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Are Risk Factors for Growth of Choroidal Nevi Associated With Malignant Transformation? Assessment With a Validated Genomic Biomarker

Published:September 06, 2018DOI:https://doi.org/10.1016/j.ajo.2018.08.045

      Purpose

      To test the hypothesis that widely used clinical risk factors for growth of choroidal nevi are associated with malignant transformation.

      Methods

      Fine needle biopsy for assignment of gene expression profile (class 1 or class 2) was performed in 207 choroidal melanocytic tumors < 3.5 mm in thickness. The class 2 profile was employed as a validated biomarker for malignant transformation. The following data were collected: patient age and sex, tumor diameter and thickness, distance of posterior tumor margin from the optic disc, and the presence or absence of serous retinal detachment, orange lipofuscin pigment, drusen, retinal pigment epithelial fibrosis, retinal pigment epithelial atrophy, visual symptoms, and documented tumor growth.

      Results

      Clinical features associated with the class 2 profile included patient age > 60 years and tumor thickness > 2.25 mm (Fisher exact test, P = .002 for both). Documented growth was not associated with the class 2 profile (P = .5). The odds ratio of a tumor having the class 2 profile was 2.8 (95% confidence interval 1.3–5.9) for patient age > 60 years and 3.5 (95% confidence interval 1.4–8.8) for tumor thickness > 2.25 mm. For patients with both risk factors, the “number needed to treat” to identify 1 patient with a class 2 tumor was 4.3 (P = .0002). No other clinical feature or combination of features was associated with the class 2 profile.

      Conclusions

      None of the widely used choroidal nevus risk factors for tumor growth, nor documented growth itself, is pathognomonic of malignant transformation as defined by class 2 gene expression profile. Patient age and tumor thickness may be helpful for identifying small choroidal melanocytic tumors that are more likely to have the class 2 profile. Observation for growth prior to treatment continues to be reasonable for most patients with suspicious choroidal nevi. NOTE: Publication of this article is sponsored by the American Ophthalmological Society.

      Introduction

       Distinguishing Choroidal Nevi From Small Choroidal Melanomas

      Choroidal melanocytic tumors comprise a spectrum from small flat benign nevi to large elevated high-risk melanomas, with lesions at either extreme being relatively easy to diagnose. However, it has been a long-standing challenge to distinguish between large choroidal nevi that can be safely observed and small choroidal melanomas that should be treated promptly.
      • Augsburger J.J.
      • Correa Z.M.
      • Trichopoulos N.
      • Shaikh A.
      Size overlap between benign melanocytic choroidal nevi and choroidal malignant melanomas.
      Many names have been proposed for this equivocal category of lesions, including nevoma, indeterminate melanocytic lesion, indeterminate pigmented choroidal tumor, dormant melanoma, and suspicious choroidal nevus.
      • Shields J.A.
      • Augsburger J.J.
      • Brown G.C.
      • Stephens R.F.
      The differential diagnosis of posterior uveal melanoma.
      • Augsburger J.J.
      • Schroeder R.P.
      • Territo C.
      • Gamel J.W.
      • Shields J.A.
      Clinical parameters predictive of enlargement of melanocytic choroidal lesions.
      • Butler P.
      • Char D.H.
      • Zarbin M.
      • Kroll S.
      Natural history of indeterminate pigmented choroidal tumors.
      • Gragoudas E.
      • Lane A.M.
      • Kim I.
      Indeterminate melanocytic lesions of the choroid.
      In his XXXIII Edward Jackson Memorial lecture, Gass referred to these lesions as “melanocytic choroidal tumors of uncertain biologic activity,” and he was among the first to describe clinical features predictive of tumor growth.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanoma. XXXIII Edward Jackson Memorial lecture.
      The “Doctor Gass” risk factors include visual symptoms, increased tumor size (thickness and diameter), orange lipofuscin pigment, serous retinal detachment, intrinsic tumor vessels, fluorescein angiography hot spots, and the absence of chronic changes induced by the tumor such as drusen, retinal pigment epithelial atrophy and fibrosis, intraretinal pigment migration, choroidal neovascularization, and overlying cystic retina degeneration (Table 1). Remarkably, 40 years after Gass's pioneering work, we still use most of these risk factors. While many subsequent studies have validated Gass's findings, few have identified new risk factors.
      Table 1“Doctor Gass” Clinical Features Predictive of Growth of Suspicious Choroidal Nevi
      Mnemonic LetterClinical FeatureFavorable or Unfavorable
      DDrusenFavorable
      OOverlying retinal degenerationFavorable
      CChronic RPE changes
      Chronic RPE changes include atrophy, fibrosis, and choroidal neovascularization. The widely used thickness threshold of 2 mm was suggested by Gass and is somewhat arbitrary, whereas this study indicates an optimal threshold of 2.25 mm. Reflectivity refers to low internal reflectivity, also called internal acoustic quiet zone or acoustic hollowness. Symptoms attributable to the tumor can include blurring, photopsia, metamorphopsia, and scotoma. All of these features were described by Gass, except low internal reflectivity.6,7
      Favorable
      TThickness > 2 mmUnfavorable
      OOrange lipofuscin pigmentUnfavorable
      RReflectivity (low)Unfavorable
      GGirth (diameter)Unfavorable
      AAngiographic hot spotsUnfavorable
      SSubretinal fluidUnfavorable
      SSymptomsUnfavorable
      RPE = retinal pigment epithelium.
      a Chronic RPE changes include atrophy, fibrosis, and choroidal neovascularization. The widely used thickness threshold of 2 mm was suggested by Gass and is somewhat arbitrary, whereas this study indicates an optimal threshold of 2.25 mm. Reflectivity refers to low internal reflectivity, also called internal acoustic quiet zone or acoustic hollowness. Symptoms attributable to the tumor can include blurring, photopsia, metamorphopsia, and scotoma. All of these features were described by Gass, except low internal reflectivity.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanoma. XXXIII Edward Jackson Memorial lecture.
      Augsburger and associates evaluated 197 “melanocytic choroidal lesions,” 39 of which grew during follow-up, for a 5-year actuarial growth rate of 26%.
      • Augsburger J.J.
      • Schroeder R.P.
      • Territo C.
      • Gamel J.W.
      • Shields J.A.
      Clinical parameters predictive of enlargement of melanocytic choroidal lesions.
      Features predictive of growth included increased tumor thickness, serous retinal detachment, tumor margin within 2 disc diameters of the optic disc, presence of symptoms, and presence of orange pigment. The best combination of features for prediction of lesion growth included tumor thickness, serous retinal detachment, and symptoms. The 5-year rate of growth was 5.8% when none of these factors was present, and 90.6% when all features were present.
      Butler and associates undertook a detailed statistical analysis of 293 “indeterminate pigmented choroidal tumors,” 98 of which grew on follow-up, representing a 36% 5-year actuarial growth rate. Greater tumor thickness, presence of symptoms, orange pigment, internal acoustic quiet zone on B-scan, and hot spots on fluorescein angiography all were significant predictors of tumor enlargement (relative risk of detectable tumor growth, >1.8).
      • Butler P.
      • Char D.H.
      • Zarbin M.
      • Kroll S.
      Natural history of indeterminate pigmented choroidal tumors.
      Interestingly, these investigators found that low internal reflectivity (“internal acoustic quiet zone”) was associated with tumor growth, whereas Gass did not find any ultrasound characteristics that were predictive of growth.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanoma. XXXIII Edward Jackson Memorial lecture.
      This may be owing to Butler's having access to better equipment than was available in 1977.
      The Collaborative Ocular Melanoma Study (COMS) performed a prospective observational study of “small choroidal melanomas.”
      • COMS
      Factors predictive of growth and treatment of small choroidal melanoma: COMS Report No. 5. The Collaborative Ocular Melanoma Study Group.
      Among 188 patients enrolled in this study, subsequent growth was documented in 46 cases, with an actuarial growth rate of 21% at 2 years and 31% at 5 years. Features associated with growth included increased tumor thickness and diameter, presence of orange pigment, absence of drusen, and absence of chronic retinal pigment epithelial changes.
      Shields and associates analyzed 2514 “choroidal nevi” and observed growth in 2%, 9%, and 13% at 1, 5, and 10 years, respectively.
      • Shields C.L.
      • Furuta M.
      • Berman E.L.
      • et al.
      Choroidal nevus transformation into melanoma: analysis of 2514 consecutive cases.
      Factors predictive of growth included tumor thickness > 2 mm, serous retinal detachment symptoms, orange pigment, tumor margin within 3 mm of the optic disc, ultrasonographic “hollowness” (low internal reflectivity), and the absence of a “halo” (ring of depigmentation around the tumor). The median hazard ratio associated with 1–2 features was 3; for 3–4 features was 5; for 5–6 features was 9; and for all 7 features was 21. The 2 “new” features in this report—ultrasonographic hollowness and halo—were previously described by Butler and associates.
      • Butler P.
      • Char D.H.
      • Zarbin M.
      • Kroll S.
      Natural history of indeterminate pigmented choroidal tumors.
      As mentioned above, Butler found that ultrasonographic hollowness was associated with tumor growth, but the absence of a halo was not associated with growth. Since a halo is found in only about 6% of choroidal nevi,
      • Butler P.
      • Char D.H.
      • Zarbin M.
      • Kroll S.
      Natural history of indeterminate pigmented choroidal tumors.
      and is of disputed prognostic value, we have not found this feature to be of clinical utility.

