Publications by authors named "Evangelos S Gragoudas"

91 Publications

Targeting the YAP/TAZ Pathway in Uveal and Conjunctival Melanoma With Verteporfin.

Invest Ophthalmol Vis Sci 2021 Apr;62(4)

Department of Ophthalmology, Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, United States.

Purpose: The purpose of this study was to determine whether YAP/TAZ activation in uveal melanoma (UM) and the susceptibility of melanoma cell lines to YAP/TAZ inhibition by verteporfin (VP) is related to the tumor's genetic background.

Methods: Characteristics of 144 patients with enucleated UM were analyzed together with mRNA expression levels of YAP/TAZ-related genes (80 patients from the The Cancer Genome Atlas [TCGA] project and 64 patients from Leiden, The Netherlands). VP was administered to cell lines 92.1, OMM1, Mel270, XMP46, and MM28 (UM), CRMM1 and CRMM2 (conjunctival melanoma), and OCM3 (cutaneous melanoma). Viability, growth speed, and expression of YAP1-related proteins were assessed.

Results: In TCGA data, high expression of YAP1 and WWTR1 correlated with the presence of monosomy 3 (P = 0.009 and P < 0.001, respectively) and BAP1-loss (P = 0.003 and P = 0.001, respectively) in the primary UM; metastasis development correlated with higher expression of YAP1 (P = 0.05) and WWTR1 (P = 0.003). In Leiden data, downstream transcription factor TEAD4 was increased in cases with M3/BAP1-loss (P = 0.002 and P = 0.006) and related to metastasis (P = 0.004). UM cell lines 92.1, OMM1, and Mel270 (GNAQ/11-mutation, BAP1-positive) and the fast-growing cell line OCM3 (BRAF-mutation) showed decreased proliferation after exposure to VP. Two slow-growing UM cell lines XMP46 and MM28 (GNAQ/11-mutation, BAP1-negative) were not sensitive to VP, and neither were the two conjunctival melanoma cell lines (BRAF/NRAS-mutation).

Conclusions: High risk UM showed an increased expression of YAP/TAZ-related genes. Although most UM cell lines responded in vitro to VP, BAP1-negative and conjunctival melanoma cell lines did not. Not only the mutational background, but also cell growth rate is an important predictor of response to YAP/TAZ inhibition by VP.
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http://dx.doi.org/10.1167/iovs.62.4.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024781PMC
April 2021

Proton beam irradiation of uveal melanoma involving the iris, ciliary body and anterior choroid without surgical localisation (light field).

Br J Ophthalmol 2020 Dec 21. Epub 2020 Dec 21.

Retina Service, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA

Aims: To assess treatment outcomes after proton beam irradiation (PBI) without surgical localisation of uveal melanomas involving the iris, ciliary body and anterior choroid.

Methods: Retrospective chart review of 125 patients evaluated at Massachusetts Eye and Ear and treated with PBI using a light field set-up without localisation surgery between November 1975 and April 2017. The tumours were characterised as follows: iris (n=18, 14.4%), ciliary body (n=12, 9.6%), iridociliary (n=58, 46.4%), ciliochoroidal (n=24, 19.2%) and iridociliochoroidal (n=13, 10.4%). The tumours were measured by transillumination and ultrasonography before treatment. Tumours with posterior margin located less than two disc diameters from the ora serrata were treated using the light field technique. Patient outcomes after PBI were evaluated.

Results: Most patients had good vision at the time of tumour diagnosis (69.6% had baseline visual acuity (VA) of ≥20/40). Median VA at last follow-up (median follow-up: 72.1 months) was 20/63. Recurrences occurred in 12 patients (9.6%) at a median time of 4.0 years post-treatment. Recurrences were treated by repeat PBI (n=5) or enucleation (n=7). Secondary enucleation was performed in 18 patients (14.4%), and 61.1% of these were due to complications. Neovascular glaucoma (NVG) developed in 21 patients (16.8%). Of seven patients who developed NVG after anti-vascular endothelial growth factor (anti-VEGF) therapies became available, five were treated with intravitreal Avastin injections (23.8% of patients with NVG). Of 69 patients diagnosed with cataract after treatment, 51 (73.9%) were characterised as radiation-related. Death from metastatic uveal melanoma occurred in 20.8% of the cohort, with a median follow-up of 10.1 years.

Conclusions: Patients treated with PBI using a light field set-up technique experience good outcomes after irradiation. Eye preservation and retention of good VA are seen in the majority of cases, and tumour recurrence is low.
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http://dx.doi.org/10.1136/bjophthalmol-2020-318063DOI Listing
December 2020

Conservative management of suspicious melanocytic lesions of the iris.

Graefes Arch Clin Exp Ophthalmol 2019 Jun 27;257(6):1319-1324. Epub 2019 Mar 27.

Ocular Melanoma Center, Retina Service, Mass Eye and Ear, 243 Charles Street., Boston, MA, 02114, USA.

Purpose: The diagnosis of iris melanoma can be difficult, with no established diagnostic criteria currently available. Careful monitoring of patients with suspicious iris lesions is one approach to managing these tumors. We determined the risk of malignant transformation and melanoma-related mortality in patients under observation to evaluate the validity of this management approach.

Methods: This was a retrospective chart review of patients with suspicious iris lesions diagnosed at Massachusetts Eye and Ear Infirmary (MEE) between 1975 and 2014. All patients with an initial diagnosis of suspicious iris lesion followed and/or treated after malignant transformation at the MEE in this 39-year period were included in the cohort. Rates of malignant transformation and melanoma-related mortality were calculated. Treatment outcomes after proton beam irradiation were evaluated in patients who developed iris melanomas during observation.

