Diagnostic Cytologic Features of Uveal Melanoma Carlos A. Medina, MD,1 Charles V. Biscotti, MD,2 Nakul Singh, MS,1 Arun D. Singh, MD1 Purpose: To report the cytologic characteristics of uveal melanoma. Design: This is a prospective, single-center study of consecutive patients. Subjects: All patients with a clinical diagnosis of uveal melanoma from May 2009 to July 2013 who underwent prognostication fine-needle aspiration biopsy (FNAB) were included. Methods: The cytologic characteristics of uveal melanoma were analyzed for 150 consecutive patients with a clinical diagnosis of uveal melanoma who were treated at the Cleveland Clinic Cole Eye Institute between May 2009 and August 2012. Main Outcome Measures: Cellular features of all cases were analyzed for cell type, presence of melanin, nuclear grade, tumor-infiltrating lymphocytes, and necrosis. Cytology was then correlated with histopathology in enucleated eyes. Results: A total of 150 patients were included. Seven samples of tumor resections were excluded from the study because they were studied by impression smears. A total of 143 FNAB samples of 143 patients formed the basis for analysis. Fifty-three percent of the patients were male, and the average age for all patients was 60 years. Transcorneal (n ¼ 8), transscleral (n ¼ 71), and transvitreal (n ¼ 64) approaches were used. Of 143 samples, 131 were adequate. Among these, spindle cells were observed in 98% (63% mixed and 35% spindle only), whereas only epithelioid cells were observed in 2 samples. Melanin granules were observed in 80% of samples. Tumor nuclear grade (atypia) increased with tumor height and by tumor location (least atypia with iris tumors). Conclusions: Cytologic features such as spindle cells and melanin granules, present in 98% and 80% of samples, respectively, are important cytologic diagnostic features. Tumor nuclear grade (atypia) increased with tumor height. Iris melanoma has bland features compared with ciliary and choroidal melanoma. Ophthalmology 2015;122:15801584 ª 2015 by the American Academy of Ophthalmology
The majority of intraocular tumors can be diagnosed on the basis of clinical examination and ocular imaging studies. The Collaborative Ocular Melanoma Study reported a misdiagnosis rate of 0.48%: Of 413 eyes with a clinical diagnosis of choroidal melanoma, only 2 were found to be misdiagnosed on histopathology.1 However, the Collaborative Ocular Melanoma Study had strict inclusion and exclusion criteria, and only large and obvious melanomas were included, with the exclusion of difficult diagnostic cases, such as those with media opacification.1 In clinical practice, the diagnosis of choroidal melanoma is not always clear, particularly in the setting of a confusing clinical pattern such as an amelanotic uveal tumor, a history of carcinoma, media opacities, or when dealing with smaller or atypical tumors.2 It is estimated that 1% to 2% of all cases require diagnostic fine-needle aspiration biopsy (FNAB).3,4 Rates of complications from intraocular FNAB are relatively low. Frequently used needles range from 25 to 30 gauge.5,6 Although using a larger needle increases the likelihood of obtaining a sufficient sample, the risk of complications associated with the procedure also increases. In more than 200 000 cases of FNABs reported in the literature, there has been no evidence of local or systemic spread of tumor cells with the use of 25-gauge or smallerdiameter needles.3,7e12 Transient localized subretinal and
1580
2015 by the American Academy of Ophthalmology Published by Elsevier Inc.
vitreous hemorrhage at the biopsy site are the most frequent complication.3,13 An experienced cytopathologist can achieve a high concordance of 96% between cytologic and histologic diagnosis.3,6,8,14 Nevertheless, cytologic characteristics that are diagnostic of uveal melanoma have not been described. The purpose of this study is to report the diagnostic cytologic characteristics of uveal melanoma.