       Growth Versus Malignant Transformation

      A common assumption regarding these choroidal nevus risk factors is that growth is equivalent to malignant transformation, and that these risk factors can identify small choroidal melanomas that should be promptly treated rather than initially observed.
      • Shields C.L.
      • Furuta M.
      • Berman E.L.
      • et al.
      Choroidal nevus transformation into melanoma: analysis of 2514 consecutive cases.
      • Shields J.A.
      Treating some small melanocytic choroidal lesions without waiting for growth.
      • Kaiserman I.
      • Kaiserman N.
      • Pe'er J.
      Long term ultrasonic follow up of choroidal naevi and their transformation to melanomas.
      But is this a reasonable assumption?
      In a review of 2627 cases of choroidal melanoma over a 40-year period, Zimmerman and McLean concluded that there was “no support for the concept that dissemination leading to metastasis begins with the onset of … local growth.”
      • Zimmerman L.E.
      • McLean I.W.
      Do growth and onset of symptoms of uveal melanomas indicate subclinical metastasis?.
      Other studies have demonstrated that histopathologically benign choroidal melanocytic tumors can grow.
      • Abramson D.H.
      Growing melanocytic tumor is not always cancer.
      • Elner V.M.
      • Flint A.
      • Vine A.K.
      Histopathology of documented growth in small melanocytic choroidal tumors.
      Furthermore, the definitions of growth used in the literature are arbitrary and inadequate. Why, for example, would lesion enlargement of 0.5 mm be indicative of malignant transformation,
      • Shields C.L.
      • Furuta M.
      • Berman E.L.
      • et al.
      Choroidal nevus transformation into melanoma: analysis of 2514 consecutive cases.
      but 0.4 mm would not? The rate of growth is also important but has rarely been considered in published studies. For example, an increase in thickness of 0.5 mm over 1 month would be more concerning for malignancy than the same amount of growth occurring over 10 years. Taken together, these lines of evidence raise doubts about the use of tumor growth as an indicator of malignant transformation. Yet, it is not feasible to use metastasis—the defining feature of malignant transformation—as the endpoint in studies of choroidal nevi owing to the low rate of metastasis and long latency period.

       Gene Expression Profiling

      Fortunately, recent discoveries in the molecular genetics of uveal melanoma provide a more accurate means of assessing the malignant potential of a choroidal melanocytic tumor. In 2004, we reported that gene expression profiling could be used to predict the risk of metastasis in uveal melanomas treated by enucleation.
      • Onken M.D.
      • Worley L.A.
      • Ehlers J.P.
      • Harbour J.W.
      Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death.
      Tumors with the class 1 profile had a low metastatic risk, whereas those with the class 2 profile had a high metastatic risk. Subsequently, our group and others showed that gene expression profiling was superior to clinical, histopathologic, and chromosome markers for predicting which uveal melanoma would metastasize.
      • Worley L.A.
      • Onken M.D.
      • Person E.
      • et al.
      Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma.
      • Petrausch U.
      • Martus P.
      • Tonnies H.
      • et al.
      Significance of gene expression analysis in uveal melanoma in comparison to standard risk factors for risk assessment of subsequent metastases.
      • Singh A.D.
      • Sisley K.
      • Xu Y.
      • et al.
      Reduced expression of autotaxin predicts survival in uveal melanoma.
      • van Gils W.
      • Lodder E.M.
      • Mensink H.W.
      • et al.
      Gene expression profiling in uveal melanoma: two regions on 3p related to prognosis.
      We refined and migrated the gene expression profile to a highly accurate and reproducible microfluidics real-time polymerase chain reaction (PCR) platform comprising 12 discriminating genes and 3 control genes suitable for analysis of clinical specimens obtained by fine needle biopsy.
      • Onken M.D.
      • Worley L.A.
      • Davila R.M.
      • Char D.H.
      • Harbour J.W.
      Prognostic testing in uveal melanoma by transcriptomic profiling of fine needle biopsy specimens.
      • Onken M.D.
      • Worley L.A.
      • Tuscan M.D.
      • Harbour J.W.
      An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma.
      Further work has shown that the class 1 profile is indicative of a differentiated melanocytic phenotype that is seen not only in low-grade uveal melanomas, but also in benign uveal nevi, melanocytomas, and even normal choroidal melanocytes.
      • Onken M.D.
      • Ehlers J.P.
      • Worley L.A.
      • Makita J.
      • Yokota Y.
      • Harbour J.W.
      Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.
      • de Alba M.A.
      • Villegas V.M.
      • Gold A.S.
      • et al.
      Clinical findings and genetic expression profiling of three pigmented lesions of the optic nerve.
      In contrast, tumors with the class 2 profile exhibit features of cytologic de-differentiation and expression of cancer stem cell markers.
      • Onken M.D.
      • Ehlers J.P.
      • Worley L.A.
      • Makita J.
      • Yokota Y.
      • Harbour J.W.
      Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.
      • Chang S.H.
      • Worley L.A.
      • Onken M.D.
      • Harbour J.W.
      Prognostic biomarkers in uveal melanoma: evidence for a stem cell-like phenotype associated with metastasis.
      A large prospective multicenter clinical trial performed by the Collaborative Ocular Oncology Group (COOG) validated the prognostic accuracy of the 15-gene clinical-grade profile, and confirmed its superior prognostic accuracy compared to clinical, histopathologic, and chromosomal features.
      • Onken M.D.
      • Worley L.A.
      • Char D.H.
      • et al.
      Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma.
      With the subsequent availability and expanding use of the commercially available version of the 15-gene profile (DecisionDx-UM), ocular oncologists have found that accurate molecular prognostic results can readily be obtained by fine needle biopsy in almost all cases, even for very small tumors in which biopsy yielded insufficient material for cytopathologic examination.
      • Correa Z.M.
      • Augsburger J.J.
      Sufficiency of FNAB aspirates of posterior uveal melanoma for cytologic versus GEP classification in 159 patients, and relative prognostic significance of these classifications.
      • Plasseraud K.M.
      • Wilkinson J.K.
      • Oelschlager K.M.
      • et al.
      Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics.
      Thus, for purposes of this thesis, we have defined malignant transformation as having occurred if a choroidal melanocytic tumor exhibits the class 2 gene expression profile.