Results: Two hundred thirty-four patients had a diagnosis of suspicious iris lesion (median follow-up, 5.8 years). Malignant transformation occurred in 16 (6.8%) patients with suspicious lesions during the observation period (median follow-up, 9.9 years). All patients diagnosed with iris melanomas were treated with proton beam irradiation (PBI). Complications after treatment included cataract (18.8%), secondary glaucoma (6.3%), and neovascular glaucoma (12.5%). Two of 16 patients (12.5%) who developed iris melanomas died of metastatic melanoma 32.6 months and 10 years after treatment with PBI. Both cases had been followed regularly to monitor for malignant transformation of their suspicious lesions (8.2 years and 3.2 years before melanoma diagnosis, respectively).

Conclusions: These data suggest that suspicious iris lesions have low malignant potential, and a conservative approach to the management of these lesions is appropriate. Survival does not appear to be compromised with an observational approach, and there is potential for preservation of good visual function because vision-threatening treatments can be avoided.
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http://dx.doi.org/10.1007/s00417-019-04296-0DOI Listing
June 2019

Survival Rates in Patients After Treatment for Metastasis From Uveal Melanoma.

JAMA Ophthalmol 2018 09;136(9):981-986

Ocular Melanoma Center, Retina Service, Massachusetts Eye and Ear Infirmary, Boston.

Importance: Despite high rates of local tumor control in patients who are treated for uveal melanoma, most patients will eventually die of metastasis. When metastasis develops, the liver is involved in most cases, and hepatic metastases are particularly refractory to treatment. Finding effective treatments has been challenging. A comparison of survival rates in patients who were treated for metastasis over approximately 30 years may offer insights into progress that has been made in prolonging survival.

Objective: To compare survival after treatment for metastasis in a cohort of patients who were treated for uveal melanoma at the Massachusetts Eye and Ear Infirmary (MEE) during an approximately 30-year period with an earlier analysis to determine if there was meaningful improvement in survival rates after treatment for metastasis.

Design, Setting, And Participants: This review included patients (n = 661) who received a diagnosis of metastasis from uveal melanoma who were identified from a cohort of 3063 patients treated at MEE between January 1982 and December 2009 and followed up through December 2011. They were compared with findings from a previous study of patients treated between 1975 and 1987.

Main Outcomes And Measures: Survival rates in patients who received treatment for metastasis were compared with those who did not receive treatment. The differences in survival rates were compared with an earlier analysis that was completed at MEE. A comparison of patients with hepatic metastases and extrahepatic metastases was also completed. Kaplan-Meier analysis was used to calculate survival rates and the log rank test was used to test for statistically significant differences between the groups.

Results: Of 620 patients with race information available, 615 (97.3%) were white; the mean (SD) age of patients was 59.71 (13.23) years and 307 (47.3%) were women. The median time from the initial treatment of the tumor to metastasis was 3.45 years (interquartile range [IQR], 2.0-5.57). Overall, the median survival time was poor (3.9 months [IQR, 1.6-10.1]). Patients who received treatment fared better than those who did not receive treatment (median survival after metastasis diagnosis, 6.3 months [IQR, 2.96-14.41] vs 1.7 months [IQR, 0.66-3.5]). This finding was similar to that of our earlier study in which median survival was 5.2 months and 2 months for treated and untreated patients, respectively.

Conclusions And Relevance: These findings suggest that advances in treatments that lead to clinically meaningful improvements in survival times have not been realized. Similar survival rates in patients who were treated for metastasis were observed in this recent analysis compared with our earlier study. Adjuvant therapies that are initiated at the time of melanoma diagnosis may be the most effective way to prolong survival.
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http://dx.doi.org/10.1001/jamaophthalmol.2018.2466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142974PMC
September 2018

Genetic Risk Factors for Radiation Vasculopathy.

Invest Ophthalmol Vis Sci 2018 03;59(3):1547-1553

Ocular Melanoma Center and Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, United States.

Purpose: The purpose of this study was to perform a genome-wide scan for polymorphisms associated with risk of vision loss from radiation complications in patients treated with proton beam irradiation for choroidal melanoma.

Methods: We identified a cohort of 126 patients at high risk of radiation complications due to tumor location within 2 disc diameters of the optic nerve and/or fovea who provided a blood sample to the Massachusetts Eye and Ear Uveal Melanoma Repository. Controls (n = 76) were defined as patients with visual acuity 20/40 or better 3 years after treatment. Cases (n = 50) were selected as patients with visual acuity 20/200 or worse due to radiation damage 3 years after treatment. Genotyping of these samples was performed using the Omni 2.5 chip (Illumina, Inc.).

Results: Hypertension (odds ratio [OR] = 3.749, P = 0.0009), visual acuity at diagnosis of choroidal melanoma (OR = 1.031, P = 0.002), tumor distance to fovea (OR = 0.341, P = 6.52E-05), tumor distance to optic disc (OR = 0.481, P = 5.41E-05), and height of tumor (OR = 1.704, P = 0.0069) were associated with poor vision (20/200 or worse). Individual single nucleotide polymorphism (SNP) analysis was performed controlling for the risk factors identified using stepwise regression and the first principal component. Although this analysis determined that there were 74,529 nominally significant SNPs (P < 0.05), there were no SNPs that reached genome-wide significance (P < 5E-08). The SNP reaching the highest significance level (P < 1E-04) was rs11678387, located on chromosome 2, intergenic between EPB41L5/RALB (P = 4.43E-05).

Conclusions: Visual loss from radiation vasculopathy after treatment for choroidal melanoma is not only related to tumor location but may be influenced by hypertension and possibly genetic factors.
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http://dx.doi.org/10.1167/iovs.17-22791DOI Listing
March 2018

Evaluation of choroidal lesions with swept-source optical coherence tomography.

Br J Ophthalmol 2019 01 31;103(1):88-93. Epub 2018 Mar 31.

