- Email: [email protected]
Photodynamic Therapy of Pigmented Choroidal Melanomas of Greater than 3-mm Thickness
Photodynamic Therapy of Pigmented Choroidal Melanomas of Greater than 3--mm Thickness Rosa Y. Kim, MD, Li~Kuan Hu, MD, Bradley S. Foster, MD, Evangelos S. Gragoudas, MD, Lucy H. Y. Young, MD, PhD Purpose: The purpose of the study is to determine the effect of photodynamic therapy in the destruction of experimental pigmented choroidal melanomas ~ 3 mm in thickness using a liposomal preparation of benzoporphyrin derivative, verteporfin. Methods: Pigmented choroidal tumors were established in 32 New Zealand albino rabbit eyes. Animals were treated with daily injections of cyclosporine, and tumor growth was followed by serial fundus examinations and ultrasonography. When a tumor ex ceeded 3 mm in thickness (tumor height ranged from 3.1-4.6 mm), the authors adminis tered benzoporphyrin derivative intravenously (1 mg/kg) and irradiated the tumor at 692 nm through an argon-pumped dye laser at different total light doses ranging from 60 to 120 J/cm2 • Control animals were treated with light or benzoporphyrin derivative only. Each animal then was followed-up for 4 to 6 weeks by fundus photography, fluorescein angiography, and ultrasonography. Results: All animals treated with benzoporphyrin derivative and light at fluences of ~ao J/cm2 showed complete tumor arrest. In contrast, both control groups showed continu ous tumor growth in all animals with tumors filling most of the vitreous cavity by 3 weeks. Histologic examination results of tumors treated with dye plus light immediately after treat ment showed prominent vascular closure. No vascular changes were noted in the control eye treated with light or dye alone. Examination results of the eyes that showed tumor regression after a 4-week follow-up period showed tumor necrosis and extensive infiltration of mononuclear cells and pigment-laden macrophages at the tumor site. Conclusion: These data suggest that photodynamic therapy may have a role in the management of pigmented choroidal melanomas. Ophthalmology 1996; 103:2029-2036
Photodynamic therapy (PDT) involves the systemic ad ministration of photosensitizers in combination with light of appropriate wavelength for the treatment of abnormal Originally received: March 22, 1996. Revision accepted: August 2, 1996. From Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston. Presented in part at the ARVO Annual Meeting, Ft. Lauderdale, May 1995. Supported in part by NIH EY10975-0l, Cancer Research Institute, Re search to Prevent Blindness, Inc, New York, New York, and Massachu setts Lions Eye Research Fund, Inc, Northboro, Massachusetts. The Massachusetts Eye and Ear Infirmary has a proprietary interest in this technology under a research agreement with Coherent Inc, Palo Alto, California, and as part of a patent application. Reprint requests to Lucy H. Y. Young, MD, PhD, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, 243 Charles St, Bos ton, MA 02114.
tissue. It is one of the newest strategies for the local treatment of malignant tumors. 1' 2 Phase III clinical trials for cancer of the lung and phase II trials for cancer of the bladder with porfimer sodium (photofrin, QLT Photo therapeutics, Inc, Vancouver, BC), a purified preparation of hematoporphyrin derivative (HPD), have been com pleted.3-5 Photofrin has been accepted recently by the U.S. Food and Drug Administration for the treatment of advanced esophageal cancer. Porphyrins have been used in PDT because of their ability to generate oxygen radi -cals wl;J.en irradiated with light. 6 - 8 However, PDT using these compounds has been limited because of skin photo sensitivity lasting up to 2 months and poor tissue penetra tion of light at 630 nm, the wavelength used to activate HPD. 9 Recently, PDT has gained much interest with the de velopment of second-generation photosensitizers. 10' 11 Benzoporphyrin derivative (BPD) is one such photosensi
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tizer whose peak absorption occurs at 692 nm, allowing improved tissue penetration and significantly decreased skin phototoxicity. In addition, BPD exhibits 10 to 70 times more cytotoxicity than does HPD in vitro. 12 Because of its absorption and emission peaks in the infrared region, BPD has been effective for PDT in experimental murine tumors 13 and choroidal melanomas 14 and currently is in phase II clinical trials for the treatment of cutaneous le sions. Furthermore, it also holds some promise as a bone marrow purging agent in the treatment of leukemia, 15 as 16 an antiviral agent for the decontamination of blood, and as a treatment for nonmalignant ocular diseases (e.g., subretinal neovascularization). 17 Previous studies using various second-generation pho tosensitizers have treated experimental choroidal melano mas. 18 - 2 1 In these studies, the Greene hamster amelanotic melanoma cell line was used. Even though these results are encouraging, data obtained with amelanotic melano mas are not applicable to clinical practice because more than 90% of human ocular melanomas are pigmented. Recently, we developed an animal model of pigmented choroidal melanoma in our laboratory,22 and earlier expe rience in treating small tumors less than 2.5 mm in height was quite successful. 23 In this study, we set out to evaluate the effects of PDT in the destruction of tumors ~ 3 mm in thickness using a liposomal preparation of BPD.