       Hypothesis

      The term “malignant transformation” indicates that a primary tumor has undergone a complex series of genetic, molecular, and cellular changes associated with the ability to metastasize.
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      Since gene expression profiling of choroidal melanocytic tumors “captures a functional ‘snapshot’ of the tumor's microenvironment,” we argue that the presence of the class 2 gene expression profile is the most accurate method currently available for assessing whether the complex processes associated with malignant transformation have occurred in a given tumor. Contrariwise, it seems unlikely that such complex changes can be adequately accounted for by simple clinical features. Therefore, we hypothesized that most clinical risk factors for growth of suspicious choroidal melanocytic tumors are not associated with malignant transformation. To test this hypothesis, we analyzed each clinical risk factor, individually and in combination, for statistically significant association with the class 2 gene expression profile. This approach allowed us to overcome the difficulties associated with tumor growth as an indicator of malignant transformation, and it avoided the unfeasibly long time and large number of patients that would be required to use metastasis as the endpoint of a prospective study.

      Methods

       Data Sources

      Data sources included patients from the ocular oncology practice of J.W.H. at Washington University (enrolled between November 22, 2006, and November 12, 2011), the ocular oncology practice of J.W.H. at Bascom Palmer Eye Institute (enrolled between January 23, 2013 and June 20, 2017), and the ocular oncology practice of J.J.A. and Z.M.C. at the University of Cincinnati (enrolled between October 26, 2007, and May 19, 2015). The institutional review boards of Washington University, University of Miami, and University of Cincinnati approved this retrospective study before data collection began at each respective institution. Informed consent was obtained from each patient for clinical treatment and participation in research. Patient information was accessed in compliance with the Health Insurance Portability and Accountability Act.
      The study was limited to patients who underwent fine needle biopsy for lesions diagnosed as suspicious choroidal nevus or small choroidal melanoma with tumor thickness ≤ 3.5 mm that was predominantly (>90%) confined to the choroid. This thickness cut-off was chosen to allow objective determination of the optimal thickness threshold associated with increased risk of malignant transformation, rather than using an arbitrary threshold. Melanocytic tumors confined to the iris and/or ciliary body were excluded because they do not typically exhibit the choroidal nevus clinical features that were the subject of this study. The decision to biopsy was based on either (1) documented growth or (2) the presence of multiple clinical risk factors such that the risk of malignancy was deemed sufficiently high that biopsy was performed without waiting for growth. The following data were collected: patient age at biopsy, patient sex, tumor diameter (measured by indirect ophthalmoscopy and B-scan ultrasonography, whichever was larger), tumor thickness (measured using A- or B-scan ultrasonography), and distance of posterior tumor margin from the optic disc (measure by indirect ophthalmoscopy and/or fundus photography). The presence or absence of the following features was assessed by indirect ophthalmoscopy and fundus biomicroscopy in every case, and also by optical coherence tomography (OCT), fundus autofluorescence, and fluorescein angiography as deemed necessary for confirmation in selected cases: serous retinal detachment, orange lipofuscin pigment, drusen, retinal pigment epithelial fibrosis, and retinal pigment epithelial atrophy. The presence of low internal reflectivity was determined using A- or B-scan ultrasonography, and was defined as at least one third of the tumor interior demonstrating average reflectivity below one third that of the retina. Symptoms that were judged to be attributable to the tumor by the treating ocular oncologist (J.W.H., J.J.A., or Z.M.C.) were noted, including blurred vision, metamorphopsia, micropsia, photopsia, and/or floaters. Tumor growth and metastasis were not included as endpoints in this study, owing to the inherent inaccuracies associated with these variables, as discussed in the Introduction. Features described by Gass that were not analyzed in this study include intraretinal pigment migration, choroidal neovascularization, overlying retinal degeneration, and fluorescein angiographic hot spots (Table 1). Intraretinal pigment migration was not routinely noted, and choroidal neovascularization
      • Callanan D.G.
      • Lewis M.L.
      • Byrne S.F.
      • Gass J.D.
      Choroidal neovascularization associated with choroidal nevi.
      was not common enough to be useful. We have shown that overlying retinal degeneration detected by OCT is prognostically favorable,
      • Espinoza G.
      • Rosenblatt B.
      • Harbour J.W.
      Optical coherence tomography in the evaluation of retinal changes associated with suspicious choroidal melanocytic tumors.
      but OCT was not performed routinely throughout the entire study period. Similarly, fluorescein angiography is not part of our routine evaluation of choroidal nevi.

       Data Analysis Methods

      The method for performing the 15-gene expression profile has been described in detail elsewhere.
      • Harbour J.W.
      A prognostic test to predict the risk of metastasis in uveal melanoma based on a 15-gene expression profile.
      Briefly, a needle biopsy of the tumor is performed, and the aspirated contents are immediately expelled into an empty tube. Extraction buffer (200 μL) is drawn up into the needle to flush the hub, and is then expelled into the tube containing the tumor sample. RNA is isolated, reverse transcribed into cDNA, preamplified, loaded onto microfluidics cards, and analyzed by real-time quantitative PCR. Ct values are calculated for each of the 15 genes in the profile (Table 2), and the results are analyzed by a machine learning algorithm that assigns each new sample to class 1 or class 2. Prior to September 1, 2010, samples were analyzed in the Harbour Laboratory at Washington University. Thereafter, all samples were analyzed at the College of American Pathologists–accredited Clinical Laboratory Improvement Amendments–certified laboratory of Castle Biosciences (Friendswood, TX), under the trade name DecisionDx-UM.
      • Plasseraud K.M.
      • Wilkinson J.K.
      • Oelschlager K.M.
      • et al.
      Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics.
      • Harbour J.W.
      • Chen R.
      The DecisionDx-UM gene expression profile test provides risk stratification and individualized patient care in uveal melanoma.
      Table 2Genes Included in the 15-Gene Expression Profile
      Gene SymbolGene NameDirection in Class 2
      CDH1E-cadherinUp
      ECM1Extracellular matrix protein 1Up
      HTR2B5-Hydroxytryptamine (serotonin) receptor 2BUp
      RAB31RAB31, member RAS oncogene familyUp
      EIF1BEukaryotic translation initiation factor 1BDown
      FXR1Fragile X mental retardation, autosomal homolog 1Down
      ID2Inhibitor of DNA binding 2Down
      LMCD1LIM and cysteine-rich domains 1Down
      LTA4HLeukotriene A4 hydrolaseDown
      MTUS1Microtubule-associated tumor suppressor 1Down
      ROBO1Roundabout, axon guidance receptor, 1Down
      SATB1SATB homeobox 1Down
      MRPS21Mitochondrial ribosomal protein S21Control
      RBM23RNA-binding motif protein 23Control
      SAP130Sin3A-associated protein, 130 kDaControl