Retina Service, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA

Aims: The aim of our study was to image choroidal lesions with swept-source optical coherence tomography (SS-OCT) and to identify the morphological characteristics associated with optimal visualisation.

Methods: This was a prospective, cross-sectional study. Patients with choroidal melanocytic lesions <3 mm in thickness on B-scan ultrasonography were recruited. All participants underwent SS-OCT. On SS-OCT we evaluated qualitative (eg, lesion outline, detection of scleral-choroidal interface and quality of the image) and quantitative (measurement of maximum lesion thickness and the largest basal diameter) parameters. Probability of optimal image quality was examined using ordered logistic regression models. The main outcome measure was quality of the choroidal lesion images on SS-OCT, defined as: optimal, suboptimal or poor.

Results: We included 85 choroidal lesions of 82 patients. There were 24 choroidal lesions (29%) for which image quality was classified as optimal, 31 lesions (37%) as suboptimal and 30 lesions (36%) as poor. The factors associated with optimal image quality were distance closer to the fovea (OR 0.76, p<0.001), posterior pole location (OR 3.87, p=0.05), lower ultrasonography thickness (OR 0.44, p=0.04), lighter lesion pigmentation (OR 0.12, p=0.003) and smaller lesion diameter (OR 0.73, p<0.001). In the multivariable analysis, closer distance to the fovea (OR 0.81, p=0.005), lighter lesion pigmentation (OR 0.11, p=0.01) and smaller lesion diameter (OR 0.76, p=0.006) remained statistically significant.

Conclusion: SS-OCT is useful in imaging most choroidal melanocytic lesions. Image quality is best when the choroidal lesion is closer to the fovea, has a smaller diameter and a lighter choroidal pigmentation.
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http://dx.doi.org/10.1136/bjophthalmol-2017-311586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365049PMC
January 2019

Rare Variant, Gene-Based Association Study of Hereditary Melanoma Using Whole-Exome Sequencing.

J Natl Cancer Inst 2017 12;109(12)

Melanoma Genetics Program, MGH Cancer Center, MGH, Boston, MA.

Background: Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM).

Methods: Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three human melanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of human melanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided.

Results: Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10-6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10-4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10-4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10-5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels.

Conclusions: The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene.
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http://dx.doi.org/10.1093/jnci/djx083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5939858PMC
December 2017

Issues with the Specificity of Immunological Reagents for NLRP3: Implications for Age-related Macular Degeneration.

Sci Rep 2018 01 11;8(1):461. Epub 2018 Jan 11.

Department of Ophthalmology, Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, 02114, USA.

Contradictory data have been presented regarding the implication of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in age-related macular degeneration (AMD), the leading cause of vision loss in the Western world. Recognizing that antibody specificity may explain this discrepancy and in line with recent National Institutes of Health (NIH) guidelines requiring authentication of key biological resources, the specificity of anti-NLRP3 antibodies was assessed to elucidate whether non-immune RPE cells express NLRP3. Using validated resources, NLRP3 was not detected in human primary or human established RPE cell lines under multiple inflammasome-priming conditions, including purported NLRP3 stimuli in RPE such as DICER1 deletion and Alu RNA transfection. Furthermore, NLRP3 was below detection limits in ex vivo macular RPE from AMD patients, as well as in human induced pluripotent stem cell (hiPSC)-derived RPE from patients with overactive NLRP3 syndrome (Chronic infantile neurologic cutaneous and articulate, CINCA syndrome). Evidence presented in this study provides new data regarding the interpretation of published results reporting NLRP3 expression and upregulation in RPE and addresses the role that this inflammasome plays in AMD pathogenesis.
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http://dx.doi.org/10.1038/s41598-017-17634-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5764999PMC
January 2018

LONG-TERM OBSERVATION OF MULTIFOCAL METASTATIC INTRAOCULAR CARCINOID WITH ACQUIRED IRIS HETEROCHROMIA.

Retin Cases Brief Rep 2020 ;14(3):265-267

Department of Ophthalmology, Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts.

Background/purpose: To report a unique case of a pulmonary carcinoid tumor unilaterally metastatic to the iris and ciliary body and bilaterally to the choroid that was conservatively followed.

Methods: A 46-year-old woman presented with bilateral choroidal lesions and a left iris tumor. Ultrasound biomicroscopy disclosed a ciliary body component. A diagnosis of metastatic carcinoid tumor was made based on the clinical features. Rather than an excision, photodynamic therapy, or radiation treatment, as has been reported in all previous cases of carcinoid tumor metastatic to the iris, the patient was observed.

Results: Excellent vision was maintained for 8 years. The iris tumor gradually enlarged, but the choroidal lesions remained unchanged. The iris with the carcinoid tumor gradually acquired a brown pigmentation; this is the first reported case of acquired iris heterochromia in the setting of carcinoid tumor.

Conclusion: We conclude, in cases of metastatic carcinoid in which visual acuity is excellent and the patient is asymptomatic, that observation of the ocular lesions is an acceptable course of action. The iris heterochromia is believed to have been caused by secretory factors produced by the tumor.
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http://dx.doi.org/10.1097/ICB.0000000000000690DOI Listing
January 2020

Long-term Outcomes After Proton Beam Irradiation in Patients With Large Choroidal Melanomas.

JAMA Ophthalmol 2017 11;135(11):1191-1196

Retina Service, Massachusetts Eye and Ear, Harvard Medical School, Boston.

Importance: Although radiotherapy has been used more frequently in past decades for the management of large melanomas, long-term efficacy of proton beam irradiation (PBI) of large choroidal melanomas has not been reported.

Objective: To evaluate long-term outcomes in patients who underwent PBI for the treatment of large choroidal melanomas.

