H. Boutrid

Targeting Hypoxia, a Novel Treatment for Advanced Retinoblastoma

PURPOSE The purpose of this study was to evaluate the presence and extent of hypoxia in murine retinoblastoma tumors and the feasibility of targeting hypoxic cells as a novel therapeutic strategy. METHODS Hypoxic and vascular areas in LH(BETA)T(AG) mouse retinal tumors were measured using immunohistochemistry. The glycolytic inhibitor 2-deoxy-d-glucose (2-DG) was used to test the efficacy of targeting hypoxic cells in retinoblastoma. Sixteen-week-old LH(BETA)T(AG) mice received injections of saline, carboplatin (31.25 microg/20 microL), 2-DG (500 mg/kg), and carboplatin (31.25 microg/20 microL) + 2-DG (500 mg/kg). Carboplatin was administered through biweekly subconjunctival injections to right eyes only for 3 weeks. 2-DG was administered through intraperitoneal injection three times a week for 5 weeks. Saline was administered using both methods. Eyes were enucleated at 21 weeks of age and examined for residual tumor. RESULTS Hypoxic regions were observed in tumors larger than 3.28 mm(2). When 2-DG was combined with carboplatin, a marked decrease in tumor burden was observed that was significantly more pronounced than when either agent was given alone. The hypoxic tumor cell population as measured by pimonidazole was markedly reduced by carboplatin + 2-DG (P < 0.01) and by 2-DG alone (P < 0.01), but not by carboplatin alone, indicating that 2-DG effectively killed hypoxic retinoblastoma cells in vivo. CONCLUSIONS Treatment with glycolytic inhibitors as adjuvants to chemotherapy has the potential to increase the efficacy of chemotherapy in advanced retinoblastoma. This approach may have benefits for children with this disease and should be further investigated.

Supraselective injection of intraarterial melphalan as the primary treatment for late presentation unilateral multifocal stage VB retinoblastoma

Purpose: The purpose of this study was to report a case of a 7-year-old girl with unilateral, multifocal Reese Ellsworth Stage Vb retinoblastoma who was successfully treated using intraarterial chemotherapy infusion as the primary therapy. Methods: This is an interventional case report. A 7-year-old girl presented with advanced unilateral retinoblastoma. The patient received intraarterial melphalan infusion therapy as the primary treatment. Results: Complete tumor resolution was seen at 1 month after intraarterial melphalan infusion. Conclusion: This case of advanced retinoblastoma in a 7-year-old girl was successfully treated with intraarterial melphalan infusion alone. Treatment resulted in complete resolution of the tumor 1 month after treatment. In comparison with systemic chemotherapy, intraarterial melphalan infusion therapy may be a less toxic and more effective primary treatment option in the future management of advanced retinoblastoma.

Blood vessel maturation in retinoblastoma tumors: Spatial distribution of neovessels and mature vessels and its impact on ocular treatment

PURPOSE The purposes of this study were to evaluate the spatial distribution of neovessels versus mature vessels in both human retinoblastoma (RB) and LH(BETA)T(AG) tumors, assess similarities and differences between the animal model and the human RB specimens, and determine whether vessel maturation is associated with risk factors for metastasis. METHODS Immunohistochemical analyses were performed on human (n = 10) and LH(BETA)T(AG) (n = 11) enucleation specimens to evaluate the spatial distribution of neovessels and mature vessels. In human RB, vessel maturation was correlated with treatment history and metastatic risk factors. RESULTS In human RB, the percentage of neovessels was higher in the periphery of the tumor than in the center (P = 0.021). This finding was mostly attributed to the distribution of large-caliber vessels (i.e., neovessels were higher in the periphery for large [P = 0.050]- and medium [P = 0.032]-caliber vessels; and mature vessels were higher in the center for large-caliber vessels [P = 0.032]). In this small series, vessel maturation did not correlate with risk for metastasis. Similar results were observed in LH(BETA)T(AG) tumors. The percentage of large-caliber neovessels was higher in the periphery than in the center (P = 0.038). CONCLUSIONS There is a spatially distributed, heterogeneous vessel population containing neovessels and mature vessels in advanced RB disease. There is a significantly higher concentration of mature, large-caliber vessels in the center of tumors that is similar in human RB and LH(BETA)T(AG) retinal tumors. From these data the authors hypothesize that tumor vessel maturation in RB initiates in central regions of the tumor and radiates toward the periphery.