       Statistical Analysis

      All statistical analyses were performed in MedCalc software (version 15.8; MedCalc Software, Ostend, Belgium). The main study endpoint was class 2 gene expression profile. The optimal cut-off for continuous variables was determined by receiver operating characteristics analysis. Evaluation of clinical variables for statistically significant association with class 2 gene expression profile was performed using Fisher exact test. The best fit model for describing the relationship between dichotomous clinical variables and class 2 gene expression profile was determined using logistic regression. A P value of ≤ .05 was interpreted as statistically significant.

      Results

       Summary Data

      Baseline clinical characteristics are summarized in Table 3. We identified 207 patients who met the study inclusion criteria—112 (54.1%) female and 95 (45.9%) male—with a median age of 62 years (mean 61 years, range 15–88 years). Gene expression profile status was class 1 in 163 (78.7%) and class 2 in 39 (21.3%) cases. Median tumor thickness was 2.5 mm (mean 2.4 mm, range 1.0–3.5 mm), and LBD was 9.5 mm (mean 9.8 mm, range 2.5–17.0 mm). The tumor was located ≤ 3 mm from the disc in 112 (54.1%) cases. Visual symptoms attributable to the tumor were present in 120 (58.0%) cases. Subretinal fluid was detected in 136 (65.7%) cases, orange lipofuscin pigment in 121 (58.4%) cases, low acoustic internal reflectivity in 60 (29.0%) cases, drusen in 44 (21.0%) cases, retinal pigment epithelial fibrosis in 25 (12.3%) cases, and retinal pigment epithelial atrophy in 44 (21.3%) cases. Biopsy was performed owing to documented growth in 95 (45.9%) cases and the presence of multiple clinical risk factors in 112 (54.1%) cases.
      Table 3Clinical Features of Patients With Suspicious Choroidal Nevus
      VariableSummary Data (N = 207)
      Age at diagnosis, years
       Mean61
       Median (range)62 (15–88)
      Sex, n (%)
       Female112 (54.1)
       Male95 (45.9)
      Largest basal diameter (mm)
       Mean9.8
       Median (range)9.5 (2.5–17.0)
       ≤12, n (%)162 (78.2)
       >12, n (%)45 (21.7)
      Thickness (mm)
       Mean2.4
       Median (range)2.5 (1.0–3.5)
       ≤2.25, n (%)68 (32.9)
       >2.25, n (%)139 (67.1)
      Subretinal fluid, n (%)
       Yes137 (66.1)
       No49 (23.7)
       Not available22 (10.6)
      Visual symptoms, n (%)
       Yes120 (58.0)
       No86 (41.5)
       Not available1 (0.4)
      Orange pigment, n (%)
       Yes121 (58.4)
       No84 (40.5)
       Not Available2 (1.0)
      Tumor margin < 3 mm from optic disc, n (%)
       Yes112 (54.1)
       No94 (45.4)
       Not available1 (0.4)
      Drusen, n (%)
       Yes44 (21.3)
       No161 (77.8)
       Not available2 (1.0)
      RPE fibrosis, n (%)
       Yes25 (12.1)
       No179 (86.5)
       Not available3 (1.4)
      RPE atrophy, n (%)
       Yes44 (21.3)
       No159 (76.8)
       Not Available4 (2.0)
      Low acoustic internal reflectivity, n (%)
       Yes60 (29.0)
       No24 (11.6)
       Not available123 (59.4)
      Gene expression profile class, n (%)
       Class 1163 (78.7)
      Class 1A87 (42.0)
      Class 1B40 (19.3)
      Subclassification not available36 (17.4)
       Class 244 (21.3)
      Documented growth, n (%)
       Class 1
      Yes77 (37.2)
      No86 (41.5)
       Class 2
      Yes18 (8.7)
      No26 (12.6)
      RPE = retinal pigment epithelium.

       Receiver Operating Characteristics Analysis

      Receiver operating characteristics analysis was performed to identify optimal cut-offs for continuous variables (patient age, tumor largest basal diameter [LBD] and thickness) (Figure 1). The optimal discriminant threshold for patient age was >62 years (P = .002, Youden index J = 0.277), and for tumor thickness was >2.25 mm (P = .003, Youden index J = 0.244). The area under the curve for LBD did not reach statistical significance (P = .3, Youden index J = .120). For statistical analysis, we used an age cut-off of >60 years. For LBD, we selected 12 mm, since this cut-off was previously shown to be a significant threshold for metastatic risk in choroidal melanomas.
      • Walter S.D.
      • Chao D.L.
      • Feuer W.
      • Schiffman J.
      • Char D.H.
      • Harbour J.W.
      Prognostic implications of tumor diameter in association with gene expression profile for uveal melanoma.
      • Correa Z.M.
      • Augsburger J.J.
      Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas.
      Figure thumbnail gr1
      Figure 1Receiver operating characteristics analysis of (Left) patient age, (Middle) tumor thickness, and (Right) largest basal tumor diameter vs class 2 gene expression profile. The indicated point for age and thickness represent the optimal thresholds for sensitivity and specificity using the Youden J index.

       Clinical Features Associated With Class 2 Profile

      The Fisher exact test was performed to identify choroidal nevus clinical features associated with the class 2 gene expression profile (Table 4). In addition to the established choroidal nevus clinical features associated with lesion growth, we analyzed patient age and sex. The only features associated with class 2 gene expression profile were patient age > 60 years (P = .002) and tumor thickness > 2.25 mm (P = .002) (Figure 2). Indeed, documented tumor growth itself was not associated with the class 2 profile (P = .5). Using logistic regression analysis, the only feature associated with class 2 gene expression profile was age > 60 years (P = .006) (Table 5). The odds ratio of a tumor having the class 2 profile was 2.8 (95% confidence interval 1.3–5.9) for patient age > 60 years and 3.5 (95% confidence interval 1.4–8.8) for tumor thickness > 2.25 mm. A 2-factor logistic regression model using age > 60 years and tumor thickness > 2.25 mm was highly predictive of class 2 gene expression profile (P = .0001). For patients with both of these features vs those with 1 or none of these features, the odds ratio was 3.9 (95% confidence interval 1.9–7.9, P = .0001), and the number needed to treat to identify 1 patient with a class 2 tumor was 4.3 (95% confidence interval 2.9–8.1, P = .0002).
      Table 4Fisher Exact Test for Association Between Choroidal Nevus Features and Class 1 Versus Class 2 Gene Expression Profile
      VariableClass 1

      N (%)
      Class 2

      N (%)
      P Value
      Age > 60 yearsNo: 88 (42.5)

      Yes: 75 (36.2)
      No: 12 (5.7)