Design, Setting, And Participants: Data were obtained at a single Boston, Massachusetts, academic tertiary referral practice for this retrospective cohort study. In total, 336 patients with large tumors treated over a 13-year period from January 1, 1985, to December 31, 1997, and followed up until the end points were reached or until December 31, 2008, were included. Data analyses were initially completed in February 2017 and finalized in July 2017. Large tumors were those with a height 10 mm or greater or a longest linear diameter greater than 16 mm or a height greater than 8 mm when the optic nerve was involved.

Intervention: Proton beam irradiation (total 70 Gy) delivered in 5 equal fractions.

Main Outcomes And Measures: The primary outcomes of rates of visual acuity retention, eye retention, tumor recurrence, and melanoma-related mortality were calculated using Kaplan-Meier estimates, and Cox proportional hazards regression analyses were completed to evaluate risk factors for tumor recurrence and melanoma-related mortality.

Results: In this cohort of 336 patients with large tumors, 150 were women and 329 were white; mean (SD) age was 60.0 (14.0) years. Of 178 patients without optic nerve involvement (tumor >1 disc diameter from optic nerve), the mean (SD) largest basal diameter was 18.1 (1.9) mm and mean height was 8.2 (2.7) mm. Optic nerve involvement and tumors greater than 8 mm were observed in 109 patients (32.4% of the cohort). Baseline visual acuity of 20/200 or better was observed in 244 patients (72.6%), and worse than 20/800 in 52 (15.5%). Ten-year rates of visual acuity retention were 8.7% (95% CI, 4.1%-15.6%) for at least 20/200 and 22.4% (95% CI, 15.4%-30.4%) for at least counting fingers. Ten years after PBI therapy, the eye was retained (70.4%; 95% CI, 61.5%-77.6%) and tumor controlled (87.5%; 95% CI, 76.8%-93.5%) in most patients. The 10-year all-cause mortality rate was 60.7% (95% CI, 55.5%-65.9%). Approximately half of the patients died of metastatic uveal melanoma (10-year rate, 48.5%; 95% CI, 43.0%-54.4%).

Conclusions And Relevance: This study demonstrates that eye conservation is possible in most cases, with ambulatory vision retained in a small proportion of patients 10 years after PBI. Tumor recurrence rates were low and mortality rates were comparable to those observed after enucleation.
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http://dx.doi.org/10.1001/jamaophthalmol.2017.3805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710395PMC
November 2017

Systematic genomic and translational efficiency studies of uveal melanoma.

PLoS One 2017 8;12(6):e0178189. Epub 2017 Jun 8.

Department of Systems Biology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

To further our understanding of the somatic genetic basis of uveal melanoma, we sequenced the protein-coding regions of 52 primary tumors and 3 liver metastases together with paired normal DNA. Known recurrent mutations were identified in GNAQ, GNA11, BAP1, EIF1AX, and SF3B1. The role of mutated EIF1AX was tested using loss of function approaches including viability and translational efficiency assays. Knockdown of both wild type and mutant EIF1AX was lethal to uveal melanoma cells. We probed the function of N-terminal tail EIF1AX mutations by performing RNA sequencing of polysome-associated transcripts in cells expressing endogenous wild type or mutant EIF1AX. Ribosome occupancy of the global translational apparatus was sensitive to suppression of wild type but not mutant EIF1AX. Together, these studies suggest that cells expressing mutant EIF1AX may exhibit aberrant translational regulation, which may provide clonal selective advantage in the subset of uveal melanoma that harbors this mutation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178189PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464544PMC
September 2017

Introducing Joan W. Miller, the 2015 Recipient of the Weisenfeld Award.

Invest Ophthalmol Vis Sci 2016 12;57(15):6910

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http://dx.doi.org/10.1167/iovs.15-18205DOI Listing
December 2016

Ranibizumab for the Prevention of Radiation Complications in Patients Treated With Proton Beam Irradiation for Choroidal Melanoma.

Trans Am Ophthalmol Soc 2016 Aug;114:T2

Massachusetts Eye and Ear, Retina Service, Department of Ophthalmology, Harvard Medical School, Boston, MA.

Purpose: To investigate the safety and potential efficacy of ranibizumab for prevention of radiation complications in patients treated with proton irradiation for choroidal melanoma.

Methods: Forty patients with tumors located within 2 disc diameters of the optic nerve and/or macula were enrolled in this open-label study. Participants received ranibizumab 0.5 mg or 1.0 mg at tumor localization and every 2 months thereafter for the study duration of 24 months. The incidence of adverse events, visual acuity, and other measures of ocular morbidity related to radiation complications were assessed. Historical controls with similar follow-up meeting the eligibility criteria for tumor size, location, and baseline visual acuity were assembled for comparison.

Results: Fifteen patients with large tumors and 25 patients with small/medium tumors were enrolled. Thirty-two patients completed the month 24 visit. No serious ocular or systemic adverse events related to ranibizumab were observed. At 24 months, the proportion of patients with visual acuity ≥ 20/200 was 30/31 (97%) in the study group versus 92/205 (45%) in historical controls (P < .001). The proportion of patients with visual acuity ≥20/40 was 24/31 (77%) in the study group versus 46/205 (22%) in controls at 24 months (P<.001). Clinical evidence of radiation maculopathy at month 24 was seen in 8/24 (33%) patients with small/medium tumors versus 42/62 (68%) of controls (P = .004). Three patients with large tumors developed metastases.

Conclusions: In this small pilot study, prophylactic ranibizumab appears generally safe in patients treated with proton irradiation for choroidal melanoma. High rates of visual acuity retention were observed through 2 years.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5012854PMC
August 2016

CHRONICALLY PROGRESSIVE SARCOID GRANULOMA IN AN ASYMPTOMATIC PATIENT.

Retin Cases Brief Rep 2017 Spring;11(2):183-185

Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts.