Methods All patients with a clinical diagnosis of uveal melanoma who underwent prognostication biopsy from May 2009 to July 2013 were included. Institutional review board approval was obtained (local institutional review board no.: CASE 5608-CC666) for this prospective, single-center study of consecutive patients, and clinical and diagnostic cytologic features of uveal melanoma were analyzed. Factors such as suspected diagnosis, size, presence of retinal detachment, visibility, location, and patient and surgeon preference factored in determining whether the patient underwent prognostic biopsy and was subsequently included in the study. All patients who underwent prognostic biopsy were included in this study. All patients provided appropriate consent, and approval from the institutional review board was obtained. Biopsy technique and instrumentation varied by the location of the tumor. Biopsy was obtained by a transcorneal, transscleral, or http://dx.doi.org/10.1016/j.ophtha.2015.04.013 ISSN 0161-6420/15
Medina et al
Diagnostic Cytologic Features of Uveal Melanoma
transvitreal approach. A 25-gauge needle attached to short tubing and a 5-ml syringe was used for all approaches. For transcorneal biopsy, a 1-mm incision was created at the limbus and viscoelastic material was injected over the biopsy site. Care was taken to avoid the lens, particularly in phakic patients. After FNAB, the intraocular pressure was increased with balanced salt solution to control hemorrhage. A single 10-0 nylon suture was used to close the wounds. A short 25-gauge needle was used for tumors anterior to the equator where a transscleral approach was used. A partialthickness 80% scleral flap was created with a crescent blade. For tumors posterior to the equator, the biopsy was obtained via a long 25-gauge needle. The needle was inserted through the pars plana and into the tumor under visualization by indirect ophthalmoscopic control. As with all 3 techniques, once the needle is inserted into the tumor, gentle aspiration was applied by slowly withdrawing the syringe plunger up to the 3-ml mark. Finally, the needle was then carefully withdrawn following the original path of insertion.15 The contents of the needle, tubing, and syringe were then rinsed out with Cytolyt solution (Hologic Inc, Marlborough, MA) for ThinPrep (Hologic Inc.) processing. No immediate adequacy check was performed except for visual inspection of debris in the sample. The sample was then subjected to 1 or more centrifugation and concentration steps. The pellet obtained was resuspended in a cellpreserving solution (PreservCyt; Hologic Inc.) for additional processing. The ThinPrep processor mixed the sample, and then, using a gentle vacuum, collected cells on a filter in an evenly dispersed manner that approaches a monolayer. The filter was then inverted, and its cellular contents were transferred to a microscope slide.16 The method used optimized cellular yield and preservation while standardizing slide preparation for interpretation. One of the authors (C.V.B.) examined Papanicolaou-stained ThinPrep slides from the aspirate samples of all of the cases for the following cellular features: cell type, presence of melanin, nuclear grade, tumor-infiltrating lymphocytes, and necrosis. Cytology was then correlated with histopathology in enucleated eyes. Cell type was classified as spindle cell type if all of the tumor cells had spindleshaped cytoplasm, epithelioid type if none of the tumor cells had spindle-shaped cytoplasm, and mixed cell type for all others. Grade 3 nuclear atypia had conspicuous nucleoli when observed through the 10 objective, 4-fold nuclear pleomorphism, hyperchromasia, and coarse chromatin. Grade 1 nuclei had none of these features. All other tumors were regarded as nuclear grade 2. Ultimately, grade 3 nuclear atypia was referred to as high grade, and grades 1 and 2 were grouped into low grade for the purposes of statistical analysis. Tumorinfiltrating lymphocytes were classified as present or absent. Lymphocytes infiltrating aggregates of melanoma cells or intimately associated with melanoma cell cytoplasm were required for the presence of tumor-infiltrating lymphocytes. The presence or absence of melanin and necrosis was recorded. Statistical analysis to determine factors influencing adequacy of samples was then performed. To test associations between categoric variables, the Fisher exact test was used. For hypothesis testing related to continuous outcomes, equal variance assumptions were made on the basis of examination of boxplots. For associations between continuous variables and 2 categories where equal variance was not assumed, Welch’s t test was used. Associations between continuous variables and multiple categories were tested by analysis of variance. In those multiple categories, significant differences in means were assessed using Tukey’s correction for multiple comparisons of means. R version 3.02 was used for all analyses.
impression cytology was used. A total of 143 FNAB samples from 143 patients formed the basis of the study. This included 76 samples (55%) from the right eye and 67 samples (47%) from the left eye. A total of 76 patients (53%) were male, and 67 patients (47%) were female. The average age of all patients was 60 years, with the youngest aged 26 years and the oldest aged 92 years. Tumor location included 4 iris (3%), 4 iridociliary (3%), 4 ciliary (3%), 25 ciliochoroidal (18%), and 106 choroidal (74%) tumors. Biopsy was performed using a transcorneal approach in 8 patients (5%), and 71 patients (50%) and 64 patients (45%) underwent transscleral and transvitreal biopsy, respectively. All patients were treated after FNAB. A total of 102 patients (71%) underwent plaque brachytherapy, and 41 patients (29%) underwent enucleation. Average follow-up was 24 months, with a range of 0.5 to 51 months. At study closure (July 1, 2013), 128 patients (90%) were alive. Eleven patients (8%) died with known metastatic disease, and 4 patients (3%) died of unrelated causes (Table 1). Six patients (4%) were alive with known metastatic disease. Adequate samples were defined as those with sufficient cellularity to ascertain a diagnosis. Of 143 samples, 131 adequate samples were obtained (92% true positive). Of the remaining 12 inadequate samples, 10 contained nondiagnostic atypical cells and 2 lacked even atypical cells. One contained benign squamous cells and columnar cells suggestive of conjunctival contamination, and one contained rare histiocytes. Both height and largest basal dimension were correlated with nuclear grade. High-grade tumors with an average height of 8.1 mm were significantly higher than low-grade tumors (both grades 1 and 2), with an average height of 5.6 mm. Basal diameter for highTable 1. Diagnostic Cytologic Features of Uveal Melanoma: Profile of Study Patients (n ¼ 143) Parameter
Results
Sex Male Female Age (yrs) Range Average Eye Right Left Location Iris Iridociliary Ciliary body Ciliochoroidal Choroidal FNAB approach Transcorneal Transscleral Transvitreal Treatment Plaque Enucleation Follow-up (mos) Average Range Status Alive without metastases Alive with metastases Dead without metastases Dead with metastases
A total of 150 samples from 150 patients were obtained. Seven samples were excluded because these contained tumor resections where
FNAB ¼ fine-needle aspiration biopsy.