      Yes: 32 (15.5)
      .002
      Thickness > 2.25 mmNo: 62 (30.0)

      Yes: 101 (49.0)
      No: 6 (3.0)

      Yes: 38 (18.3)
      .002
      Absence of fibrous metaplasiaNo: 139 (68.1)

      Yes: 22 (10.8)
      No: 40 (19.6)

      Yes: 3 (1.5)
      .3
      Largest basal diameter > 12 mmNo: 129 (62.3)

      Yes: 34 (16.4)
      No: 33 (16.0)

      Yes: 11 (5.3)
      .5
      Visual symptomsNo: 69 (33.5)

      Yes: 93 (45.2)
      No: 16 (7.8)

      Yes 28 (13.6)
      .5
      Presence of subretinal fluidNo: 39 (21.0)

      Yes: 103 (55.4)
      No: 10 (5.4)

      Yes: 34 (18.3)
      .7
      Absence of RPE atrophyNo: 126 (62.0)

      Yes: 34 (16.7)
      No: 33 (16.0)

      Yes: 10 (5.0)
      .8
      Absence of any chronic featureNo: 100 (48.5)

      Yes: 62 (30.1)
      No: 28 (13.6)

      Yes: 16 (7.7)
      .8
      Tumor margin < 3 mm from discNo: 74 (36.0)

      Yes: 88 (42.8)
      No: 20 (9.7)

      Yes: 24 (11.7)
      1.0
      Absence of drusenNo: 126 (61.5)

      Yes: 35 (17.1)
      No: 35 (17.1)

      Yes: 9 (4.4)
      1.0
      Female sexNo: 24 (28.0)

      Yes: 36 (41.9)
      No: 10 (11.6)

      Yes: 16 (18.6)
      1.0
      Presence of orange pigmentNo: 66 (32.2)

      Yes: 95 (46.3)
      No: 18 (8.8)

      Yes: 26 (12.7)
      1.0
      Low acoustic internal reflectivityNo: 17 (20.2)

      Yes: 42 (50.0)
      No: 7 (8.3)

      Yes: 18 (21.4)
      1.0
      Documented tumor growthNo: 86 (41.5)

      Yes: 77 (37.2)
      No: 26 (12.6)

      Yes: 18 (8.7)
      .5
      RPE = retinal pigment epithelium.
      Figure thumbnail gr2
      Figure 2Box-and-whiskers plots of (Left) patient age and (Right) tumor thickness vs gene expression profile class. P value calculated using Fisher exact test.
      Table 5Logistic Regression Best Fit Model For Choroidal Nevus Clinical Features and Class 2 Gene Expression Profile
      Sample size
      ClassN (%)
      Class 244 (21.3%)
      Class 1163 (78.7%)
      Overall Model Fit
      Null model −2 Log Likelihood214.174
      Full model −2 Log Likelihood195.659
      χ218.515
      Significance levelP = .0001
      Coefficients and Standard Errors
      VariableCoefficientStd. ErrorWaldP Value
      Patient age > 601.035440.380077.4219.006
      Tumor thickness > 2.25 mm1.243310.474716.8596.009
      Constant−2.870330.4923733.9839<.0001
      Odds Ratios and 95% Confidence Intervals
      VariableOdds Ratio95% CI
      Patient age > 602.81631.3371–5.9321
      Tumor thickness > 2.25 mm3.46711.3673–8.7912

       Clinical Features Associated With Class 1B Profile

      With increasing application of this technology to analyze smaller tumors, and with longer follow-up, it has been recognized that class 1 tumors can be sub-classified in a biologically meaningful manner into class 1A (minimal metastatic risk) and class 1B (intermediate metastatic risk), based on the expression of 2 genes (CDH1 and RAB31) in the 15-gene profile.
      • Plasseraud K.M.
      • Wilkinson J.K.
      • Oelschlager K.M.
      • et al.
      Gene expression profiling in uveal melanoma: technical reliability and correlation of molecular class with pathologic characteristics.
      • Nguyen B.T.
      • Kim R.S.
      • Bretana M.E.
      • Kegley E.
      • Schefler A.C.
      Association between traditional clinical high-risk features and gene expression profile classification in uveal melanoma.
      Strikingly, none of the choroidal nevus clinical features showed statistically significant association with class 1B gene expression profile by Fisher exact test (Table 6), including tumor thickness > 2.25 mm (P = .4) and patient age > 60 years (P = 1.0).
      Table 6Fisher Exact Test for Association Between Choroidal Nevus Features and Class 1A Versus Class 1B Gene Expression Profile
      VariableClass 1A

      N (%)
      Class 1B

      N (%)
      P Value
      Tumor thickness > 2.25 mmNo: 35 (27.6)

      Yes: 52 (41.0)
      No: 13 (10.2)

      Yes: 27 (21.3)
      .4
      Female sexNo: 14 (27.0)

      Yes: 20 (38.5)
      No: 5 (9.61)

      Yes: 13 (25.0)
      .4
      Absence of any chronic RPE featuresNo: 54 (42.5)

      Yes: 33 (26.0)
      No: 22 (17.3)

      Yes: 18 (14.2)
      .6
      Tumor margin ≤ 3 mm from discNo: 41 (32.5)

      Yes: 45 (35.7)
      No: 22 (17.5)

      Yes: 18 (14.3)
      .6
      Visual symptomsNo: 36 (28.4)

      Yes: 51 (40.2)
      No: 18 (14.2)

      Yes: 22 (17.3)
      .7
      Absence of drusenNo: 69 (54.8)

      Yes: 18 (14.3)
      No: 30 (23.8)

      Yes: 9 (7.1)
      .8
      Absence of RPE fibrosisNo: 76 (60.3)

      Yes: 11 (8.7)
      No: 33 (26.2)

      Yes: 6 (4.8)
      .8
      Largest basal tumor diameter > 12 mmNo: 67 (52.8)

      Yes: 20 (15.8)
      No: 32 (25.2)

      Yes: 8 (6.3)
      .8
      Presence of subretinal fluidNo: 22 (19.0)

      Yes: 57 (49.1)
      No: 9 (7.8)

      Yes: 28 (24.1)
      .8
      Age > 60 yearsNo: 47 (37.1)

      Yes: 40 (31.5)
      No: 21 (16.5)

      Yes: 19 (15.0)
      1.0
      Absence of RPE atrophyNo: 67 (53.6)

      Yes: 18 (14.4)
      No: 31 (24.8)

      Yes: 9 (7.2)
      1.0
      Low acoustic internal reflectivityNo: 10 (19.6)

      Yes: 23 (45.1)
      No: 6 (11.8)

      Yes: 12 (23.5)
      1.0
      Presence of orange pigmentNo: 35 (27.8)

      Yes: 52 (41.3)
      No: 16 (12.7)

      Yes: 23 (18.3)
      1.0
      RPE = retinal pigment epithelium.