Purpose: To report 18 years of follow-up of a patient with atypical ocular sarcoidosis.

Methods: The patient was observed in clinic and with photography for over 18 years.

Results: One patient with a history of dermal melanoma and atypical ocular sarcoidosis.

Discussion: Several features presented in this case are exceedingly atypical including lack of any intraocular inflammation over 18 years of yearly observation, the preservation of excellent vision despite the choroidal disease, and lack of systemic symptoms. This case suggests a disconnect between ocular sarcoidosis and intraocular inflammation, and supports clinical observation as a prudent method in patients with extensive choroidal disease and good vision.
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http://dx.doi.org/10.1097/ICB.0000000000000325DOI Listing
September 2017

Multiple simultaneous choroidal melanomas arising in the same eye: globe salvage by radiotherapy.

Acta Ophthalmol 2016 Dec 15;94(8):e799-e802. Epub 2016 Apr 15.

Department of Ophthalmology, Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, TN, USA.

Purpose: Multiple choroidal melanomas arising in the same eye is a very rare entity, usually leading ophthalmologists to entertain other diagnoses. Historically, the only available treatment reported for this rare entity was enucleation. In this study we demonstrate in a series of patients with multiple simultaneous choroidal melanomas that eye salvage is possible using a variety of radiotherapy techniques.

Observations: Both patients presented with two simultaneous choroidal melanomas in one eye. The first patient was only 30 years old and presented with two largely amelanotic tumours with large exudative retinal detachment. Cytology from fine needle aspiration biopsies from both tumours with immunohistochemistry confirmed two separate melanomas. Sequential radioactive iodine plaque brachytherapy led to regression of both tumours. The second, older patient's two tumours both had the typical appearance of choroidal melanoma and he underwent proton beam irradiation to the entire field leading to tumour regression.

Conclusions: Multiple choroidal melanomas can rarely arise simultaneously in the same eye, and despite their variable appearance, a definitive diagnosis can be aided by cytology and immunohistochemistry in atypical-appearing cases. While all other previously reported cases have necessitated enucleation, we demonstrate that globe salvage is possible using either proton beam irradiation to the entire tumour field, or with sequential radioactive plaque brachytherapy.
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http://dx.doi.org/10.1111/aos.13034DOI Listing
December 2016

Regression of Some High-risk Features of Age-related Macular Degeneration (AMD) in Patients Receiving Intensive Statin Treatment.

EBioMedicine 2016 Mar 4;5:198-203. Epub 2016 Feb 4.

Retina Service, Department of Ophthalmology, Mass. Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA.

Importance: Age-related macular degeneration (AMD) remains the leading cause of blindness in developed countries, and affects more than 150 million worldwide. Despite effective anti-angiogenic therapies for the less prevalent neovascular form of AMD, treatments are lacking for the more prevalent dry form. Similarities in risk factors and pathogenesis between AMD and atherosclerosis have led investigators to study the effects of statins on AMD incidence and progression with mixed results. A limitation of these studies has been the heterogeneity of AMD disease and the lack of standardization in statin dosage.

Objective: We were interested in studying the effects of high-dose statins, similar to those showing regression of atherosclerotic plaques, in AMD.

Design: Pilot multicenter open-label prospective clinical study of 26 patients with diagnosis of AMD and the presence of many large, soft drusenoid deposits. Patients received 80 mg of atorvastatin daily and were monitored at baseline and every 3 months with complete ophthalmologic exam, best corrected visual acuity (VA), fundus photographs, optical coherence tomography (OCT), and blood work (AST, ALT, CPK, total cholesterol, TSH, creatinine, as well as a pregnancy test for premenopausal women).

Results: Twenty-three subjects completed a minimum follow-up of 12 months. High-dose atorvastatin resulted in regression of drusen deposits associated with vision gain (+ 3.3 letters, p = 0.06) in 10 patients. No subjects progressed to advanced neovascular AMD.

Conclusions: High-dose statins may result in resolution of drusenoid pigment epithelial detachments (PEDs) and improvement in VA, without atrophy or neovascularization in a high-risk subgroup of AMD patients. Confirmation from larger studies is warranted.
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http://dx.doi.org/10.1016/j.ebiom.2016.01.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4816836PMC
March 2016

Long-term Risk of Melanoma-Related Mortality After Uveal Melanoma--Reply.

JAMA Ophthalmol 2016 Feb;134(2):239-40

Retina Service, Massachusetts Eye and Ear Infirmary, Boston.

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http://dx.doi.org/10.1001/jamaophthalmol.2015.5235DOI Listing
February 2016

Visual Outcomes after Proton Beam Irradiation for Choroidal Melanomas Involving the Fovea.

Ophthalmology 2016 Feb 3;123(2):369-377. Epub 2015 Nov 3.

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts. Electronic address:

Purpose: To report visual outcomes in patients undergoing proton beam irradiation of tumors located within 1 disc diameter of the fovea.

Design: Retrospective review.

Participants: Patients with choroidal melanoma involving the fovea treated with proton beam therapy between 1975 and 2009.

Methods: Three hundred fifty-one patients with choroidal melanomas located 1 disc diameter (DD) or less from the fovea and more than 1 DD away from the optic nerve were included in this study. In a subgroup of 203 of the patients with small and medium choroidal melanomas, the effect of a reduced dose of radiation, 50 Gy (relative biological effectiveness [RBE]) versus 70 Gy (RBE), on visual outcomes was analyzed. The Kaplan-Meier method and Cox regression analysis were performed to calculate cumulative rates of vision loss and to assess risk factors for vision loss, respectively.

Main Outcome Measures: Visual acuity and radiation complications, which included radiation maculopathy, papillopathy, retinal detachment, and rubeosis, were assessed.