No. 76 67 26e91 60 76 67 4 4 4 25 106 7 72 64 102 41 24 0.5e51 122 6 4 11
1581
Ophthalmology Volume 122, Number 8, August 2015
Figure 1. Box plots of tumor size in millimeters (largest basal diameter [right] and height [left]) grouped by tumor nuclear grade. There was an association between tumor grade and tumor height (P ¼ 0.02, analysis of variance [ANOVA]). Using the Tukey correction for the comparison of means, tumor grades 1 and 2 are not statistically different (P ¼ 0.85), tumor grade 3 is marginally insignificantly taller (P ¼ 0.06), and tumor grade 3 is significantly taller than tumor grade 2 (P ¼ 0.03). There was an association between tumor grade and tumor basal diameter (P ¼ 0.004, ANOVA). Using the Tukey correction for the comparison of means, tumor grades 1 and 2 are not statistically different (P ¼ 0.32), and tumor grade 3 is wider than grade 1 (P ¼ 0.005) and grade 2 tumors (P ¼ 0.017).
grade tumors was 16 mm compared with 12.2 mm for low-grade tumors (Fig 1). There was no significant correlation between tumor height or basal diameter and cytologic type (Fig 2). Within diagnostically adequate samples, a combination of spindle and epithelioid cells were observed in 63% of samples (Figs 3 and 4). Pure epithelioid cells were present in only 1.75% of samples, and 35% had pure spindle cells (Table 2). Overall, spindle cells were observed in 98% of samples. Cytoplasmic melanin was present in 80% of samples (Fig 5). Tumor nuclear grade also varied by tumor location, with the least atypia seen with iris tumors. None of the iris tumors displayed high-grade nuclear atypia. Ciliary body tumors had significantly more atypia, with 36% of tumors categorized as high grade. Tumor-infiltrating lymphocytes were present in 31% of samples. Some 79% of tumors were described as having a low nuclear grade. There was 100% correlation between cytologic and histologic diagnosis in all 41 enucleated eyes.
Discussion To our knowledge, this study is the first prospective study to analyze the diagnostic cytologic features of uveal melanoma. The study sample included a broad spectrum of tumors; size and location were variable, ranging from small to large from iris to choroid. Histopathologic concordance was 100% in all specimens that subsequently underwent enucleation (n ¼ 41). Spindle cells were common among adequate samples, with 98% of samples containing pure spindle cells or a combination of these cells. Pure epithelioid cells were uncommon, with only 2 samples (1.75%) containing purely epithelioid cells. Melanin was present in 79.8% of samples, yet both
Figure 2. Box plots of tumor size (basal diameter [right] and height [left]) by cytologic cell type. There was no significant difference in tumor height (P ¼ 0.486, analysis of variance [ANOVA]) or basal diameter (P ¼ 0.077, ANOVA).
1582
Medina et al
Diagnostic Cytologic Features of Uveal Melanoma Table 2. Diagnostic Cytologic Features of Uveal Melanoma: Adequate Samples (n ¼ 131) Features Cell type/pattern Mixed Spindle Epithelial Nuclear grade Low High TILS Yes No Necrosis Yes No Distribution of melanin Yes No
Adequate Samples (n [ 131), % 63 35 1.75 79 21 31 69 4 96 79.8 20.2
samples with pure epithelioid cells had no melanin. Atypia, as in histologic analysis, was correlated with location. Iris tumors contained bland nuclear features compared with other locations. The presence or absence of tumor-infiltrating
lymphocytes was not helpful in establishing a diagnosis. The presence of spindle cells and melanin was deemed an important diagnostic feature and may be helpful in the future for establishing diagnostic criteria. We considered true-positive samples those that were positive for melanoma and those with cytologic findings consistent with the diagnosis of melanoma (92%). Our data are similar to the previously published adequacy of FNAB samples ranging from 88% to 95%.3,4 Additional analysis is underway to identify factors that may contribute to the inadequacy of FNAB samples. It is worth emphasizing that a negative cytologic diagnosis of malignancy should not be considered unequivocal proof that an intraocular malignancy does not exist.8,13,17 As FNAB gains popularity with an improved safety profile, techniques, and instrumentation, there is a need to develop descriptive cytologic diagnostic features of uveal melanoma.18e20 Because cytologic diagnosis is
Figure 4. Most of the cells in this uveal melanoma have an epithelioid appearance. Note the higher-grade nuclear atypia compared with the example in Figure 3.