      Discussion

      Forty years after Gass's landmark treatise,
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanoma. XXXIII Edward Jackson Memorial lecture.
      the choroidal nevus clinical features that he described have stood the test of time as predictors of tumor growth. Yet, he understood that growth was not equivalent to malignant transformation, and he shared the view of many experts that most choroidal nevi have “very low malignant potential” and can be safely observed for growth prior to treatment, regardless of risk factors.
      • Lane A.M.
      • Egan K.M.
      • Kim I.K.
      • Gragoudas E.S.
      Mortality after diagnosis of small melanocytic lesions of the choroid.
      • Murray T.G.
      • Sobrin L.
      The case for observational management of suspected small choroidal melanoma.
      An alternative view posits that “two or more risk factors probably represent small choroidal melanomas” and “early treatment is generally indicated” so that “better prognosis can be achieved.”
      • Shields C.L.
      • Demirci H.
      • Materin M.A.
      • Marr B.P.
      • Mashayekhi A.
      • Shields J.A.
      Clinical factors in the identification of small choroidal melanoma.
      However, there is very little high-quality evidence in the literature to support this position. To address this controversy, we used the class 2 gene expression profile as a validated biomarker for choroidal melanocytic tumors that have undergone malignant transformation, rather than relying on the crude and problematic endpoint of tumor growth. Strikingly, we found that none of the traditional risk factors for choroidal nevus growth, except for tumor thickness, was associated with the class 2 profile. Further, we found that increased patient age (not one of the traditional risk factors) was the strongest predictor of class 2 profile. These findings raise important questions.

       Should Clinical Risk Factors Be Used To Make Treatment Decisions?

      Several studies have shown that class 2 uveal melanomas with diameter less than 12 mm have much lower risk of metastasis than those with larger diameter,
      • Correa Z.M.
      • Augsburger J.J.
      Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas.

      Harbour JW. Prognostic implications of the largest basal tumor diameter vs the TNM staging system in association with the gene expression profile for uveal melanoma-Reply. JAMA Ophthalmol. https://doi.org/10.1001/jamaophthalmol.2016.5109. 2017.01.05.

      suggesting that early ablative treatment of class 2 tumors when they are still small might improve survival. However, only about 1 in 5 suspicious choroidal nevi have the class 2 profile. By stratifying patients by age > 60 years and tumor thickness > 2.25 mm, we can reduce the number of lesions needed to treat to ∼4 for each one with the class 2 profile. Consequently, when tumor location would allow ablative treatment with only a small threat to vision, this “number needed to treat” may be acceptable. On the other hand, when ablative treatment would pose a high risk for vision loss, it is less clear whether this “number needed to treat” is acceptable. In this case, a fine needle aspiration biopsy to ascertain gene expression profile class may be appropriate to avoid ablative treatment in class 1 tumors where the benefit is questionable. This risk-benefit analysis could change with the development of noninvasive tests that more accurately predict class 2 profile (ie, lower the number needed to treat) or the development of ablative therapies that pose less risk of vision loss. The use of nonablative treatment aimed at reducing symptomatic serous retinal detachment is beyond the scope of this thesis.
      • Fabian I.D.
      • Stacey A.W.
      • Papastefanou V.
      • et al.
      Primary photodynamic therapy with verteporfin for small pigmented posterior pole choroidal melanoma.
      Our results suggest that treating patients with choroidal nevi based on clinical risk factors alone could result in many patients with benign lesions receiving potentially unnecessary vision-threatening treatment. As such, we do not view any of these choroidal nevus risk factors as pathognomonic of malignant transformation, although patient age and tumor thickness may be helpful for identifying small choroidal melanocytic tumors that are more likely to have the class 2 profile.

       Should All Class 1 Tumors Be Observed Without Treatment?

      The 5-year metastatic rate for class 1 uveal melanomas > 12 mm in diameter is only about 10%.
      • Walter S.D.
      • Chao D.L.
      • Feuer W.
      • Schiffman J.
      • Char D.H.
      • Harbour J.W.
      Prognostic implications of tumor diameter in association with gene expression profile for uveal melanoma.
      For class 1 tumors < 12 mm, this metastatic rate is much lower. Thus, initial observation for growth would seem appropriate in most suspicious choroidal nevi ≤ 2.25 mm thickness and in patients ≤ 60 years old, since most will have the class 1 profile. But what if a tumor is found to have the class 1 profile, then subsequently grows? Shouldn't that tumor receive ablative treatment to prevent it from “transforming” to the class 2 profile? Recent discoveries in our laboratory have shed new light on this question (Figure 3). All types of benign choroidal melanocytic lesions (eg, nevi, melanocytomas, melanocytosis, benign diffuse uveal melanocytic proliferation) initially have the class 1 gene expression profile, which signifies melanocytic differentiation.
      • Onken M.D.
      • Ehlers J.P.
      • Worley L.A.
      • Makita J.
      • Yokota Y.
      • Harbour J.W.
      Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.
      If the nascent lesion then acquires a mutation in EIF1AX (Eukaryotic Translation Initiation Factor 1A, X-Linked) or SF3B1 (Splicing Factor 3B Subunit 1), it may progress and grow, but it retains the class 1 profile (class 1A or class 1B, respectively). Alternatively, if the tumor undergoes biallelic mutational inactivation of BAP1 (BRCA1 Associated Protein 1), the class 1 profile is overtaken by the class 2 profile, which denotes a loss of melanocytic differentiation and replacement by a cancer stem cell–like state.
      • Onken M.D.
      • Ehlers J.P.
      • Worley L.A.
      • Makita J.
      • Yokota Y.
      • Harbour J.W.
      Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas.
      • Chang S.H.
      • Worley L.A.
      • Onken M.D.
      • Harbour J.W.
      Prognostic biomarkers in uveal melanoma: evidence for a stem cell-like phenotype associated with metastasis.
      Interestingly, while BAP1 is differentially expressed in uveal melanoma (increased in class 1 and decreased in class 2),
      • Harbour J.W.
      • Onken M.D.
      • Roberson E.D.
      • et al.
      Frequent mutation of BAP1 in metastasizing uveal melanomas.
      it did not pass the rigorous filtering process to be included in the final 15-gene profile, which was optimized for dynamic range, microfluidic PCR, and other characteristics needed for optimal performance for needle biopsy samples. Further, the gene expression profile has prognostic accuracy that is superior to sequencing for BAP1 mutations because current sequencing methods cannot detect all BAP1 mutations.
      • Field M.G.
      • Durante M.A.
      • Anbunathan H.
      • et al.
      Punctuated evolution of canonical genomic aberrations in uveal melanoma.
      Figure thumbnail gr3
      Figure 3Schematic diagram depicting current understanding of how choroidal melanoma progresses. First, choroidal melanocytes acquire a Gαq mutation that leads to development of an early choroidal melanocytic neoplasm.
      • Harbour J.W.
      • Chao D.L.
      A molecular revolution in uveal melanoma: implications for patient care and targeted therapy.
      • Van Raamsdonk C.D.
      • Griewank K.G.
      • Crosby M.B.
      • et al.
      Mutations in GNA11 in uveal melanoma.
      The vast majority of such nevi are arrested by tumor suppressor and/or immune surveillance mechanisms and driven into a dormant state. Some lesions progress past this checkpoint to the “BSE node,” in which they acquire mutations in either BAP1 (BRCA1 Associated Protein 1), SF3B1 (Splicing Factor 3B Subunit 1), or EIF1AX (Eukaryotic Translation Initiation Factor 1A, X-Linked), in a mutually exclusive manner. Tumors that acquire an SF3B1 or EIF1AX mutation retain the class 1 profile (class 1A or class 1B, respectively), whereas those that undergo BAP1 inactivation acquire a class 2 profile. Gαq mutations include mutual exclusive hemizygous activating point mutations in GNAQ (G protein subunit alpha Q), GNA11 (G Protein Subunit Alpha 11), PLCB4 (Phospholipase C Beta 4), and CYSLTR2 (Cysteinyl Leukotriene Receptor 2).
      Recently, we showed that once a choroidal melanocytic tumor acquires a “BSE” (BAP1, SF3B1 or EIF1AX) mutation, its evolutionary trajectory generally becomes fixed, such that it does not acquire another BSE mutation or switch gene expression profiles.
      • Field M.G.
      • Durante M.A.
      • Anbunathan H.
      • et al.
      Punctuated evolution of canonical genomic aberrations in uveal melanoma.
      Thus, it may be unnecessary to treat an EIF1AX-mutant class 1 tumor (regardless of the presence of risk factors for growth), since its metastatic risk is minimal and its likelihood of acquiring a BAP1 mutation and switching to the class 2 profile is exceedingly low. The same argument could possibly be made for an SF3B1-mutant class 1 tumor (regardless of the presence of risk factors for growth), even though the risk of metastasis is slightly higher. The growing class 1 tumors that are most likely to benefit from treatment are those that have neither an EIF1AX or SF3B1 mutation, as these tumors are not yet evolutionarily stabilized and may go on to acquire a BAP1 mutation. We anticipate that gene expression profiling and BSE mutation profiling will play complementary roles in precision medicine for patients with suspicious choroidal nevi. However, until these concepts have been prospectively validated (see below), we still favor observation for evidence of growth for most suspicious choroidal nevi owing to the low risk of malignant transformation, regardless of the presence of clinical risk factors.

