital Italiano de Buenos Aires in Buenos Aires, Argentina. Arturo Irarrazaval, MD, is director of the Department of Ophthalmic Oncology, Consultores Oftalmológicos. Charles V. Biscotti, MD, is with the Department of Anatomic Pathology and is director of the Gynecological Pathology Subspecialty at the Robert J. Tomsich Institute of Pathology and Laboratory Medicine of the Cleveland Clinic in Cleveland, Ohio. Arun D. Singh, MD, is director of the Department of Ophthalmic Oncology at the Cole Eye Institute of the Cleveland Clinic. Dr. Singh reports advisory board membership with Aura Biosciences and Immunocore, and honoraria from Isoaid LLC and Eckert & Ziegler Medical. The remaining authors report no related disclosures. Reach Dr. Singh at singha@ccf.org. Editor’s note: This article is featured in a journal club episode of Straight From the Cutter’s Mouth: A Retina Podcast. Listen to the episode at www.retinapodcast.com .

BIOPSY TECHNIQUE

Iris

For small tumors without invasion of the angle, the small-incision guarded hydroaspiration (SINGH) technique can be considered to obtain an excisional biopsy with clear margins.2 For larger lesions, transcorneal fine-needle aspiration biopsy (FNAB) is the best approach (Figure 1). A 25- to 30-gauge needle is passed through a clear corneal incision parallel to the iris plane, near the lesion and not going across the pupil. Aspiration of tumor material is performed with the bevel up while slowly turning the needle.3,4 Some authors recommend using the bevel down, in a “vacuum cleaner” fashion.5 We prefer using the bevel up to visualize the cells entering the needle.

Figure 1. A 62-year-old female was evaluated for a growing iris nevus. Visual acuity was 20/25 in both eyes with normal intraocular pressure. The lesion was brown colored, located at the 6:30 to 8-o’clock position, and extended into the anterior-chamber angle. Ultrasound biomicroscopy confirmed a posterior extension to the pars plicata with tumor dimensions measuring 2.9 mm x 2.8 x 1.8 mm in size. A transcorneal fine-needle aspiration biopsy was performed with a short angulated 25-gauge needle. The needle was inserted at the 9-o’clock position, advanced along the iris plane, with a bevel up along the long axis of the tumor. Cytopathology revealed atypical cells derived from a melanocytic neoplasm, consistent with melanoma.
Figure 1. A 62-year-old female was evaluated for a growing iris nevus. Visual acuity was 20/25 in both eyes with normal intraocular pressure. The lesion was brown colored, located at the 6:30 to 8-o’clock position, and extended into the anterior-chamber angle. Ultrasound biomicroscopy confirmed a posterior extension to the pars plicata with tumor dimensions measuring 2.9 mm x 2.8 x 1.8 mm in size. A transcorneal fine-needle aspiration biopsy was performed with a short angulated 25-gauge needle. The needle was inserted at the 9-o’clock position, advanced along the iris plane, with a bevel up along the long axis of the tumor. Cytopathology revealed atypical cells derived from a melanocytic neoplasm, consistent with melanoma.

Ciliary body

For small tumors of unknown origin, iridocyclectomy is a good option for excisional biopsy, because it offers both diagnosis and treatment in a single procedure. However, such biopsies should be performed with particular caution for ciliary body medulloepitheliomas. Enucleation or adjuvant brachytherapy therapy may be required, because excision alone is not sufficient for local control and may result in metastatic spread.6,7 For larger tumors, trans-scleral FNAB can be performed (Figure 2). The tumor area is marked by transillumination. A 3-mm square scleral flap of 80%8 thickness is marked, and a 25- to 30-gauge needle attached to a 5-mL syringe by a short tubing, is inserted tangentially to aspirate the tumor cells.