Figure 5. The spindle-shaped melanoma cell to the upper right has prominent melanin pigmentation.
Figure 3. This uveal melanoma illustrates the characteristic cellular features. Note the spindle cell shape characterized by elongated, often caudate cytoplasm. Note also the relatively bland nuclear features, grade 2 by our criteria (Papanicolaou stain).
1583
Ophthalmology Volume 122, Number 8, August 2015 predominantly morphologically driven, automated digital methods that can identify, quantify, and characterize spindle cells in an ocular FNAB cytopathology slide can be developed for computer-assisted diagnosis.
References 1. Accuracy of diagnosis of choroidal melanomas in the Collaborative Ocular Melanoma Study. COMS report no. 1. Arch Ophthalmol 1990;108:1268–73. 2. Char DH, Kemlitz AE, Miller T. Intraocular biopsy. Ophthalmol Clin North Am 2005;18:177–85. 3. Shields JA, Shields CL, Ehya H, et al. Fine-needle aspiration biopsy of suspected intraocular tumors. The 1992 Urwick Lecture. Ophthalmology 1993;100:1677–84. 4. Char DH, Miller T. Accuracy of presumed uveal melanoma diagnosis before alternative therapy. Br J Ophthalmol 1995;79: 692–6. 5. Young TA, Burgess BL, Rao NP, et al. Transscleral fine-needle aspiration biopsy of macular choroidal melanoma. Am J Ophthalmol 2008;145:297–302. 6. Singh A, Pelayes D, Brainard J, Biscotti C. History, indications, technique and limitations. In: Biscotti C, Singh A, eds. FNA Cytology of Ophthalmic Tumors. Basel, Switzerland: Karger; 2011:2–9. 7. Pelayes DE, Zarate JO. Fine needle aspiration biopsy with liquidbased cytology and adjunct immunohistochemistry in intraocular melanocytic tumors. Eur J Ophthalmol 2010;20:1059–65. 8. Augsburger JJ, Shields JA, Folberg R, et al. Fine needle aspiration biopsy in the diagnosis of intraocular cancer. Cytologichistologic correlations. Ophthalmology 1985;92:39–49.
9. Glasgow BJ, Brown HH, Zargoza AM, Foos RY. Quantitation of tumor seeding from fine needle aspiration of ocular melanomas. Am J Ophthalmol 1988;105:538–46. 10. Karcioglu ZA, Gordon RA, Karcioglu GL. Tumor seeding in ocular fine needle aspiration biopsy. Ophthalmology 1985;92: 1763–7. 11. Schefler AC, Gologorsky D, Marr BP, et al. Extraocular extension of uveal melanoma after fine-needle aspiration, vitrectomy, and open biopsy. JAMA Ophthalmol 2013;131:1220–4. 12. Materin MA. Extraocular extension from FNAB? Cleveland: ISOO; 2013. 13. Eide N, Walaas L. Fine-needle aspiration biopsy and other biopsies in suspected intraocular malignant disease: a review. Acta Ophthalmol 2009;87:588–601. 14. Augsburger JJ. Fine needle aspiration biopsy of suspected metastatic cancers to the posterior uvea. Trans Am Ophthalmol Soc 1988;86:499–560. 15. Augsburger JJ, Shields JA. Fine needle aspiration biopsy of solid intraocular tumors: indications, instrumentation and techniques. Ophthalmic Surg 1984;15:34–40. 16. Brainard JA, Biscotti CV. Cytological preparation. Monogr Clin Cytol 2012;21:10–6. 17. Seregard S. To biopsy or not to biopsy? Acta Ophthalmol 2009;87:586–7. 18. Akgul H, Otterbach F, Bornfeld N, Jurklies B. Intraocular biopsy using special forceps: a new instrument and refined surgical technique. Br J Ophthalmol 2011;95:79–82. 19. Matthews BJ, Mudhar HS, Rennie IG. Trans-corneal fine cannula aspiration: Rycroft cannula aspiration technique for sampling iris tumours. Br J Ophthalmol 2012;96:329–31. 20. Pelayes DE, Zarate JO, Biscotti CV, Singh AD. Calibrated needle for ophthalmic fine needle aspiration biopsy. Br J Ophthalmol 2012;96:1147–8.
Footnotes and Financial Disclosures Originally received: January 26, 2015. Final revision: April 5, 2015. Accepted: April 9, 2015. Available online: May 23, 2015.
