Comprehensive clinical imaging, histopathological analysis and liquid biopsy-based surveillance of human uveal melanoma in a prolonged rabbit xenograft model.

Uveal melanoma is the most common intraocular tumor in adults. Our group has previously developed a human uveal melanoma animal model; however, adverse effects caused by the immunosuppressive agent, cyclosporine A, prevented animals from surviving more than 12 weeks. In this study, we tested multiple cyclosporine A doses over an extended disease course up to 20 weeks, providing complete clinical imaging of intraocular tumors, histopathological analysis and liquid biopsy biomarker analysis. Twenty albino rabbits were divided into four groups with different daily cyclosporine A schedules (0-10 mg/kg) and inoculated with human uveal melanoma cell lines, 92.1 or MP41, into the suprachoroidal space. Rabbits were monitored with fundoscopy, ultrasound and optical coherence tomography. Intraocular tumors (macroscopic or microscopic) were detected in all study animals. Tumor size and growth were correlated to cyclosporine A dose, with tumors regressing when cyclosporine A was arrested. All tumors expressed HMB-45 and MelanA; however, tumor size, pigmentation and cell morphology differed in 92.1 vs. MP41 tumors. Finally, across all groups, circulating tumor DNA from plasma and aqueous humor was detected earlier than tumor detection by imaging and correlated to tumor growth. In conclusion, using three clinically relevant imaging modalities (fundoscopy, ultrasonography and optical coherence tomography) and liquid biopsy, we were successfully able to monitor tumor progression in our rabbit xenograft model of human uveal melanoma.

Histopathological analysis of residual lens cells in capsular opacities after cataract surgery using objective software.

Purpose: Remnant lens epithelial cells (LECs) within the capsular bag (CB) undergo epithelial-to-mesenchymal transition (EMT) and acquire a myofibroblast phenotype, depositing extracellular matrix (ECM) components, leading to posterior capsular opacification (PCO). This study histopathologically analyzes the LEC-to-myofibroblast transition and de novo ECM component deposition (i.e., smooth muscle actin (SMA) and fibronectin (FN) expression) and determines the intraocular lens (IOL) and patient factors associated with these changes. Methods: In total, 190 CBs with IOLs were removed from donor eyes. Digital images were obtained, and PCO was graded using published software (ADOS, Medical Parachute). Automated immunohistochemistry was performed using anti-SMA to detect EMT and anti-FN to document ECM remodeling. Slides were digitized and analyzed using the Positive Pixel Count v9 algorithm. Linear regression and Poisson regression were performed (P < 0.05). Results: SMA positive expression decreased as the time of IOL implantation increased (P < 0.0001). Positivity of SMA and FN demonstrated a positive correlation (P = 0.0002). Controlling for confounding factors in Poisson regression, hydrophobic and hydrophilic materials showed higher FN and SMA expression when compared to silicone material lenses (FN; P = 0.018; P < 0.0001, SMA; P = 0.001; P = 0.003, respectively). The square optic design had 29% higher SMA positivity compared to the opti-edge design (P = 0.042). One-piece haptic lenses had higher SMA expression compared to three-piece haptic (P = 0.042). A higher risk of expression of SMA and FN was seen in patients with a history of smoking, hypertension, and glaucoma (P < 0.05). Conclusion: This study demonstrated that SMA and FN expression is different according to IOL design and patient factors, thus indicating that LEC changes depend on lens biocompatibility. Therefore, by analyzing the histopathological composition of PCO by using LECs, further insight into the characteristics of IOLs that are important for biocompatibility can be ascertained.

Circulating tumor DNA tracking through driver mutations as a liquid biopsy-based biomarker for uveal melanoma.