Figure 2. A 65-year-old male was noted to have a pigmented lesion of the peripheral iris of the right eye that extended into the trabecular meshwork. The patient recalled multiple prior eye examinations but the lesion was only noted at the recent exam. Visual acuity was 20/25 in both eyes. There was a 2 mm x 1 mm periperhal iris lesion of the right eye, the posterior extent of which could not be visualized (A). Gonioscopy revealed extension into the meshwork but there was no seeding or pigment dispersion. Ultrasound biomicroscopy confirmed extension into pars plicata (B). Fine-needle aspiration biopsy was performed to rule out melanoma. The aspirate specimen contained a monomorphic sample of widely scattered heavily pigmented cells. The nuclear detail was obscured by the cytoplasmic pigment; however, the nuclei were recognizable as relatively small and round. They varied little among the tumor cells. Note the relatively large and regular pigment granules and the low nuclear-to-cytoplasmic ratio. These features help distinguish melanocytoma from melanoma (C; Papanicolaou stain).
Figure 2. A 65-year-old male was noted to have a pigmented lesion of the peripheral iris of the right eye that extended into the trabecular meshwork. The patient recalled multiple prior eye examinations but the lesion was only noted at the recent exam. Visual acuity was 20/25 in both eyes. There was a 2 mm x 1 mm periperhal iris lesion of the right eye, the posterior extent of which could not be visualized (A). Gonioscopy revealed extension into the meshwork but there was no seeding or pigment dispersion. Ultrasound biomicroscopy confirmed extension into pars plicata (B). Fine-needle aspiration biopsy was performed to rule out melanoma. The aspirate specimen contained a monomorphic sample of widely scattered heavily pigmented cells. The nuclear detail was obscured by the cytoplasmic pigment; however, the nuclei were recognizable as relatively small and round. They varied little among the tumor cells. Note the relatively large and regular pigment granules and the low nuclear-to-cytoplasmic ratio. These features help distinguish melanocytoma from melanoma (C; Papanicolaou stain).

Choroid

For pre-equatorial tumors, biopsy technique is similar to that used in ciliary-body tumors. The scleral flap is optional, depending on the tumor position and the surgeon’s preference. In some cases, as in almost all melanomas, plaque therapy is performed at the same time as the biopsy. In those cases, the scleral flap can be omitted. In other cases, we recommend performing a scleral flap. Careful hemostasis and cautery after needle withdrawal are other measures that may improve safety and decrease the risk of tumor seeding.

In postequatorial tumors, depending on the experience of the surgeon, different techniques can be selected. For the experienced pathologist, a small sample with just a few cells can be enough for diagnosis, so FNAB can be performed either directly through the sclera or with the use of trocars 180 degrees away at the pars plana, 4 mm behind the limbus (Figure 3).9 Using a valved 25-gauge canula offers the potential advantage of isolating the needle tract and allowing retrieval of the contaminating cells.10

Figure 3. A 14-year-old female with a long-standing history of strabismus status-post surgical correction was seen for routine follow-up was found to have a decline in vision (20/30). A dome-shaped, lightly pigmented choroidal mass centered at the 7-o’clocl position just anterior to the equator measuring 16.5 mm x 15.5 mm x 7.5 mm was observed (A). There was a surrounding, shallow exudative retinal detachment. B-scan and A-scan echography of the lesion showed low-to-medium reflectivity suggestive of choroidal melanoma (B, C). Transvitreal fine-needle aspiration biopsy using 25-gauge trocar was performed. A small area of hemorrhage on the site of puncture (D) resolved during the next few weeks (E). Cytology showed atypical cells derived from a spindle-cell neoplasm consistent with melanoma.
Figure 3. A 14-year-old female with a long-standing history of strabismus status-post surgical correction was seen for routine follow-up was found to have a decline in vision (20/30). A dome-shaped, lightly pigmented choroidal mass centered at the 7-o’clocl position just anterior to the equator measuring 16.5 mm x 15.5 mm x 7.5 mm was observed (A). There was a surrounding, shallow exudative retinal detachment. B-scan and A-scan echography of the lesion showed low-to-medium reflectivity suggestive of choroidal melanoma (B, C). Transvitreal fine-needle aspiration biopsy using 25-gauge trocar was performed. A small area of hemorrhage on the site of puncture (D) resolved during the next few weeks (E). Cytology showed atypical cells derived from a spindle-cell neoplasm consistent with melanoma.