Results: Three hundred fifty-one patients were included in this study with a mean follow-up time of 68.7 months. More than one-third of patients (35.5%) retained 20/200 or better vision 5 years after proton beam irradiation. For those patients with a baseline visual acuity of 20/40 or better, 16.2% of patients retained this level of vision 5 years after proton beam irradiation. Tumor height less than 5 mm and baseline visual acuity 20/40 or better were associated significantly with a better visual outcome (P < 0.001). More than two-thirds (70.4%) of patients receiving 50 Gy (RBE) and nearly half (45.1%) of patients receiving 70 Gy (RBE) retained 20/200 or better vision 5 years after treatment, but this difference was not significant. Approximately 20% of patients with these smaller macular tumors retained 20/40 vision or better 5 years after irradiation.

Conclusions: The results of this retrospective analysis demonstrate that despite receiving a full dose of radiation to the fovea, many patients with choroidal melanoma with foveal involvement maintain useful vision. A radiation dose reduction from 70 to 50 Gy (RBE) did not seem to increase the proportion of patients who retain usable vision.
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http://dx.doi.org/10.1016/j.ophtha.2015.09.031DOI Listing
February 2016

Severe vision loss with optic disc neovascularization after hemiretinal vascular obstruction associated with optic disc melanocytoma.

JAMA Ophthalmol 2015 Oct 8;133(10):e151502. Epub 2015 Oct 8.

Retina Service, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston.

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http://dx.doi.org/10.1001/jamaophthalmol.2015.1502DOI Listing
October 2015

Clinical Characteristics of Uveal Melanoma in Patients With Germline BAP1 Mutations.

JAMA Ophthalmol 2015 Aug;133(8):881-7

Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston.

Importance: Somatic mutations in BAP1 (BRCA1-associated protein 1 gene) are frequently identified in uveal melanoma. To date, the role of germline BAP1 mutations in uveal melanoma has not been characterized.

Objective: To characterize the clinical phenotype of uveal melanoma in patients with germline BAP1 mutations.

Design, Setting, And Participants: Retrospective cohort study at an academic ophthalmology referral center among 507 patients with uveal melanoma who consented for collection of blood samples. The study dates were June 22, 1992, to December 14, 2010.

Main Outcomes And Measures: Clinical characteristics of uveal melanoma and the development of metastases. BAP1 gene sequencing from blood samples of patients with uveal melanoma was correlated with clinical characteristics.

Results: Of 507 blood samples analyzed, 25 patients (4.9%) exhibited 18 BAP1 polymorphisms, of which 9 were novel. Computational analyses predicted that 8 BAP1 mutations in 8 patients (1.6%) were likely to result in damaged BAP1 protein. Five of these 8 mutations were novel. These 8 patients were compared with 482 patients in whom no BAP1 polymorphisms were identified. In univariate analyses, patients with germline BAP1 mutations exhibited larger tumor diameters (mean, 15.9 vs 12.3 mm; P = .004) and higher rates of ciliary body involvement (75.0% vs 21.6%, P = .002) and metastases (71.4% vs 18.0%, P = .003) compared with control subjects. Patients with germline BAP1 mutations exhibited increased frequency of family history of cancer (100% vs 65.9%, P = .06), particularly cutaneous melanoma (62.5% vs 9.9%, P < .001) and ocular melanoma (25.0% vs 1.9%, P = .01). No differences were identified in age at diagnosis, sex, history of other malignant neoplasm, presenting visual acuity, distance of the tumor from the optic nerve or fovea, iris involvement, extrascleral extension, or tumor pigmentation. Germline BAP1 mutations increased risk of metastasis independent of ciliary body involvement (P = .02). Germline BAP1 mutation approached significance as an independent risk factor for metastasis (P = .09).

Conclusions And Relevance: These data suggest that germline BAP1 mutations occur infrequently in uveal melanoma and are associated with larger tumors and higher rates of ciliary body involvement, 2 known risk factors for metastasis.
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http://dx.doi.org/10.1001/jamaophthalmol.2015.1119DOI Listing
August 2015

Long-term Risk of Melanoma-Related Mortality for Patients With Uveal Melanoma Treated With Proton Beam Therapy.

JAMA Ophthalmol 2015 Jul;133(7):792-6

Retina Service, Massachusetts Eye and Ear Infirmary, Boston.

Importance: Little is known about the long-term risk of dying of uveal melanoma after treatment with radiotherapy.

Objective: To determine the long-term risk of dying of this disease, we evaluated melanoma-related mortality rates up to 25 years after proton beam therapy in a large series of patients with uveal melanoma.

Design, Setting, And Participants: In this analysis, we included 3088 patients with uveal melanoma, identified from a hospital-based cohort and treated with proton irradiation between January 1975 and December 2005. Vital status and cause of death were ascertained through active follow-up and searches of government databases (the Social Security Death Index and the National Death Index) through December 31, 2008. Cumulative rates of melanoma-related mortality were calculated using the Kaplan-Meier method. Patient and tumor characteristics of known prognostic significance for melanoma-associated death were evaluated, including patient age and tumor dimensions.

Main Outcomes And Measures: The primary outcome measure was cumulative rates of melanoma-specific mortality, and secondary measures included annual melanoma-specific mortality hazard rates and cumulative all-cause mortality rates.

Results: Of 1490 deceased patients, 620 (41.6%) died of ocular melanoma. In addition, 19 patients were alive, but their melanoma metastasized, by the end of the observation period (mean follow-up after diagnosis of metastasis, 5.3 years). All-cause mortality rates in this cohort were 49.0% (95% CI, 47.0-51.1) at 15 years, 58.6% (95% CI, 56.4%-60.8%) at 20 years, and 66.8% (95% CI, 64.2%-69.4%) at 25 years. Melanoma-related mortality rates were 24.6% (95% CI, 22.8-26.4) at 15 years after treatment, 25.8% (95% CI, 24.0-27.8) at 20 years after treatment, and 26.4% (95% CI, 24.5-28.5) at 25 years after treatment. The 20-year mortality rate was 8.6% (95% CI, 6.2-11.9) for younger patients (≤ 60 years) with small tumors (≤ 11 mm) and 40.1% (95% CI, 36.1-44.3) for older patients (>60 years) with large tumors (>11 mm).