BACKGROUND: Uveal melanoma (UM) is the most common intraocular tumor in adults. Despite good primary tumor control, up to 50% of patients develop metastasis, which is lethal. UM often presents asymptomatically and is usually diagnosed by clinical examination and imaging, making it one of the few cancer types diagnosed without a biopsy. Hence, alternative diagnostic tools are needed. Circulating tumor DNA (ctDNA) has shown potential as a liquid biopsy target for cancer screening and monitoring. The aim of this study was to evaluate the feasibility and clinical utility of ctDNA detection in UM using specific UM gene mutations. METHODS: We used the highly sensitive digital droplet PCR (ddPCR) assay to quantify UM driver mutations (GNAQ, GNA11, PLCß4 and CYSTLR2) in cell-free DNA (cfDNA). cfDNA was analyzed in six well established human UM cell lines with known mutational status. cfDNA was analyzed in the blood and aqueous humor of an UM rabbit model and in the blood of patients. Rabbits were inoculated with human UM cells into the suprachoroidal space, and mutated ctDNA was quantified from longitudinal peripheral blood and aqueous humor draws. Blood clinical specimens were obtained from primary UM patients (n = 14), patients presenting with choroidal nevi (n = 16) and healthy individuals (n = 15). RESULTS: The in vitro model validated the specificity and accuracy of ddPCR to detect mutated cfDNA from UM cell supernatant. In the rabbit model, plasma and aqueous humor levels of ctDNA correlated with tumor growth. Notably, the detection of ctDNA preceded clinical detection of the intraocular tumor. In human specimens, while we did not detect any trace of ctDNA in healthy controls, we detected ctDNA in all UM patients. We observed that UM patients had significantly higher levels of ctDNA than patients with nevi, with a strong correlation between ctDNA levels and malignancy. Noteworthy, in patients with nevi, the levels of ctDNA highly correlated with the presence of clinical risk factors. CONCLUSIONS: We report, for the first time, compelling evidence from in vitro assays, and in vivo animal model and clinical specimens for the potential of mutated ctDNA as a biomarker of UM progression. These findings pave the way towards the implementation of a liquid biopsy to detect and monitor UM tumors.

Optimizing optical coherence tomography and histopathology correlation in retinal imaging.

OBJECTIVE: To develop a methodology to correlate optical coherence tomography (OCT) images and histopathological sections from the same eye. Part 1: To determine the best fixative for optimal OCT and histopathological analysis in post-mortem eyes. Part 2: A protocol is proposed to correlate histopathological features and OCT scans from the same post-mortem eyes. DESIGN: Experimental study. PARTICIPANTS: Part 1: Twenty-three rabbit eyes and 14 post-mortem human eyes. Part 2: Nineteen post-mortem human eyes. METHODS: Part 1: Six different fixatives were tested, and specimens were evaluated on 4 criteria: globe shape, structure opacification, retinal detachment, and nuclear details. Part 2: Based on the findings from Part 1, fixed human eyes were imaged using OCT. Orientation-controlled histopathological processing was performed to obtain serial tissue sections from paraffin embedded tissue, which were matched to corresponding OCT images. RESULTS: Part 1: Of the 6 fixatives, 2% glutaraldehyde and Davidson's solution met the proposed criteria in rabbit eyes. Of these, glutaraldehyde showed similar results in human eyes and was selected for Part 2. Part 2: Using anatomical landmarks, cross-sectional histopathological sections in the same orientation as the OCT images were correlated to their corresponding OCT images. Retinal lesions such as a macular hole, an epiretinal membrane, and the presence of drusen were easily correlated, proving the reliability of our methodology. Moreover, the photoreceptor's inner/outer junction was correlated to a hyperreflective band on OCT. CONCLUSIONS: A standardized protocol was developed to correlate OCT images and histopathological findings by generating serial cross-sections of the retina, which can be used to better understand otherwise ambiguous OCT findings.

Removing Subjective Post-Mortem Grading from Posterior Capsular Opacification: A New Automated Detector Opacification Software, ADOS.

Purpose/aim: Posterior capsular opacification (PCO) is the leading complication following cataract surgery. However, PCO grading methods vary between studies and rely on subjective scoring methods; hence, they are prone to error. Therefore, software that reduces subjectivity and enhances reproducibility is needed. The aim of the study was to evaluate the reproducibility and objectivity of a novel automated custom-designed PCO detection software. MATERIALS AND METHODS: In total, 165 fixed eyes with intraocular lenses were sectioned to obtain a Miyake Apple view (MAV) image. The capsular bag (CB) with the in situ IOL was removed and imaged using the Olympus DSX110 stereoscope. Central, peripheral, and Soemmering's ring area (SRA) and intensity (SRI) were graded by two ophthalmologists using a scale of 0-4. Software was developed to detect PCO, and these values were correlated with subjective scoring. RESULTS: Intra-observer agreement for MAV and CB images ranged from poor to moderate, with very good agreement for SRI (k = 0.88, 0.86, respectively). Agreement between graders was fair to good (k = 0.31-0.77). Miyake Apple view and capsular bag images showed good correlation; central PCO showed the least correlation for both evaluators (k = 0.29, 0.72). Subjective average grading versus software correlation demonstrated very good correlation for intensity and good for area (r = 0.85, 0.61). Reproducibility of the methodology resulted in good to very good correlation. CONCLUSIONS: Software correlates with previous scoring methods and is a reliable and reproducible system. Moreover, the capsular bag view, as opposed to the Miyake Apple view, allows visualization of the capsular bag directly and avoids overlapping structures (vitreous, cornea) that may interfere with proper PCO quantification.