Different viewing systems can be used depending on surgeon experience, such as indirect biomicroscopy, microscopy with contact or no-contact viewing systems, and optical coherence tomography (OCT)-guided methods.11 Tissue samples can also be obtained by a vitrectomy-assisted approach.12

Vitreous

The most frequent indication for a vitreous biopsy in ocular oncology is primary vitreoretinal lymphoma (PVRL). In those cases, FNAB is not a good option, because of the difficulty to acquire tumor cells, so vitrectomy must be performed if cytologic diagnosis is needed. First, a sample of undiluted vitreous should be taken, with the infusion canula closed and using a low cut rate with a 5-mL syringe. Then the second sample of diluted vitreous can be submitted together with the cassette for examination.13,14 Air infusion is another elegant way of obtaining such specimens.15 In presence of subretinal infiltrates, subretinal aspiration can enhance positive yield.

SPECIMEN HANDLING

Aspirated material from FNAB samples can be processed with different methods of sample concentration. Liquid-based cytology techniques like ThinPrep (Healthdxs) processing system are widespread in many countries, as they preserve morphology and allow cells to be placed in a monolayer. This is particularly important in small samples with limited material, such as ophthalmic FNAB samples.16,17 However, if handled carefully, other methods including cytocentrifugation, Millipore filter concentration, and celloidin bag techniques18 can be used with good results and are good options in settings where access to liquid-based cytology is limited. Cell-block preparation can be performed regardless of the method used (liquid-based vs conventional cytology) in cases of sufficient sample.19 Although immunostaining is possible on cytologic smears (immunocytochemistry), most pathology laboratories prefer immunohistochemistry methods on paraffin-embedded cell blocks.20

Differential diagnosis of intraocular tumors is broad and can be separated into 2 groups: melanocytic and non-melanocytic lesions. The first step in cytological evaluation of FNAB specimens is to identify the presence of melanic pigment in the cell cytoplasm of lesional cells and to identify the presence of spindle cells (Figure 4).21,22 Melanin is finely granular and is evident in HE stained slides; however, it can be focal and subtle. Spindle cells are observed in 98% of uveal melanoma samples and are unlikely to be present in a metastatic tumor.23 Careful examination by the pathologist is mandatory, and interpretation within the clinical context is fundamental for correct diagnosis.

Figure 4. Spindle cells with melanic pigment compatible with uveal melanoma (A). Nuclear detail of melanoma cells with prominent nucleoli (B). Hematoxylin-eosin stain (400x).
Figure 4. Spindle cells with melanic pigment compatible with uveal melanoma (A). Nuclear detail of melanoma cells with prominent nucleoli (B). Hematoxylin-eosin stain (400x).

MELANOCYTIC LESIONS

Melanocytic intraocular lesions include ocular melanoma and nevi. Cytologic classification of melanoma cells is based on the modified Callender system and includes spindle type A (nevus-like) cells, spindle type B, epithelioid, and mixed spindle B and epithelioid cells.24 Spindle type A cells have oval, folded nuclei with small nucleoli and fusiform appearance. Spindle type B cells are large and the nuclei are cigar shaped with larger nucleoli and tend to have bipolar cytoplasmic processes. Epithelioid melanoma cells are large and polygonal, with irregular nuclei with coarse chromatin and distinct nucleoli. While melanomas can be composed of epithelioid and spindle cells, uveal nevi are exclusively composed of type A spindle cells. Distinction between nevi and spindle-cell melanoma in FNAB specimens can be challenging.25 Interpretation of the cytologic finding should be done in the clinical context and in communication with the cytopathologist.

NONMELANOCYTIC NEOPLASMS

Nonmelanocytic lesions include metastasis, lymphoproliferative processes, retinoblastoma, choroidal hemangioma, and medulloepithelioma. Age and clinical information are mandatory for correct assessment.

