Impact of tumor-associated macrophages in LH(BETA)T(AG) mice on retinal tumor progression: relation to macrophage subtype.

PURPOSE: To determine the distribution of tumor-associated macrophages (TAMs) during retinoblastoma tumor development, examine the contribution of bone marrow-derived TAMs in retinoblastoma tumors, and evaluate the supportive role of TAMs in tumor growth in a transgenic retinoblastoma mouse model. METHODS: The time course of macrophage infiltration in transgenic retinoblastoma tumors was assessed by immunohistochemistry at different time points in tumorigenesis. The origin of TAMs in transgenic retinoblastoma tumors was determined by transplanting 10(7) bone marrow cells from green fluorescent protein (GFP)-positive 16-week-old mice into age-matched, irradiated LH(BETA)T(AG) mice via tail vein injections. Macrophage depletion was performed by subconjunctival (SC) delivery of liposomal clodronate. RESULTS: The density of TAMs increased from 4 to 12 weeks of age in mice with small to medium tumors (P = 0.037) and remained stable in the later stages of disease (i.e., 16 weeks old with large tumors; P = 0.20). In 16-week-old mice, 38% (2.5 +/- 3.2 cells per 400x high-power field) of TAMs were GFP-positive, bone marrow-derived macrophages. Total TAM depletion was associated with a significant decrease in the expression levels of MMP-9 (P = 0.014) and mature vessels (P < 0.001) and a nonsignificant decrease in the density of neovessels (P = 0.94). The density of M2-polarized TAMs did not change significantly after TAM depletion (P = 0.68). After M1-polarized TAM depletion, the tumor burden increased (P = 0.056). CONCLUSIONS: This work extends understanding of the complex role that macrophages play in retinoblastoma. Macrophage modulation in the tumor microenvironment is a critical factor in retinoblastoma tumor progression.

Blood vessel maturation in human uveal melanoma: spatial distribution of neovessels and mature vasculature.

PURPOSE: The aims of this study are (1) to evaluate the spatial distribution of neovessels and mature vessels in human uveal melanoma tumors and (2) to determine whether vessel maturation is associated with the major indicators for poor prognosis. METHODS: Immunohistochemical analyses were performed on human tissue specimens from enucleated eyes (n = 14) to assess total vessels, neovessels, mature vessels, and cell proliferation. Tumor morphology was analyzed by hematoxylin and eosin and modified periodic acid-Schiff (PAS) staining.The spatial distribution of neovessels and mature vessels was analyzed by immunohistochemistry, and correlated with major indicators of poor prognosis (i.e., aggressive PAS patterns, epithelioid cytology, mitotic figures, extraocular extension, anterior tumor location, ciliary body involvement, large tumor size, cell proliferation, and angiogenic activity). RESULTS: Neovesseldensity was greater than mature vessel density in apical (p = 0.17), central (p = 0.036), and peripheral (p = 0.31) regions of the tumors, while mature vessel density was greater than neovessel density in basal areas of the tumor (p = 0.47). This pattern indicated that vessel maturation begins at the base of the tumor and later extends to the peripheral and apical regions. The difference between mature and neovessel densities for the apical (-0.8 +/- 1.9) and central areas (-0.8 +/- 1.3) of the tumor was significantly higher than the difference obtained for the basal area (0.3 +/- 1.6; p = 0.014 and p = 0.012, respectively), indicating a higher density of mature vessels compared to neovessels at the base. Statistical correlations were found between mature vessel density and tumor size (r = 0.48, p = 0.084), cell proliferation (r = 0.62, p = 0.042), and mitotic figures (r = 0.76, p = 0.001). CONCLUSIONS: Significant differences exist in the spatial distribution of mature versus neovessels in human uveal melanoma. Vessel maturation is associated with known clinical and pathologic indicators of poor prognosis (e.g., cell proliferation). Antiangiogenic therapy should be considered for the treatment of ocular malignancies; however, the results of this study indicate that blood vessel maturation heterogeneity may limit the efficacy of vessel targeting agents.

Echographic localization of periocular Carboplatin for treatment of advanced retinoblastoma.

PURPOSE: To study ocular ultrasonography as a means to effectively localize periocular carboplatin in patients with advanced retinoblastoma. METHODS: In a cases series, seven patients diagnosed with advanced retinoblastoma refractory to standard chemotherapy were treated with two to four periocular carboplatin injections. Echographic images were obtained before and after injection. RESULTS: The periocular carboplatin depot was a discrete homogeneous structure with lower internal reflectivity than the surrounding orbital tissue. The mean maximal juxtascleral height of the drug depot ± SD was 3.3 ±1.4 mm and was located directly posterior to the area of maximal intraocular tumor thickness in all seven patients. Moderate shadowing from calcification was present in one patient. Five patients had a pattern of a thinner pocket of drug visible after subsequent injections. CONCLUSIONS: Echography is a useful technique to study novel periocular drug delivery. It effectively images the drug in relation to the intraocular tumor, confirming the most effective drug placement for these resistant tumors.