       Why Is Age Associated With Malignant Transformation?

      To our knowledge, this is the first study to identify age as a choroidal nevus risk factor that can be used to assess the risk of malignant transformation in individual patients. A previous epidemiologic study using a public database found an association between increased patient age and the ratio of individuals in the U.S. population with a choroidal melanoma vs those with a choroidal nevus, but this was only a very rough approximation of malignant transformation.
      • Singh A.D.
      • Kalyani P.
      • Topham A.
      Estimating the risk of malignant transformation of a choroidal nevus.
      What might explain the relationship between age and the class 2 gene expression profile? The class 2 profile is strongly associated with inactivating mutations in the tumor suppressor BAP1.
      • Harbour J.W.
      • Onken M.D.
      • Roberson E.D.
      • et al.
      Frequent mutation of BAP1 in metastasizing uveal melanomas.
      Indeed, it is likely that loss of BAP1 is the trigger for development of the class 2 profile by causing a loss of melanocytic differentiation.
      • Harbour J.W.
      • Onken M.D.
      • Roberson E.D.
      • et al.
      Frequent mutation of BAP1 in metastasizing uveal melanomas.
      • Matatall K.A.
      • Agapova O.A.
      • Onken M.D.
      • Worley L.A.
      • Bowcock A.M.
      • Harbour J.W.
      BAP1 deficiency causes loss of melanocytic cell identity in uveal melanoma.
      These findings suggest that increased patient age is associated with an increased risk of BAP1 mutations in suspicious choroidal nevi, as it is in overt uveal melanomas.
      • Decatur C.L.
      • Ong E.
      • Garg N.
      • et al.
      Driver mutations in uveal melanoma: associations with gene expression profile and patient outcomes.
      One possible explanation is that BAP1 mutations occur more commonly with increasing age, perhaps owing to less efficient DNA damage repair. Although we cannot rule this out completely at the current time, our recent analysis of a large number of primary uveal melanomas using next-generation sequencing did not find an aging signature associated with BAP1 mutations.
      • Field M.G.
      • Durante M.A.
      • Anbunathan H.
      • et al.
      Punctuated evolution of canonical genomic aberrations in uveal melanoma.
      Another possible explanation is that with increasing age, the cellular microenvironment of the uveal tract becomes less able to limit the growth of a uveal melanocyte that develops a BAP1 mutation. Indeed, there is growing evidence that aging-associated tissue changes provide a substrate for previously latent mutant cells to overgrow.
      • DeGregori J.
      Challenging the axiom: does the occurrence of oncogenic mutations truly limit cancer development with age?.
      Likely contributors to this altered milieu are the resident choroidal macrophages, which have been shown to undergo senescent changes contributing to uveal melanoma
      • Ly L.V.
      • Baghat A.
      • Versluis M.
      • et al.
      In aged mice, outgrowth of intraocular melanoma depends on proangiogenic M2-type macrophages.
      and age-related macular degeneration.
      • Sene A.
      • Khan A.A.
      • Cox D.
      • et al.
      Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.
      The failure of metastatic uveal melanoma to respond to T-cell checkpoint inhibitor immunotherapy may be attributable, at least in part, to this altered immune microenvironment.
      • Heppt M.V.
      • Steeb T.
      • Schlager J.G.
      • et al.
      Immune checkpoint blockade for unresectable or metastatic uveal melanoma: a systematic review.
      Research into the interplay between aging immune cells and metastasis may lead to improved strategies for immunotherapy in uveal melanoma,
      • Chandran S.S.
      • Somerville R.P.T.
      • Yang J.C.
      • et al.
      Treatment of metastatic uveal melanoma with adoptive transfer of tumour-infiltrating lymphocytes: a single-centre, two-stage, single-arm, phase 2 study.
      • Rothermel L.D.
      • Sabesan A.C.
      • Stephens D.J.
      • et al.
      Identification of an immunogenic subset of metastatic uveal melanoma.
      as the current immunotherapeutic approaches have been less effective in uveal melanoma compared to cutaneous melanoma.