Author Contributions: Conception and design: Biscotti, A.D. Singh Manuscript no. 2015-136.
1
Department of Ophthalmic Oncology, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio.
2
Department of Anatomic Pathology, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Supported by the FALK Trust (Chicago, IL), the Ratner Foundation (NY, NY), and in part by the Cole Eye Institute and a Research to Prevent Blindness unrestricted grant.
1584
Data collection: Medina, Biscotti, A.D. Singh Analysis and interpretation: Medina, Biscotti, A.D. Singh, N. Singh Obtained funding: Not applicable Overall responsibility: Medina, Biscotti, A.D. Singh, N. Singh Abbreviations and Acronyms: FNAB ¼ fine-needle aspiration biopsy. Correspondence: Arun D. Singh, MD, Department of Ophthalmic Oncology, Cleveland Clinic Cole Eye Institute, 9500 Euclid Avenue, Desk i32, Cleveland, OH 44195. E-mail: [email protected].
The majority of intraocular tumors can be diagnosed on the basis of clinical examination and ocular imaging studies. The Collaborative Ocular Melanoma Study reported a misdiagnosis rate of 0.48%: Of 413 eyes with a clinical diagnosis of choroidal melanoma, only 2 were found to be misdiagnosed on histopathology.1 However, the Collaborative Ocular Melanoma Study had strict inclusion and exclusion criteria, and only large and obvious melanomas were included, with the exclusion of difficult diagnostic cases, such as those with media opacification.1 In clinical practice, the diagnosis of choroidal melanoma is not always clear, particularly in the setting of a confusing clinical pattern such as an amelanotic uveal tumor, a history of carcinoma, media opacities, or when dealing with smaller or atypical tumors.2 It is estimated that 1% to 2% of all cases require diagnostic fine-needle aspiration biopsy (FNAB).3,4 Rates of complications from intraocular FNAB are relatively low. Frequently used needles range from 25 to 30 gauge.5,6 Although using a larger needle increases the likelihood of obtaining a sufficient sample, the risk of complications associated with the procedure also increases. In more than 200 000 cases of FNABs reported in the literature, there has been no evidence of local or systemic spread of tumor cells with the use of 25-gauge or smallerdiameter needles.3,7e12 Transient localized subretinal and
1580
2015 by the American Academy of Ophthalmology Published by Elsevier Inc.
vitreous hemorrhage at the biopsy site are the most frequent complication.3,13 An experienced cytopathologist can achieve a high concordance of 96% between cytologic and histologic diagnosis.3,6,8,14 Nevertheless, cytologic characteristics that are diagnostic of uveal melanoma have not been described. The purpose of this study is to report the diagnostic cytologic characteristics of uveal melanoma.
Methods All patients with a clinical diagnosis of uveal melanoma who underwent prognostication biopsy from May 2009 to July 2013 were included. Institutional review board approval was obtained (local institutional review board no.: CASE 5608-CC666) for this prospective, single-center study of consecutive patients, and clinical and diagnostic cytologic features of uveal melanoma were analyzed. Factors such as suspected diagnosis, size, presence of retinal detachment, visibility, location, and patient and surgeon preference factored in determining whether the patient underwent prognostic biopsy and was subsequently included in the study. All patients who underwent prognostic biopsy were included in this study. All patients provided appropriate consent, and approval from the institutional review board was obtained. Biopsy technique and instrumentation varied by the location of the tumor. Biopsy was obtained by a transcorneal, transscleral, or http://dx.doi.org/10.1016/j.ophtha.2015.04.013 ISSN 0161-6420/15
Medina et al
Diagnostic Cytologic Features of Uveal Melanoma
transvitreal approach. A 25-gauge needle attached to short tubing and a 5-ml syringe was used for all approaches. For transcorneal biopsy, a 1-mm incision was created at the limbus and viscoelastic material was injected over the biopsy site. Care was taken to avoid the lens, particularly in phakic patients. After FNAB, the intraocular pressure was increased with balanced salt solution to control hemorrhage. A single 10-0 nylon suture was used to close the wounds. A short 25-gauge needle was used for tumors anterior to the equator where a transscleral approach was used. A partialthickness 80% scleral flap was created with a crescent blade. For tumors posterior to the equator, the biopsy was obtained via a long 25-gauge needle. The needle was inserted through the pars plana and into the tumor under visualization by indirect ophthalmoscopic control. As with all 3 techniques, once the needle is inserted into the tumor, gentle aspiration was applied by slowly withdrawing the syringe plunger up to the 3-ml mark. Finally, the needle was then carefully withdrawn following the original path of insertion.15 The contents of the needle, tubing, and syringe were then rinsed out with Cytolyt solution (Hologic