Pretreatment of RPE Cells with Lutein Can Mitigate Bevacizumab-Induced Increases in Angiogenin and bFGF.

PURPOSE: To determine whether pretreatment of retinal pigmented epithelial (RPE) cells with lutein can affect the response of cells to bevacizumab therapy. METHODS: One human RPE cell line (ARPE-19) was used for all experiments. The cells were treated with lutein in different concentrations (0.01, 0.1, 1, 10, or 100 µg/ml). After 24 h, all plates were treated with bevacizumab (0.25 mg/ml). Media were harvested 24 h later for sandwich ELISA-based angiogenesis arrays. A Quantibody Human Angiogenesis Array was used in order to quantify the secretion of the following 10 proangiogenic cytokines: angiogenin, ANG2, EGF, bFGF, HB-EGF, PDGF-BB, leptin, PIGF, HGF and VEGF. RESULTS: Treatment with bevacizumab alone led to a significant decrease in VEGF, as well as a significant increase in angiogenin and bFGF. Pretreatment with 0.1 and 1.0 µg/ml of lutein led to significant decreases in both bFGF and angiogenin following treatment with bevacizumab compared to bevacizumab treatment alone. Lutein alone did not modify the secretion of proangiogenic cytokines. CONCLUSIONS: Pretreatment of human RPE cells in culture with specific doses of lutein prior to bevacizumab treatment mitigated the increase in bFGF and angiogenin caused by bevacizumab monotherapy.

Multiple pregnancies and its relationship with the development of retinopathy of prematurity (ROP).

BACKGROUND: The influence of multiple gestation on the occurrence of retinopathy of prematurity (ROP) is still not completely understood. OBJECTIVES: To verify the incidence of any stage of ROP and threshold ROP in singletons and in multiple gestation among preterm infants. METHODS: This was an institutional, prospective, and descriptive cohort study, which included preterm newborns with birth weight (BW) of 1500 g or less and/or gestational age (GA) of 32 weeks or less, as admitted to the neonatal units at Laranjeiras and Amparo Hospitals in Rio de Janeiro, Brazil, between January 2001 and July 2005, and whom remained hospitalized for at least 28 days. There were no exclusion criteria. Patients were divided into two groups: Group 1 included multiples; and Group 2 consisted of singletons. RESULTS: A total of 159 infants that remained in neonatal unit care for at least 28 days were included in this study. Group 1 comprised 56 (35%) multiples; and Group 2 comprised 103 (65%) singletons. Mean BW was 1072 g ± 272 and 1089 g ± 282 in Groups 1 and 2, respectively (analysis of variance [ANOVA] P > 0.05). Mean GA among multiple gestation (Group 1) was 29 weeks ± 2.1; and 29 weeks ± 2.4 among singletons (Group 2) (ANOVA P > 0.05). Days in oxygen therapy ranged from 0 to 188 days. Median among Group 1 was 15 days, while median in Group 2 was 10 days (Kruskal-Wallis P > 0.05). Any stage ROP was detected in 66 (41.5%) of the whole cohort comprising 159 babies. Among the 56 multiples, 30 (53.6%) achieved any stage ROP, and among 103 singletons, 36 (35%) achieved any stage ROP (Chi-square test P < 0.05). Threshold ROP occurred in 12 (7.5%) of the 159 patients included. Three (5.3%) patients from Group 1 and nine (8.7%) patients in Group 2 reached threshold ROP needing laser treatment (Fisher's exact test P > 0.05). CONCLUSION: This study showed higher frequency of any stage of ROP in twins and triplets but not regarding threshold disease. Because of the relatively small number of patients in this sample, other studies are necessary to determine if gemelarity plays a role in the occurrence of ROP.