Pediatric nasopharyngeal carcinoma with retinal metastasis.

PURPOSE: To report an unusual case of nasopharyngeal carcinoma (NPC) metastatic to the lungs and retina. To our knowledge, this is the first report of retinal metastasis from NPC. DESIGN: Observational case report. METHODS: We studied the case of a 15-year-old boy who had been recently diagnosed with metastatic NPC and was referred for evaluation of a suspicious retinal lesion in the right eye. RESULTS: Clinical examination and fundus photography showed an amelanotic, 1-disk-area infiltration in the retina along the inferotemporal arcade that was suggestive of metastatic disease. The lesion was small and irregularly shaped by echographic examination, and a nodular area of retinal thickening was seen by optical coherence tomography. Pathologic analysis of lung biopsy and nasopharynx biopsy specimens revealed undifferentiated NPC. CONCLUSIONS: Retinal metastases need to be considered in the differential diagnosis of patients presenting with NPC and vision changes.

Photodynamic therapy and vitrectomy for a large optic nerve hemangioma with neovascularization and retinal detachment: a clinicopathologic correlation.

PURPOSE: To report the clinicopathologic correlation of a young man with a von Hippel-Lindau disease-associated peripapillary hemangioblastoma and its satisfactory response to a combination of photodynamic therapy (PDT) and vitrectomy. DESIGN: Clinicopathologic correlation. METHODS: We studied the case of a 14-year-old boy with an optic nerve mass and large inferior exudative retinal detachment complicated by a significant tractional component from extensive secondary neovascularization over the lesion. RESULTS: A juxtapapillary hemangioblastoma with secondary neovascularization was documented by clinical examination, fundus photography, and optical coherence tomography. A von Hippel-Lindau gene mutation was detected. The patient responded satisfactorily to a combination of PDT and vitrectomy. CONCLUSIONS: A staged approach to treatment of peripapillary hemangioblastoma with a combination of PDT and vitrectomy may be favorable to therapy with one modality.

Tumor volume reduction using combined phacoemulsification and intravitreal triamcinolone injection for the management of cataract with treated uveal melanoma and atypical nevi.

PURPOSE: To study the reduction in tumor size and the safety and efficacy of combined phacoemulsification and intravitreal triamcinolone acetonide injection (phaco-IVTA) in patients with treated melanoma and atypical nevi. SETTING: Bascom Palmer Eye Institute, Miami, Florida, USA. METHODS: The medical records of 49 consecutive patients (51 eyes) with treated melanoma or atypical nevi treated with phaco-IVTA were evaluated retrospectively for changes in Snellen visual acuity, tumor volume, and frequency of complications. Main outcome measures included a postsurgical change in tumor size greater than or equal to 0.5 mm of height or 1.0 mm of basal diameter by echographic analysis, improvement in visual acuity at 6 months and final follow-up, and complications including endophthalmitis, cystoid macular edema, epiretinal membrane, increased intraocular pressure, and persistent corneal edema. RESULTS: The median baseline visual acuity was 20/80 in the affected eye. At the 6-month follow-up examination, 13 (68%) of 19 eyes had achieved better than 20/40 visual acuity. Treated uveal melanomas (n=30) and atypical choroidal nevi (n=21) were stable with combined therapy, and echographic measurements improved in 12 eyes. Intraocular pressure increased from baseline to 25 mm Hg or more postoperatively in 4 of 51 eyes (8%). No other significant complications occurred. CONCLUSIONS: Combined phacoemulsification and IVTA was reasonably safe in patients with treated melanoma and atypical nevi. Tumors remained stable or decreased slightly in size. Intravitreal triamcinolone acetonide injection at the time of cataract surgery in patients with treated melanoma or nevus may reduce rates of tumor progression in these patients.

Basic fibroblast growth factor impact on retinoblastoma progression and survival.

PURPOSE: Chemotherapy resistance is a problem in the treatment of advanced retinoblastoma (RB). Since basic fibroblast growth factor (bFGF) is a survival factor for neural precursor cells, bFGF was evaluated as a growth and chemoresistance factor in RB. METHODS: bFGF expression was analyzed in the LH-betaTag transgenic mouse model of RB and human RB cell lines by immunofluorescence, RT-PCR, and Western blot. Proliferation and apoptosis (TUNEL) assays were performed. RESULTS: bFGF levels significantly increased during tumorigenesis in transgenic RB, as a function of tumor status (P = 0.005). PCR and confocal microscopy confirmed that the human cell lines and primary tumors expressed bFGF. bFGF was localized to vascular and tumor cells and rarely to glial cells. Exogenous 18-kDa bFGF induced proliferation in two RB cell lines (WERI and Y79). Western blot analysis demonstrated 34-, 22-, and 18-kDa isoforms in transgenic RB and both cell lines. In TUNEL assays, chemoresistance to carboplatin-induced apoptosis was observed in the Y79 line, which expressed a higher ratio of high (34 kDa)- to low-molecular-weight bFGF isoforms, compared with the WERI line. Similar to other bFGF tumor studies, exogenous low-molecular-weight (18 kDa) bFGF (1 ng) significantly enhanced carboplatin-induced apoptosis in the more chemosensitive WERI, but not the chemoresistant Y79 line. CONCLUSIONS: RB tumors produce significant amounts of bFGF, and the differential production and response to isoforms of bFGF may have implications for invasive tumor growth and chemoresistance.