Conclusions And Relevance: In this large series of patients with ocular melanoma treated conservatively with proton beam irradiation, the cumulative melanoma-related mortality rates continued to increase up to 23 years after treatment. Annual rates decreased considerably (to <1%) 14 years after treatment. Information regarding the long-term risk of dying of uveal melanoma may be useful to clinicians when counseling patients.
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http://dx.doi.org/10.1001/jamaophthalmol.2015.0887DOI Listing
July 2015

Immunohistochemical investigations of adult intraocular medulloepitheliomas.

Clin Exp Ophthalmol 2015 May-Jun;43(4):379-85. Epub 2014 Oct 22.

Retina Service, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, USA.

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http://dx.doi.org/10.1111/ceo.12436DOI Listing
January 2016

Effects of metformin on retinoblastoma growth in vitro and in vivo.

Int J Oncol 2014 Dec 11;45(6):2311-24. Epub 2014 Sep 11.

Retina Service, Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, MA, USA.

Recent studies suggest that the anti-diabetic drug metformin may reduce the risk of cancer and have anti-proliferative effects for some but not all cancers. In this study, we examined the effects of metformin on human retinoblastoma cell proliferation in vitro and in vivo. Two different human retinoblastoma cell lines (Y79, WERI) were treated with metformin in vitro and xenografts of Y79 cells were established in nu/nu immune-deficient mice and used to assess the effects of pharmacological levels of metformin in vivo. Metformin inhibited proliferation of the retinoblastoma cells in vitro. Similar to other studies, high concentrations of metformin (mM) blocked the cell cycle in G0‑G1, indicated by a strong decrease of G1 cyclins, especially cyclin D, cyclin-dependent kinases (4 and 6), and flow cytometry assessment of the cell cycle. This was associated with activation of AMPK, inhibition of the mTOR pathways and autophagy marker LC3B. However, metformin failed to suppress growth of xenografted tumors of Y79 human retinoblastoma cells in nu/nu mice, even when treated with a maximally tolerated dose level achieved in human patients. In conclusion, suprapharmacological levels (mM) of metformin, well above those tolerated in vivo, inhibited the proliferation of retinoblastoma cells in vitro. However, physiological levels of metformin, such as seen in the clinical setting, did not affect the growth of retinoblastoma cells in vitro or in vivo. This suggests that the potential beneficial effects of metformin seen in epidemiological studies may be limited to specific tumor types or be related to indirect effects/mechanisms not observed under acute laboratory conditions.
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http://dx.doi.org/10.3892/ijo.2014.2650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215581PMC
December 2014

Proton beam irradiation for non-AMD CNV: 2-year results of a randomised clinical trial.

Br J Ophthalmol 2014 Sep 12;98(9):1212-7. Epub 2014 May 12.

Retina Service, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.

Aims: To evaluate safety and visual outcomes after proton beam irradiation (PBI) therapy for subfoveal choroidal neovascularisation (CNV) secondary to causes other than age-related macular degeneration (AMD).

Methods: This study is a prospective, unmasked and randomised clinical trial using two dosage regimens, conducted in the Massachusetts Eye and Ear Infirmary. The study included 46 patients with CNV secondary to non-AMD and best-corrected visual acuity of 20/320 or better. Patients were randomly assigned to receive 16 or 24 cobalt gray equivalents (CGE) of PBI in two equal fractions. Complete ophthalmological examinations, fundus photography and fluorescein angiography were performed at baseline and 6, 12, 18 and 24 months after treatment.

Results: At 1 year after treatment, 82% and 72% lost fewer than 1.5 lines of vision in the 16 CGE and in 24 CGE groups, respectively. At 2 years after therapy, 77% in the lower dose group and 64% in the higher dose group lost fewer than 1.5 lines of vision. Mild radiation complications such as radiation vasculopathy developed in 17.6% of patients.

Conclusions: PBI is a safe and efficacious treatment for subfoveal CNV not due to AMD. The data with respect to visual outcomes and radiation complications trend in favour of the 16 CGE group, although differences do not reach statistical significance. PBI may be considered as an alternative to current therapies.
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http://dx.doi.org/10.1136/bjophthalmol-2013-304761DOI Listing
September 2014

Choroidal Metastases From EML4-ALK-Positive Non-Small-Cell Lung Adenocarcinoma.

J Clin Oncol 2015 Oct 14;33(30):e112-4. Epub 2014 Apr 14.

Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA.

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http://dx.doi.org/10.1200/JCO.2013.50.2740DOI Listing
October 2015

Aggressive skull base metastasis from uveal melanoma: a clinicopathologic study.

Eur J Ophthalmol 2014 Sep-Oct;24(5):811-3. Epub 2014 May 12.

Retina Service, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts - USA.

Purpose: We present the clinical, pathologic, and genetic findings of the first reported case of choroidal melanoma that developed a late recurrence and aggressive metastasis to the skull base without evidence of hepatic involvement.

Methods: Retrospective chart review and clinicopathologic correlation of ocular and brain tissue, including sequencing of BAP1 for mutations.

Results: A 55-year-old woman was diagnosed with choroidal melanoma and treated with proton radiotherapy. Six years later, she developed a rapidly growing local recurrence involving the ciliary body and iris. Upon enucleation, histopathology revealed an iris and ciliary body epithelioid melanoma that was contiguous with the previously treated, regressed spindle cell choroidal melanoma. Imaging was initially negative for brain involvement. Two months later, she developed cranial neuropathies and was found to have a large skull base lesion that required surgical debulking for pain palliation. Histopathology confirmed the lesion to be metastatic melanoma. Both ocular and brain tumor specimens were wild-type for BAP1. Throughout her course, she developed no hepatic metastases.