Prevalência de diabetes mellitus tipo 2 e outros fatores de risco associados em pacientes com glaucoma / Prevalence of type 2 diabetes mellitus and other associated risk factors in glaucoma patients

OBJETIVO: Avaliar a prevalência de diabéticos em uma amostra de pacientes com glaucoma; verificar se existe associação entre diabetes mellitus e glaucoma na amostra estudada; verificar outros fatores de risco associados. MÉTODOS: Foram analisados de forma retrospectiva os prontuários de 50 pacientes com diagnóstico de glaucoma. Os dados registrados foram sexo, idade, raça, história familiar de glaucoma e história pessoal de diabetes mellitus tipo 2. RESULTADOS: Do total de pacientes avaliados, 5 (10 por cento) apresentavam diabetes mellitus tipo 2. Destes, 3 eram mulheres e 2 eram homens, com mediana de idade de 81 anos (71-88). A prevalência de diabetes nos pacientes com glaucoma não mostrou diferenças significativas (OR: 1,476; Intervalo de Confiança 95 por cento: 0,4438 a 4,910; p= 0,5352) quando comparada à prevalência de diabetes mellitus tipo 2 na população geral brasileira (7,6 por cento). CONCLUSÃO: Nesta amostra de pacientes com glaucoma, a prevalência de diabetes mellitus tipo 2 foi pouco mais elevada que a da população. Entretanto, nenhuma associação foi encontrada entre diabetes mellitus e glaucoma.

PURPOSE: To evaluate prevalence of diabetes mellitus in a group of patients with glaucoma; to verify if there is association between diabetes mellitus and glaucoma; to verify other associated risk factors. METHODS: Fifty(50) glaucoma patients had their medical records analyzed in a retrospective way. Registered data included sex, age, ethnic group, family history of glaucoma and personal history of type 2 diabetes mellitus. RESULTS: Five (10 percent) of all evaluated patients had type 2 diabetes mellitus. 3 of them were female and 2 were male, median age of 81 years old (71-88). Prevalence of diabetes in glaucoma patients did not show significant differences. (OR: 1.476; 95 percent Confidence Interval: 0.4438 to 4.910; p= 0.5352) when compared to the prevalence of type 2 diabetes mellitus in general brazilian population (7.6 percent). CONCLUSION: In this group of patients with glaucoma, prevalence of type 2 diabetes mellitus was slightly higher than in general population. However, no association was found between diabetes mellitus and glaucoma.

Comparison between obtained mydriasis in type 2 diabetics and non-diabetic patients.

BACKGROUND/AIMS: To evaluate and compare obtained mydriasis with phenylephrine 10% associated with tropicamide 1% in type 2 diabetics and non-diabetic patients. METHODS: A total of 50 patients (100 eyes) scheduled for fundoscopy were dilated with phenylephrine 10% and yropicamide 1% (group 0: n = 20 type 2 diabetic patients, 40 eyes, and group 1: n = 30 non-diabetic patients, 60 eyes). Only one drop per eye of each drug was administered. In both groups, pupil diameter was measured after 40 minutes of eye drops instillation. RESULTS: Both groups were similar regarding age (p = 0.06, Mann-Whitney test). Mean pupil diameter in group 0 was 8.57 and 8.73 in group 1. There was no statistic difference between both groups (p = 0.44). Pupil diameter was greater than 7 mm in all patients (100%). CONCLUSION: When an appropriate drug combination is used, diabetic patients can achieve mydriasis as satisfactory as non-diabetic patients, allowing adequate fundus examination and/or retinopathy treatment.

Mydriatic and cardiovascular effects of phenylephrine 2.5% versus phenylephrine 10%, both associated with tropicamide 1%.

BACKGROUND/AIMS: To evaluate and compare cardiovascular effects (blood pressure and heart rate) of phenylephrine 2.5% versus phenylephrine 10%, and compare pupil diameter before and after instillation of eyedrops. METHODS: A total of 58 patients scheduled for funduscopy were dilated with either phenylephrine 2.5% and tropicamide 1% (group 0, n = 29 patients, 58 eyes) or phenylephrine 10% and tropicamide 1% (group 1, n = 29 patients, 58 eyes). Only one drop per eye of each drug was administered. In both groups, pupil diameter, blood pressure and heart rate were measured before and 40 min after eyedrop instillation. RESULTS: We did not observe significant changes in blood pressure or heart rate. Mean pupil diameter in group 0, before the instillation of eyedrops, was 3.51 mm and, in group 1, 2.66 mm. After medication, mean values were 7.38 mm in group 0 and 7.42 mm in group 1. Mean variation was 3.87 mm in group 0 and 4.76 mm in group 1 (p < 0.001). CONCLUSION: Our results corroborate the finding that one single drop of either 2.5 or 10% phenylephrine is safe and, when 1% tropicamide is combined, satisfactory pupil dilation is achieved.