Increased hypoxia following vessel targeting in a murine model of retinoblastoma.

PURPOSE The purpose of this study was to evaluate the effects of vessel targeting and chemotherapy agents on inducing hypoxic regions in LH(BETA)T(AG) murine retinal tumors. METHODS. Twelve- and 16-week-old LH(BETA)T(AG) transgenic retinoblastoma mice were treated with periocular injections to the right eye only of saline (n = 42), anecortave acetate (a single injection; 300 microg/20 microL; n = 42), or carboplatin (two injections per week for 3 weeks; 62.5 microg/20 microL; n = 42). Eyes were enucleated 1 day, 1 week, and 1 month after injection. To assess hypoxia, mice received 60 mg/kg pimonidazole via intraperitoneal injection. Eyes were enucleated, and tumor sections were analyzed. RESULTS Levels of hypoxia significantly increase in 16-week-old animals 1 day and 1 week after treatment with anecortave acetate, a known angiostatic agent. Eyes treated with anecortave acetate showed a 28% (P < 0.001) increase in hypoxic regions in comparison with the saline-treated control group 1 day after injection and a 17% (P < 0.001) increase 1 week after injection. In early tumors of 12-week-old animals, levels of hypoxia increased by 2.0% (P = 0.011) 1 day after anecortave acetate injection compared to controls. Levels of hypoxia significantly decrease in 16-week-old animals 1 week and 1 month after treatment with carboplatin, a chemotherapeutic agent. Eyes treated with carboplatin showed a 21.7% (P = 0.017) decrease in hypoxic regions in comparison with the saline-treated control group 1 week after injection and a 4.51% (P < 0.001) decrease 1 month after injection. In early tumors of 12-week-old animals, levels of hypoxia decreased by 0.0429% (P < 0.001) 1 month after carboplatin injection compared with controls. CONCLUSIONS Treatment with a vessel-targeting agent results in changes in the tumor microenvironment as early as 1 day after treatment. By increasing hypoxia in tumors, vessel-targeting agents can be combined with glycolytic inhibitors which have been shown previously to target hypoxic regions in this transgenic model. This approach may have benefits for children with this disease and should be further investigated.

Focal, Periocular Delivery of 2-Deoxy-d-Glucose as Adjuvant to Chemotherapy for Treatment of Advanced Retinoblastoma

PURPOSE The aim of this study was to evaluate the changes in tumor burden and hypoxia in the LH(BETA)T(AG) retinal tumors after treatment with a focal, single-modality, and combination therapy using periocular carboplatin and 2-deoxy-d-glucose (2-DG). METHODS Seventeen-week-old LH(BETA)T(AG) transgenic mice (n = 25) were treated with periocular injections of varying doses of 2-DG (62.5, 125, 250, 500 mg/kg) to obtain a dose response. Same-aged mice (n = 30) received periocular injections of saline, carboplatin, and 2-DG. Mice were divided into six groups: saline; carboplatin (31.25 μg/20 μL, subtherapeutic dose); 2-DG (250 mg/kg); 2-DG (500 mg/kg); carboplatin (31.25 μg/20 μL) and 2-DG (250 mg/kg); and carboplatin (31.25 μg/20 μL) and 2-DG (500 mg/kg). Injections were administered twice weekly for three consecutive weeks. Eyes were enucleated at 20 weeks of age, snap frozen, and analyzed for hypoxic regions and tumor volume. RESULTS The difference in percentage of hypoxia after treatment with 500 mg/kg 2-DG was statistically significant from the other dose groups (P < 0.015). The difference in tumor burden was statistically significant from the 250 mg/kg dose (P < 0.015) and 500 mg/kg dose (P < 0.001). Highly significant differences were found between the treatment types for tumor burden, percentage of hypoxia, and pimonidazole intensity (P < 0.001). Tumor burden decreased significantly after all forms of treatment (P < 0.001); however, tumor burden became significantly more reduced after treatment with combination therapy of carboplatin and 2-DG than with either treatment alone (P < 0.001). The percentage of hypoxia and pimonidazole intensity decreased after treatment with 2-DG alone and in combination with carboplatin (P < 0.001) in all treatment groups using 2-DG regardless of the 2-DG dose used. There was no percentage reduction of hypoxia after treatment with carboplatin alone (P = 0.25). CONCLUSIONS This study demonstrates the efficacy of focal, periocular 2-DG as an adjunct to carboplatin chemotherapy to decrease both intratumoral hypoxia and tumor burden. Hypoxia is increasingly present in advanced disease of LH(BETA)T(AG) retinal tumors. The use of glycolytic inhibitors as a therapeutic strategy has the potential to enhance current retinoblastoma treatments.