Conclusions: Uveal melanoma may metastasize to the skull base. The present case was characterized by delayed onset and unusual aggressiveness of the metastatic disease, and lack of BAP1 mutation. The unusual course highlights a unique phenotype that may reflect an alternate molecular mechanism for metastatic disease.
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http://dx.doi.org/10.5301/ejo.5000468DOI Listing
November 2014

Biopsy of an anterior episcleral nodule as an aid in managing a ciliary body melanocytic tumor.

Cornea 2013 Aug;32(8):1174-7

David G. Cogan Laboratory of Ophthalmic Pathology, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114, USA.

Purpose: To demonstrate the value of a diagnostic biopsy of a fixed episcleral nodule overlying a uveal mass.

Method: Clinicopathologic report with immunohistochemical investigations.

Results: B-scan ultrasonographic biomicroscopy disclosed in a 67-year-old man an asymptomatic placoid ciliary body tumor measuring 1.28 mm in thickness underlying a poorly pigmented, fixed episcleral nodule 0.56 mm in thickness. Biopsy of the episcleral nodule displayed benign nevus-type spindle cells with small nuclei, punctate nucleoli, no mitoses, and scattered melanophages. Immunohistochemistry demonstrated that the tumor cells were Ki67 negative (proliferation index, 0) and MART-1, HMB-45, and microphthalmia-associated transcription factor positive, all melanocytic markers. The melanophages but not the tumor cells were CD68 positive, a histiocytic marker.

Conclusions: The results from biopsying an episcleral nodule can help to select among therapeutic options in managing an associated ciliary body tumor. A 1-year follow-up study and 3 sequential ultrasonographic studies in the current patient have failed to document the growth of the intraocular tumor, further confirming that it is a nevus. The excised epibulbar tumor has not recurred.
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http://dx.doi.org/10.1097/ICO.0b013e31829131f8DOI Listing
August 2013

Aminoimidazole carboxamide ribonucleotide (AICAR) inhibits the growth of retinoblastoma in vivo by decreasing angiogenesis and inducing apoptosis.

PLoS One 2013 3;8(1):e52852. Epub 2013 Jan 3.

Angiogenesis Laboratory, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Retina Service, Harvard Medical School, Boston, Massachusetts, United States of America.

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR), an analog of AMP is widely used as an activator of AMP-kinase (AMPK), a protein that regulates the responses of the cell to energy change. Recently, we showed that AICAR-induced AMPK activation inhibits the growth of retinoblastoma cells in vitro by decreasing cyclins and by inducing apoptosis and S-phase arrest. In this study, we investigated the effects of AMPK activator AICAR on the growth of retinoblastoma in vivo. Intraperitoneal injection of AICAR resulted in 48% growth inhibition of Y79 retinoblastoma cell tumors in mice. Tumors isolated from mice treated with AICAR had decreased expression of Ki67 and increased apoptotic cells (TUNEL positive) compared with the control. In addition, AICAR treatment suppressed significantly tumor vessel density and macrophage infiltration. We also showed that AICAR administration resulted in AMPK activation and mTOR pathway inhibition. Paradoxically observed down-regulation of p21, which indicates that p21 may have a novel function of an oncogene in retinoblastoma tumor. Our results indicate that AICAR treatment inhibited the growth of retinoblastoma tumor in vivo via AMPK/mTORC1 pathway and by apoptogenic, anti-proliferative, anti-angiogenesis mechanism. AICAR is a promising novel non-chemotherapeutic drug that may be effective as an adjuvant in treating Retinoblastoma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052852PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3536763PMC
August 2013

High throughput mass spectrometry-based mutation profiling of primary uveal melanoma.

Invest Ophthalmol Vis Sci 2012 Oct 9;53(11):6991-6. Epub 2012 Oct 9.

Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114-3096, USA.

Purpose: We assessed for mutations in a large number of oncogenes and tumor suppressor genes in primary uveal melanomas using a high-throughput profiling system.

Methods: DNA was extracted and purified from 134 tissue samples from fresh-frozen tissues (n = 87) or formalin-fixed, paraffin-embedded tissues (n = 47) from 124 large uveal melanomas that underwent primary treatment by enucleation. DNA was subjected to whole genome amplification and MALDI-TOF mass spectrometry-based mutation profiling (>1000 mutations tested across 120 oncogenes and tumor suppressor genes) using the OncoMap3 platform. All candidate mutations, as well as commonly occurring mutations in GNAQ and GNA11, were validated using homogeneous mass extension (hME) technology.

Results: Of 123 samples, 97 (79%, representing 89 unique tumors) were amplified successfully, passed all quality control steps, and were assayed with the OncoMap platform. A total of 58 mutation calls was made for 49 different mutations across 26 different genes in 34/98 (35%) samples. Of 91 tumors that underwent hME validation, 83 (91%) harbored mutations in the GNAQ (47%) or GNA11 (44%) genes, while hME validation revealed two tumors with mutations in EGFR. These additional mutations occurred in tumors that also had mutations in GNAQ or GNA11.

Conclusions: The vast majority of primary large uveal melanomas harbor mutually-exclusive mutations in GNAQ or GNA11, but very rarely have the oncogenic mutations that are reported commonly in other cancers. When present, these other mutations were found in conjunction with GNAQ/GNA11 mutations, suggesting that these other mutations likely are not the primary drivers of oncogenesis in uveal melanoma.
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http://dx.doi.org/10.1167/iovs.12-10427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471553PMC
October 2012