Aspectos atuais no diagnóstico do edema macular diabético: [revisão] / Recent aspects on diagnosis of diabetic macular edema: [review]

O diagnóstico do edema macular diabético deve ser feito precocemente, a fim de evitar perda visual significativa. Para tal, contamos com exames complementares cada vez mais desenvolvidos e específicos. Os autores fazem uma revisão dos principais métodos existentes para diagnosticar a retinopatia diabética.

Diabetic macular edema must be assessed as early as possible, to avoid significant vision loss. To achieve this goal, there are many specific diagnostic tests that can be performed. The authors review the most common methods in diagnosing diabetic retinopathy.

Aspectos atuais na fisiopatologia do edema macular diabético: [revisão] / Recent aspects on physiopathology of diabetic macular edema: [review]

O edema macular é a principal causa de baixa visual em pacientes diabéticos. Seu mecanismo de formação é complexo e envolve alterações bioquímicas e estruturais. Os autores fazem uma revisão e atualização dos conceitos fisiopatológicos envolvidos na maculopatia diabética.

Macular edema is the leading cause of poor vision in diabetic patients.The mechanism of edema formation is complex and involves biochemical and structural changes. The authors review and update the physiopathologic concepts related to diabetic maculopathy.

Telangiectasia retiniana parafoveal unilateral / Unilateral parafoveal retinal telangiectasis

Telangiectasia retiniana é um termo inicialmente proposto por Reese, em 1956. Caracteriza-se por uma anormalidade vascular retiniana, com dilatação irregular e incompetência dos vasos. E.L.S., 44 anos, sexo masculino, branco, do Rio de Janeiro, com queixa de perda progressiva da visão em olho esquerdo (OE). Ao exame observamos acuidade visual (AV) corrigida de 20/20 em olho direito (OD) e 20/100 em OE. À fundoscopia, OD normal e OE apresentando dilatação capilar e exsudatos duros com aspecto circinado na região macular. Angiografia fluoresceínica e, após o resultado, injeção intravítrea de triancinolona em OE (4mg/0,1ml). Após um mês da injeção de triancinolona a AV em OE passou a 20/40, os exsudatos duros tornaram-se mais visíveis e a angiografia mostrou diminuição do vazamento do corante. Foi possível a realização de fotocoagulação com laser. A AV final corrigida foi de 20/20 em ambos os olhos. O tratamento com laser está amplamente difundido na literatura, porém optamos inicialmente pela injeção intravítrea de triancinolona. Pode-se considerar a injeção intravítrea de triancinolona em casos de telangiectasia retiniana parafoveal com redução importante da visão e alterações muito próximas à região avascular foveal. Entretanto, os resultados em longo prazo não são conhecidos e precisam ser confrontados com as outras opções de tratamento.

Retinal telangiectasis was first described by Reese, in 1956. It is an abnormality of the retinal vessels, with irregular dilation and vessel failure. ELS, 44 years old, male, white, from Rio de Janeiro. His complaining was progressive visual loss in the left eye (OS). Corrected visual acuity (VA) was 20/20 in the right eye (OD) and 20/100 in OS. Fundoscopy in OD was normal, and in OS showed capillaries dilation and hard exsudates (circinate) in macular area. After fluorescein angiogram, we performed intravitreal injection of triamcinolone acetonide (4mg/0.1ml) in OS. One month after injection, VA in OS was 20/40, hard exsudates became more visible and angiogram showed a reduction in dye leakage. Due to better visibility, we were able to perform laser photocoagulation on the affected area. Final best corrected VA was 20/20 in both eyes. Although laser treatment is widely recommended, we chose intravitreal triamcinolone injection initially due to the fact that damaged capillaries were too close to foveal avascular zone. Intravitreal triamcinolone injection can be considered in cases of parafoveal retinal telangiectasis with significant decreased vision and changes near foveal avascular zone. However, long term results are not known yet and need to be confronted with other available treatments.