Inc, Marlborough, MA) for ThinPrep (Hologic Inc.) processing. No immediate adequacy check was performed except for visual inspection of debris in the sample. The sample was then subjected to 1 or more centrifugation and concentration steps. The pellet obtained was resuspended in a cellpreserving solution (PreservCyt; Hologic Inc.) for additional processing. The ThinPrep processor mixed the sample, and then, using a gentle vacuum, collected cells on a filter in an evenly dispersed manner that approaches a monolayer. The filter was then inverted, and its cellular contents were transferred to a microscope slide.16 The method used optimized cellular yield and preservation while standardizing slide preparation for interpretation. One of the authors (C.V.B.) examined Papanicolaou-stained ThinPrep slides from the aspirate samples of all of the cases for the following cellular features: cell type, presence of melanin, nuclear grade, tumor-infiltrating lymphocytes, and necrosis. Cytology was then correlated with histopathology in enucleated eyes. Cell type was classified as spindle cell type if all of the tumor cells had spindleshaped cytoplasm, epithelioid type if none of the tumor cells had spindle-shaped cytoplasm, and mixed cell type for all others. Grade 3 nuclear atypia had conspicuous nucleoli when observed through the 10 objective, 4-fold nuclear pleomorphism, hyperchromasia, and coarse chromatin. Grade 1 nuclei had none of these features. All other tumors were regarded as nuclear grade 2. Ultimately, grade 3 nuclear atypia was referred to as high grade, and grades 1 and 2 were grouped into low grade for the purposes of statistical analysis. Tumorinfiltrating lymphocytes were classified as present or absent. Lymphocytes infiltrating aggregates of melanoma cells or intimately associated with melanoma cell cytoplasm were required for the presence of tumor-infiltrating lymphocytes. The presence or absence of melanin and necrosis was recorded. Statistical analysis to determine factors influencing adequacy of samples was then performed. To test associations between categoric variables, the Fisher exact test was used. For hypothesis testing related to continuous outcomes, equal variance assumptions were made on the basis of examination of boxplots. For associations between continuous variables and 2 categories where equal variance was not assumed, Welch’s t test was used. Associations between continuous variables and multiple categories were tested by analysis of variance. In those multiple categories, significant differences in means were assessed using Tukey’s correction for multiple comparisons of means. R version 3.02 was used for all analyses.
impression cytology was used. A total of 143 FNAB samples from 143 patients formed the basis of the study. This included 76 samples (55%) from the right eye and 67 samples (47%) from the left eye. A total of 76 patients (53%) were male, and 67 patients (47%) were female. The average age of all patients was 60 years, with the youngest aged 26 years and the oldest aged 92 years. Tumor location included 4 iris (3%), 4 iridociliary (3%), 4 ciliary (3%), 25 ciliochoroidal (18%), and 106 choroidal (74%) tumors. Biopsy was performed using a transcorneal approach in 8 patients (5%), and 71 patients (50%) and 64 patients (45%) underwent transscleral and transvitreal biopsy, respectively. All patients were treated after FNAB. A total of 102 patients (71%) underwent plaque brachytherapy, and 41 patients (29%) underwent enucleation. Average follow-up was 24 months, with a range of 0.5 to 51 months. At study closure (July 1, 2013), 128 patients (90%) were alive. Eleven patients (8%) died with known metastatic disease, and 4 patients (3%) died of unrelated causes (Table 1). Six patients (4%) were alive with known metastatic disease. Adequate samples were defined as those with sufficient cellularity to ascertain a diagnosis. Of 143 samples, 131 adequate samples were obtained (92% true positive). Of the remaining 12 inadequate samples, 10 contained nondiagnostic atypical cells and 2 lacked even atypical cells. One contained benign squamous cells and columnar cells suggestive of conjunctival contamination, and one contained rare histiocytes. Both height and largest basal dimension were correlated with nuclear grade. High-grade tumors with an average height of 8.1 mm were significantly higher than low-grade tumors (both grades 1 and 2), with an average height of 5.6 mm. Basal diameter for highTable 1. Diagnostic Cytologic Features of Uveal Melanoma: Profile of Study Patients (n ¼ 143) Parameter
Results
Sex Male Female Age (yrs) Range Average Eye Right Left Location Iris Iridociliary Ciliary body Ciliochoroidal Choroidal FNAB approach Transcorneal Transscleral Transvitreal Treatment Plaque Enucleation Follow-up (mos) Average Range Status Alive without metastases Alive with metastases Dead without metastases Dead with metastases
A total of 150 samples from 150 patients were obtained. Seven samples were excluded because these contained tumor resections where
FNAB ¼ fine-needle aspiration biopsy.