Metastases are the most common intraocular malignancy.26 Most patients have known history of cancer; however, identification of the ocular tumor may precede a cancer diagnosis or result in the identification of a recurrence. Breast cancer represents almost half of metastasis, followed by lung and gastrointestinal tract cancer.27 Cytological characteristics are of those of the primary tumor. In some cases, immunohistochemistry on cell-block preparation may be needed for characterization of primary origin. In presence of a lesion suspicious of metastasis, computed tomography scanning of the chest, abdomen, and pelvis should be performed before intraocular tumor biopsy is attempted, because a biopsy area outside of the choroid with more straightforward surgical access is usually identified.

Lymphoproliferative processes may secondarily affect the uveal tract and retina. Patient history and monomorphic and discohesive nature of cells should raise suspicion. Primary vitreoretinal lymphomas are almost always diffuse, large, B-cell type characterized by cells with relatively large nuclei with prominent nucleoli, associated with necrosis.28 Cellular features usually suffice to establish the diagnosis. Immunohistochemistry can be helpful in problematic cases. Specifically, immunostains can confirm the lymphoid B-cell phenotype. A positive flow cytometry result is helpful because it establishes light chain restriction (monoclonality). Importantly, a negative flow cytometry result can be misleading because widespread necrosis, common in these specimens, often causes false-negative flow cytometry results.

FNAB should not be performed in patients with suspicion of retinoblastoma because of the significant risk of tumor seeding.29-31 Cytological diagnosis can also be done in cerebrospinal fluid or extraocular metastasis. Smears show small undifferentiated cells with markedly hyperchromatic nuclei, frequent apoptotic debris and mitotic figures.32 Presence of calcifications is a useful clue in retinoblastoma. In well-differentiated tumors, cytoplasmic processes and Flexner-Wintersteiner rosettes may be seen.

Medulloepithelioma is a rare neoplasm that occurs in children. Cytologic preparations show single or grouped primitive looking cells with high nuclear–cytoplasmic ratio, nuclear molding, and rosette formation.33

COMPLICATIONS

More invasive procedures, such as iridocyclectomy, are associated with a higher number of complications such as glare, monocular diplopia, hyphema, cataract, infection, intraocular pressure fluctuations, and postsurgical astigmatism.

Techniques that are less invasive than FNAB have fewer and more minor complications. The most common complication is a small hemorrhage at the site of retinal puncture (Figure 3).9,34 To prevent massive hemorrhage, vascular tumors such as hemangiomas should not be biopsied and vascular structures of solid tumors should be avoided. If active bleeding is seen during the procedure, pressure should be raised either by pressing the entrance site with a cotton tip, injecting saline solution, or increasing the pars plana infusion pressure, if available.

Only 2 cases of endophthalmitis have been reported.35,36 It is essential to perform the procedure under sterile conditions after asepsis and antisepsis with povidone–iodine 5%. Further studies have not shown any case of endophthalmitis.37 Retinal detachment after FNAB for choroidal tumors is extremely rare. The retinal break created at the biopsy site is small and self-sealed by the hemorrhagic clot. The tumor also acts as a “scleral buckle” indenting on the retinal site, preventing detachment.

Seeding has always been one of the main concerns for intraocular biopsies. Although the literature has reported seeding on the needle tract,38 the number of tumor cells was less than that associated with tumor growth in experimental models.39 In clinical practice, the rate of extraocular extension after FNAB is low, and a few cases of extraocular extension of uveal melanoma have been reported. Special care should be taken in avoiding diagnostic biopsy in cases that could harbor unsuspected retinoblastoma, because of the high risk of seeding the tumor outside the eye.29-31

CONCLUSION

Although diagnosis on most intraocular tumors can be done based on clinical examination and imaging studies, for atypical cases, diagnostic biopsy is a safe method.40 However, a negative cytologic diagnosis should not be considered proof that an intraocular malignancy does not exist. In addition to use of careful surgical techniques, proper sample handling and consultation with an expert cytopathologist are essential to reaching the correct diagnosis. RP

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