Heterogeneous tumor vasculature in retinoblastoma: implications for vessel targeting therapy.

PURPOSE: The aim of this study is to correlate tumor size of retinoblastoma tumor samples with blood vessel maturation to assess how these factors may affect vessel targeting therapy. METHODS: Analysis was performed on retinoblastoma tumor specimens (n = 5) enucleated as primary treatment from May 2005 to February 2006. Tumor size was measured as the largest cross sectional area of the tumor, measured during pathologic assessment. Vessel density and heterogeneity was measured by immunohistochemical analysis. Total microvessel density was detected by staining endothelial cells using a lectin from Bandeira simplicifolia; novel vasculature was detected with the endothelial cell marker endoglin (CD105). Blood vessel basement membrane was detected with an antibody against type IV collagen. Vessel maturation was assessed by pericyte recruitment, detected with alpha smooth muscle actin (alpha-sma). RESULTS: A statistically significant correlation was detected between tumor burden and age at enucleation (P = 0.008). All retinoblastoma tumor samples harbored a high degree of blood vessel heterogeneity containing both immature neovessels as well as pericyte-committed mature vasculature. There was a statistically significant correlation between type IV collagen and age at enucleation (P = 0.045). CONCLUSIONS: This study provides a framework for a better understanding of tumor and vessel development in retinoblastoma. Results of this study provide insight into the relationship between age and tumor burden in these tumors. Knowledge of the degree of heterogeneity detected in these tumors will aid in the selection of novel blood vessel targeting strategies for children with this disease and other diseases with pathologic neovascularization.

Bilateral ocular manifestations of aggressive cutaneous metastatic melanoma.

PURPOSE: To report an unusual case of aggressive cutaneous malignant melanoma metastatic to both eyes. The case is also remarkable for differences from the bilateral diffuse uveal melanocytic proliferation syndrome (BDUMP). DESIGN: Observational case report. METHODS: The authors studied the case of a 56-year-old man with decreased vision and floaters in the left eye. He recently had been diagnosed with metastatic cutaneous malignant melanoma. RESULTS: Clinical examination, fundus photography, and ultrasonography revealed widespread metastases to the choroid, ciliary body, iris, and eyelid. A-scan revealed medium internal reflectivity of a collar-button lesion in the ciliary body, consistent with metastatic disease. CONCLUSIONS: Despite the rarity of BDUMP and bilateral intraocular malignant cutaneous or uveal melanoma metastases, these diagnoses should be considered when diffuse bilateral melanocytic lesions are present. Ultrasonography and metastatic workup are critically important to help distinguish these disease entities.

Mechanism of retinoblastoma tumor cell death after focal chemotherapy, radiation, and vascular targeting therapy in a mouse model.

PURPOSE: To evaluate the mechanism and timing of retinal tumor cell death in the LH(BETA)T(AG) mouse model of retinoblastoma after treatment with vascular targeting therapies and conventional therapies (focal chemotherapy and radiation). METHODS: For vascular targeting therapy, 12- or 16-week-old mice were treated with a single subconjunctival injection of either anecortave acetate (300 microg) or combretastatin A4 (1.5 mg). Eyes were analyzed at 1 day and 1 week after treatment. Tumor cell death was evaluated using TUNEL assays or immunofluorescence analysis of activated caspase 3 to detect apoptosis. Histopathologic analysis was performed to identify areas of necrosis. For conventional therapy, LH(BETA)T(AG) mice were treated with six serial subconjunctival injections of focally delivered carboplatin chemotherapy (100 microg/delivery) or hyperfractionated external beam radiotherapy (EBRT; 15 Gy total dose). Cell death was analyzed by TUNEL assay. RESULTS: The highest levels of apoptotic cell death were seen 1 day after treatment in all treatment groups compared with vehicle controls. At 1 week after treatment, apoptotic cell death remained significantly elevated in the EBRT and carboplatin groups, but not after vessel targeting therapy. No significant necrosis was detected by histology in tumors of treated or of control eyes. CONCLUSIONS: Conventional therapies (focal carboplatin chemotherapy and EBRT) and vascular targeting agents significantly increase cell death through apoptosis, while not having a significant effect on necrosis in this murine model of retinoblastoma. These studies will aid in the optimization of delivery schemes of combined treatment modalities.