No. 76 67 26e91 60 76 67 4 4 4 25 106 7 72 64 102 41 24 0.5e51 122 6 4 11
1581
Ophthalmology Volume 122, Number 8, August 2015
Figure 1. Box plots of tumor size in millimeters (largest basal diameter [right] and height [left]) grouped by tumor nuclear grade. There was an association between tumor grade and tumor height (P ¼ 0.02, analysis of variance [ANOVA]). Using the Tukey correction for the comparison of means, tumor grades 1 and 2 are not statistically different (P ¼ 0.85), tumor grade 3 is marginally insignificantly taller (P ¼ 0.06), and tumor grade 3 is significantly taller than tumor grade 2 (P ¼ 0.03). There was an association between tumor grade and tumor basal diameter (P ¼ 0.004, ANOVA). Using the Tukey correction for the comparison of means, tumor grades 1 and 2 are not statistically different (P ¼ 0.32), and tumor grade 3 is wider than grade 1 (P ¼ 0.005) and grade 2 tumors (P ¼ 0.017).
grade tumors was 16 mm compared with 12.2 mm for low-grade tumors (Fig 1). There was no significant correlation between tumor height or basal diameter and cytologic type (Fig 2). Within diagnostically adequate samples, a combination of spindle and epithelioid cells were observed in 63% of samples (Figs 3 and 4). Pure epithelioid cells were present in only 1.75% of samples, and 35% had pure spindle cells (Table 2). Overall, spindle cells were observed in 98% of samples. Cytoplasmic melanin was present in 80% of samples (Fig 5). Tumor nuclear grade also varied by tumor location, with the least atypia seen with iris tumors. None of the iris tumors displayed high-grade nuclear atypia. Ciliary body tumors had significantly more atypia, with 36% of tumors categorized as high grade. Tumor-infiltrating lymphocytes were present in 31% of samples. Some 79% of tumors were described as having a low nuclear grade. There was 100% correlation between cytologic and histologic diagnosis in all 41 enucleated eyes.
Discussion To our knowledge, this study is the first prospective study to analyze the diagnostic cytologic features of uveal melanoma. The study sample included a broad spectrum of tumors; size and location were variable, ranging from small to large from iris to choroid. Histopathologic concordance was 100% in all specimens that subsequently underwent enucleation (n ¼ 41). Spindle cells were common among adequate samples, with 98% of samples containing pure spindle cells or a combination of these cells. Pure epithelioid cells were uncommon, with only 2 samples (1.75%) containing purely epithelioid cells. Melanin was present in 79.8% of samples, yet both
Figure 2. Box plots of tumor size (basal diameter [right] and height [left]) by cytologic cell type. There was no significant difference in tumor height (P ¼ 0.486, analysis of variance [ANOVA]) or basal diameter (P ¼ 0.077, ANOVA).
1582
Medina et al
Diagnostic Cytologic Features of Uveal Melanoma Table 2. Diagnostic Cytologic Features of Uveal Melanoma: Adequate Samples (n ¼ 131) Features Cell type/pattern Mixed Spindle Epithelial Nuclear grade Low High TILS Yes No Necrosis Yes No Distribution of melanin Yes No
Adequate Samples (n [ 131), % 63 35 1.75 79 21 31 69 4 96 79.8 20.2
samples with pure epithelioid cells had no melanin. Atypia, as in histologic analysis, was correlated with location. Iris tumors contained bland nuclear features compared with other locations. The presence or absence of tumor-infiltrating
lymphocytes was not helpful in establishing a diagnosis. The presence of spindle cells and melanin was deemed an important diagnostic feature and may be helpful in the future for establishing diagnostic criteria. We considered true-positive samples those that were positive for melanoma and those with cytologic findings consistent with the diagnosis of melanoma (92%). Our data are similar to the previously published adequacy of FNAB samples ranging from 88% to 95%.3,4 Additional analysis is underway to identify factors that may contribute to the inadequacy of FNAB samples. It is worth emphasizing that a negative cytologic diagnosis of malignancy should not be considered unequivocal proof that an intraocular malignancy does not exist.8,13,17 As FNAB gains popularity with an improved safety profile, techniques, and instrumentation, there is a need to develop descriptive cytologic diagnostic features of uveal melanoma.18e20 Because cytologic diagnosis is
Figure 4. Most of the cells in this uveal melanoma have an epithelioid appearance. Note the higher-grade nuclear atypia compared with the example in Figure 3.
Figure 5. The spindle-shaped melanoma cell to the upper right has prominent melanin pigmentation.
Figure 3. This uveal melanoma illustrates the characteristic cellular features. Note the spindle cell shape characterized by elongated, often caudate cytoplasm. Note also the relatively bland nuclear features, grade 2 by our criteria (Papanicolaou stain).
1583
Ophthalmology Volume 122, Number 8, August 2015 predominantly morphologically driven, automated digital methods that can identify, quantify, and characterize spindle cells in an ocular FNAB cytopathology slide can be developed for computer-assisted diagnosis.