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.

Novel retinoblastoma treatment avoids chemotherapy: the effect of optimally timed combination therapy with angiogenic and glycolytic inhibitors on LHBETATAG retinoblastoma tumors

PURPOSE The purpose of this study was to evaluate the effect of optimally timed combination treatment with angiogenic and glycolytic inhibitors on tumor burden, hypoxia, and angiogenesis in advanced retinoblastoma tumors. METHODS LH(BETA)T(AG) mice (n =30) were evaluated. Mice were divided into 5 groups (n =6) and received injections at 16 weeks of age (advanced tumors) with a) saline, b) anecortave acetate (AA), c) 2-deoxyglucose (2-DG), d) AA +2-DG (1 day post-AA treatment), or e) AA +2-DG (1 week post-AA treatment). Eyes were enucleated at 21 weeks and tumor sections were analyzed for hypoxia, angiogenesis, and tumor burden. RESULTS Eyes treated with 2-DG 1 day post-AA injection showed a 23% (P =0.03) reduction in tumor burden compared with 2-DG alone and a 61% (P < 0.001) reduction compared with saline-treated eyes. Eyes treated with 2-DG 1 week post-AA injection showed no significant decrease in tumor burden compared with 2-DG alone (P = 0.21) and a 56% (P < 0.001) decrease in comparison with saline-treated eyes. 2-DG significantly reduced the total density of new blood vessels in tumors by 44% compared to saline controls (P < 0.001), but did not affect the density of mature vasculature. CONCLUSIONS Combination therapy with angiogenic and glycolytic inhibitors significantly enhanced tumor control. Synergistic effects were shown to be dependent on the temporal course of treatment, emphasizing optimal timing. 2-DG was shown to reduce the density of neovessels, demonstrating an antiangiogenic effect in vivo. As a result, angiogenic and glycolytic inhibitors may have significant potential as alternative therapies for treating children with retinoblastoma.

Impact of Tumor-Associated Macrophages in LHBETATAG 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.

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.

Bilateral orbital vasculature alterations after systemic chemotherapy and external beam radiation therapy treatment of advanced retinoblastoma: implications for intraarterial chemotherapy management.

PURPOSE The purpose of this study was to report significant alterations in orbital vasculature after combined systemic chemoreduction with consolidating external beam radiotherapy treatment in a case of advanced retinoblastoma and to discuss implications for intraarterial melphalan rescue chemotherapy. METHODS We studied the case of a 22-month-old child who had been treated recently with intraarterial melphalan perfusion after multimodal therapy for advanced intraocular retinoblastoma. RESULTS Orbital angiography of both eyes, performed before the intraarterial melphalan injection, documented an atypical attenuated orbital vasculature. The right ophthalmic artery angiogram showed loss of a normal dominant central retinal artery. Instead, multiple dysplastic angiogenic vessels from the ophthalmic artery were found to supply the retina. A single long ciliary artery was noted with collateral vessels supplying the anterior globe. The left ophthalmic artery angiogram showed a stenotic artery with minimal anterograde flow. CONCLUSION Orbital vasculature may be impacted by combined systemic chemotherapy and external beam radiation therapy. These changes need to be considered in the use of intraarterial melphalan for the treatment of patients with advanced retinoblastoma.