       Limitations and Further Work

      One limitation of this work is that it did not include some newer diagnostic modalities that could potentially increase the ability to distinguish class 2 tumors noninvasively. We and others have shown that OCT can accurately differentiate serous retinal detachment from chronic overlying retinal cystoid degeneration, which have different prognostic implications,
      • Espinoza G.
      • Rosenblatt B.
      • Harbour J.W.
      Optical coherence tomography in the evaluation of retinal changes associated with suspicious choroidal melanocytic tumors.
      but routine performance of OCT on all patients was not standard care at the time of this study. Further, the OCT technology available today is more advanced than at the time some of the earlier patients in this study were treated. For example, swept-source OCT angiography can now elucidate intricate details of the choroidal circulation that may have prognostic value in choroidal nevi,
      • Michalewska Z.
      • Michalewski J.
      • Nawrocki J.
      Swept source optical coherence tomography of choroidal nevi.
      but this technique is only now becoming widely available. Fundus autofluorescence may improve our ability to detect orange lipofuscin pigment and to distinguish it from orange coloration that can be attributable to RPE atrophy.
      • Lavinsky D.
      • Belfort R.N.
      • Navajas E.
      • Torres V.
      • Martins M.C.
      • Belfort Jr., R.
      Fundus autofluorescence of choroidal nevus and melanoma.
      Studies are now under way in our center and others to evaluate the prognostic value of these and other emerging diagnostic imaging methods. It is important in future studies to use validated molecular biomarkers of malignant transformation, such as the class 2 gene expression profile, rather than problematic surrogates for malignant transformation such as tumor growth.
      Another limitation is the lack of uniform criteria for biopsy and the potential bias in patient selection, both of which are common shortcomings of retrospective studies. Of the 207 patients in the study, 93 (45%) were observed for growth prior to biopsy, and all patients were biopsied based on the presence of 1 or more risk factors. In cases that were observed for growth prior to biopsy, documented growth was the principal reason for biopsy in most cases. In the 55% of cases in which biopsy was performed without waiting for growth, the constellation of clinical risk factors was deemed to be of sufficiently high risk not to observe first. As such, the factors determining which patients would undergo biopsy were not standardized. However, since the patients were managed by a small number of ocular oncologists using uniform criteria, systematic bias should be small. Importantly, older patients were not observed for a longer period of time on average than younger patients, which could be a confounding factor in our finding of increased age as a factor predictive of class 2 gene expression profile. Our findings in this study point out the critical need for prospective randomized controlled studies to further address the management of small suspicious choroidal melanocytic tumors.
      A potential criticism of this study is that we used the 15-gene expression profile as a marker for malignant transformation. It has been claimed that malignant transformation in choroidal melanoma can be assessed by tumor growth or by histopathologic examination,
      • Shields C.L.
      • Furuta M.
      • Berman E.L.
      • et al.
      Choroidal nevus transformation into melanoma: analysis of 2514 consecutive cases.
      • Naumann G.
      • Yanoff M.
      • Zimmerman L.E.
      Histogenesis of malignant melanomas of the uvea. I. Histopathologic characteristics of nevi of the choroid and ciliary body.
      but these claims have been disputed by Gass, Zimmerman, and others.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanomas. The XXXIII Edward Jackson Memorial Lecture.
      • Gass J.D.
      Problems in the differential diagnosis of choroidal nevi and malignant melanoma. XXXIII Edward Jackson Memorial lecture.
      • Abramson D.H.
      Growing melanocytic tumor is not always cancer.
      • Elner V.M.
      • Flint A.
      • Vine A.K.
      Histopathology of documented growth in small melanocytic choroidal tumors.
      The one unequivocal manifestation of malignant transformation in uveal melanoma is distant metastasis, and the 15-gene expression profile is the only biomarker for metastasis in uveal melanoma that has been validated in a prospective, multicenter study and is more accurate for this purpose than clinical, histopathologic, or chromosomal markers.
      • Onken M.D.
      • Worley L.A.
      • Char D.H.
      • et al.
      Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma.
      • Correa Z.M.
      • Augsburger J.J.
      Independent prognostic significance of gene expression profile class and largest basal diameter of posterior uveal melanomas.
      Thus, we submit that the class 2 gene expression profile is the best available biomarker for malignant transformation in suspicious choroidal melanocytic tumors, as argued in another recent study that showed similar findings.
      • Nguyen B.T.
      • Kim R.S.
      • Bretana M.E.
      • Kegley E.
      • Schefler A.C.
      Association between traditional clinical high-risk features and gene expression profile classification in uveal melanoma.
      Nevertheless, we continue to search for increasingly more accurate biomarkers for malignant transformation and metastatic risk in choroidal melanocytic tumors. To this end, we have initiated a new multicenter prospective clinical study called the Collaborative Ocular Oncology Group Study Number 2 (COOG2). This is a National Cancer Institute–funded study being conducted at over 20 leading ocular oncology centers in North America, with J. William Harbour as the Principal Investigator. Inclusion criteria include a clinical diagnosis of uveal melanoma arising in the choroid and/or ciliary body (including small suspicious choroidal melanocytic tumors) that will be treated with standard methods. The primary tumors undergo fine needle biopsy prior to treatment, and the biopsy sample is subjected to gene expression profiling (class 1A, 1B, or 2), PRAME expression status,
      • Field M.G.
      • Decatur C.L.
      • Kurtenbach S.
      • et al.
      PRAME as an independent biomarker for metastasis in uveal melanoma.
      • Field M.G.
      • Durante M.A.
      • Decatur C.L.
      • et al.
      Epigenetic reprogramming and aberrant expression of PRAME are associated with increased metastatic risk in Class 1 and Class 2 uveal melanomas.
      • Gezgin G.
      • Luk S.J.
      • Cao J.
      • et al.
      PRAME as a potential target for immunotherapy in metastatic uveal melanoma.
      and mutation profiling for all common uveal melanoma driver mutations.
      • Decatur C.L.
      • Ong E.
      • Garg N.
      • et al.
      Driver mutations in uveal melanoma: associations with gene expression profile and patient outcomes.
      Patients are monitored with careful systemic surveillance for detection of metastatic disease. The primary outcome measure is metastasis, and the secondary outcome measure is melanoma-specific mortality. The study goal is to identify the optimal combinatorial use of these molecular prognostic biomarkers, all of which are obtained from a single fine needle biopsy, for precision medicine in these patients.

      Summary

      There are several practical applications of these findings. First, our findings indicate that no clinical feature or combination of features is pathognomonic for malignant transformation, defined as class 2 gene expression profile, in small suspicious choroidal melanocytic tumors. Thus, caution is urged in making treatment decisions based on these features in the absence of documented growth. Nevertheless, these features (especially tumor thickness, subretinal fluid, and orange pigment) are valuable for gauging the frequency of visits to monitor for growth. Second, the only commonly used choroidal nevus risk factor that was associated with malignant transformation was tumor thickness, emphasizing the importance of careful thickness measurements by an experienced echographer. Since there is some inter- and intra-observer variability in ultrasound thickness measurements, it may be prudent to recheck tumors with borderline significant thickness (2.0–2.5 mm) prior to making a treatment decision. Third, although the risk of a small tumor having the class 2 profile increased with age, it is important to monitor younger patients periodically for tumor growth. Finally, our results suggest that the vast majority of small suspicious choroidal melanocytic tumors can be observed safely without treatment, as long as growth is not documented. Future improvements in noninvasive imaging may improve the ability to detect small choroidal melanocytic tumors that have undergone malignant transformation without prognostic biopsy.
      Funding/Support: This work was supported by grants from the National Cancer Institute to Dr Harbour ( R01 CA125970 , Research to Prevent Blindness, Inc Senior Scientific Investigator Award, Melanoma Research Foundation , Melanoma Research Alliance , Ocular Melanoma Foundation , and the Sylvester Comprehensive Cancer Center), and to the Bascom Palmer Eye Institute ( NIH Core Grant P30EY014801 , Research to Prevent Blindness Unrestricted Grant, and Department of Defense Grant # W81XWH-09-1-0675 ). Financial Disclosures: J. William Harbour is the inventor of intellectual property related to the gene expression profile technology used in the study and intellectual property related to the discovery of BAP1 mutations in uveal melanoma. He is also a paid consultant for Castle Biosciences, which licensed this intellectual property, and he receives royalties from its commercialization. He is a member of the scientific advisory board for Aura Biosciences and Immunocore, Ltd. The following authors have no financial disclosures: Manuel Paez-Escamilla, Louis Cai, Scott D. Walter, James J. Augsburger, and Zelia M. Correa. All authors attest that they meet the current ICMJE criteria for authorship.
      Other Acknowledgments: The authors thank Christina E. Decatur, BS for administrative support for the study and William J. Feuer, MS for statistical assistance.

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