References 1. Accuracy of diagnosis of choroidal melanomas in the Collaborative Ocular Melanoma Study. COMS report no. 1. Arch Ophthalmol 1990;108:1268–73. 2. Char DH, Kemlitz AE, Miller T. Intraocular biopsy. Ophthalmol Clin North Am 2005;18:177–85. 3. Shields JA, Shields CL, Ehya H, et al. Fine-needle aspiration biopsy of suspected intraocular tumors. The 1992 Urwick Lecture. Ophthalmology 1993;100:1677–84. 4. Char DH, Miller T. Accuracy of presumed uveal melanoma diagnosis before alternative therapy. Br J Ophthalmol 1995;79: 692–6. 5. Young TA, Burgess BL, Rao NP, et al. Transscleral fine-needle aspiration biopsy of macular choroidal melanoma. Am J Ophthalmol 2008;145:297–302. 6. Singh A, Pelayes D, Brainard J, Biscotti C. History, indications, technique and limitations. In: Biscotti C, Singh A, eds. FNA Cytology of Ophthalmic Tumors. Basel, Switzerland: Karger; 2011:2–9. 7. Pelayes DE, Zarate JO. Fine needle aspiration biopsy with liquidbased cytology and adjunct immunohistochemistry in intraocular melanocytic tumors. Eur J Ophthalmol 2010;20:1059–65. 8. Augsburger JJ, Shields JA, Folberg R, et al. Fine needle aspiration biopsy in the diagnosis of intraocular cancer. Cytologichistologic correlations. Ophthalmology 1985;92:39–49.
9. Glasgow BJ, Brown HH, Zargoza AM, Foos RY. Quantitation of tumor seeding from fine needle aspiration of ocular melanomas. Am J Ophthalmol 1988;105:538–46. 10. Karcioglu ZA, Gordon RA, Karcioglu GL. Tumor seeding in ocular fine needle aspiration biopsy. Ophthalmology 1985;92: 1763–7. 11. Schefler AC, Gologorsky D, Marr BP, et al. Extraocular extension of uveal melanoma after fine-needle aspiration, vitrectomy, and open biopsy. JAMA Ophthalmol 2013;131:1220–4. 12. Materin MA. Extraocular extension from FNAB? Cleveland: ISOO; 2013. 13. Eide N, Walaas L. Fine-needle aspiration biopsy and other biopsies in suspected intraocular malignant disease: a review. Acta Ophthalmol 2009;87:588–601. 14. Augsburger JJ. Fine needle aspiration biopsy of suspected metastatic cancers to the posterior uvea. Trans Am Ophthalmol Soc 1988;86:499–560. 15. Augsburger JJ, Shields JA. Fine needle aspiration biopsy of solid intraocular tumors: indications, instrumentation and techniques. Ophthalmic Surg 1984;15:34–40. 16. Brainard JA, Biscotti CV. Cytological preparation. Monogr Clin Cytol 2012;21:10–6. 17. Seregard S. To biopsy or not to biopsy? Acta Ophthalmol 2009;87:586–7. 18. Akgul H, Otterbach F, Bornfeld N, Jurklies B. Intraocular biopsy using special forceps: a new instrument and refined surgical technique. Br J Ophthalmol 2011;95:79–82. 19. Matthews BJ, Mudhar HS, Rennie IG. Trans-corneal fine cannula aspiration: Rycroft cannula aspiration technique for sampling iris tumours. Br J Ophthalmol 2012;96:329–31. 20. Pelayes DE, Zarate JO, Biscotti CV, Singh AD. Calibrated needle for ophthalmic fine needle aspiration biopsy. Br J Ophthalmol 2012;96:1147–8.
Footnotes and Financial Disclosures Originally received: January 26, 2015. Final revision: April 5, 2015. Accepted: April 9, 2015. Available online: May 23, 2015.
Author Contributions: Conception and design: Biscotti, A.D. Singh Manuscript no. 2015-136.
1
Department of Ophthalmic Oncology, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio.
2
Department of Anatomic Pathology, Cleveland Clinic Cole Eye Institute, Cleveland, Ohio. Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article. Supported by the FALK Trust (Chicago, IL), the Ratner Foundation (NY, NY), and in part by the Cole Eye Institute and a Research to Prevent Blindness unrestricted grant.
1584
Data collection: Medina, Biscotti, A.D. Singh Analysis and interpretation: Medina, Biscotti, A.D. Singh, N. Singh Obtained funding: Not applicable Overall responsibility: Medina, Biscotti, A.D. Singh, N. Singh Abbreviations and Acronyms: FNAB ¼ fine-needle aspiration biopsy. Correspondence: Arun D. Singh, MD, Department of Ophthalmic Oncology, Cleveland Clinic Cole Eye Institute, 9500 Euclid Avenue, Desk i32, Cleveland, OH 44195. E-mail: [email protected].