Materials and Methods Cell Line B16F10 mouse melanoma cells (obtained from Dr. R. Haming) were passaged in vitro in Dulbecco modified Eagle medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Hyclone Laboratories, Logan, UT). Two weeks before subchoroidal implantation, 106 B16F10 cells in 0.1-m\ phosphate-buffered saline were inoculated subcutaneously into the flank area of a C57B 1/6 mouse. With this procedure, a palpable tumor usually was estab lished within 10 to 14 days of implantation. Tumor Model All animals were treated in accordance with the Associa tion for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Re search. The establishment of choroidal tumors was carried out as described. 22 Briefly, female New Zealand albino rabbits weighing 1.8 to 2.5 kg were immunosuppressed with daily injections of cyclosporine (Sandimmune) at a concentration of 20 mg/kg for the first week and 'r5 mg/ kg for the remainder of the experiment. Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg) before tumor implantation. The donor mouse was killed immediately before implantation, and the tumor was dissected free and stored in cold normal saline solu tion. Two limbal 7-0 polygalactin traction sutures were
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placed to rotate the globe of the rabbit superiorly. An inferior conjunctival peritomy was performed, and a 2 mm circumferential scleral incision was made with a number 64 blade (Beaver Surgical Products, Waltham, MA) 6-mm posterior to the limbus to expose the choroid. A 0.5-mm3 tumor fragment was aspirated into the blunted tip of a 22-gauge spinal needle and deposited into the subchoroidal space approximately 2 disc diameters infe rior to the optic nerve. The scleral incision was closed with 8-0 nylon sutures. Indirect ophthalmoscopy was used to verify tumor position through a pupil dilated with a mixture of 2.5% phenylephrine hydrochloride and 1o/o tropicamide. Animals were examined every 3 days by indirect ophthalmoscopy starting 1 week after implanta tion. Tumor heights were determined by B-scan ultraso nography (Quantum 200; Siemens, Issaquah, W A). The range of tumor heights at the time of entry into the treat ment groups was 3.1 to 4.6 mm (mean height, 3.3 mm). Fundus photographs were taken with a fundus camera (CF-60ZA; Canon, Lake Success, NY) before and after treatment and at 1-week intervals after treatment. Fluores cein angiography was performed immediately after treat ment using 10% sodium fluorescein (0.1 mg/kg) injected into the marginal ear vein. Photosensitizer Freeze-dried lyophilized liposomal BPD (QLT Photo Therapeutics, Inc, Vancouver, British Columbia) was re constituted with sterile water to an isotonic solution of 1.47 mg/ml immediately before use and administered in travenously at a dose of 1 mg/kg through an ear vein 30 minutes before light exposure. The animals were housed in the dark for 24 hours to protect them against skin sensitivity and to ensure that all observed effects were caused by laser light alone. Photodynamic Therapy Laser radiation was produced by an argon-pumped dye laser (Coherent Medical Laser, Coherent, Inc, Palo Alto, CA) tuned to emit light at 692 nm. The laser fluence delivered was determined by a power meter (Field Master, Coherent, Inc) before and after each treatment. The light was delivered via a 200-p,m fiberoptic cable coupled to a slit lamp (Zeiss, Carl Zeiss, Oberkochen, Germany). The beam diameter on the rabbit retina was 2 mm, and the desired irradiance was achieved by calculating the power output in relation to the area covered by the light after correction for the optics of the rabbit eye and the fundus contact lens (Ogfa-FA, Ocular Instruments, Belleview, WA). Estimated fluences ranging between 60 and 120 J/cm 2 were provided by the application of over lapping spots until the tumor was completely treated; a half-spot size margin was given around the tumor. Irradi ances of 300, 600, and 1000 mW/cm 2 were used. Thirty-two tumor-bearing animals were included in the study. Twenty-five animals were treated with light and dye. Control animals were treated with light only (three animals), dye only (two animals), or observation (two
Kim et al · PDT of Pigmented Choroidal Melanomas
Figure 1. a, pretreatment fundus photograph of a pigmented choroidal melanoma. The height of this tumor is 4.2 min and the diameter is 7.6 mm. b, pretreatment ultrasonogram of the tumor shown in a. c, post-treatment fundus photograph of the same tumor 4 weeks after photodynamic therapy using 100 J/cm2• The tumor is less heavily pigmented, and adjacent areas of atrophy are seen. d, post-treatment ultrasonogram shows the height of the same tumor at this stage to be 1. 7 mm.
animals). All animals treated with light and dye were followed for 4 to 6 weeks, except for the two rabbits killed immediately after treatment for histologic examination. Follow-up for the control animals was limited to 2 weeks because of rapid tumor growth. Histopathologic Studies After the animals were killed, the eyes were enucleated, fixed in 10% buffered formaldehyde solution (formalin, Sigma Chemical Co, St. Louis, MO), and processed for paraffin sectioning. Seven micron-thick sections were stained with hematoxylin-eosin and examined by light microscopy (BHS System, Olympus Optical Co Ltd, Tokyo, Japan).
Results Treatment Response Thirty-two rabbits bearing heavily pigmented tumors of heights 2:: 3 mm were used in our study. Figures 1a and 1b show an example of a tumor 4.2 mm in thickness. Twenty~five tumor-bearing animals were treated with light and dye, and their dimensions and responses are summarized in Table 1. Two of these animals were killed immediately for histologic examination, and the re maining 23 animals were followed-up for 4 to 6 weeks. Seven control animals were included in this study. Two animals were treated with BPD alone, and three animals were treated with light alone (120 J/cm2 , 150 J/cm 2). Fur
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Table 1. Comparison of Fluence to Treatment Outcome for Animals Treated with Dye and Light
Fluence (J/cm 2 )
No. of Animals
60 80 100 120
5 9 5* 6 25
Total
Height (mm)
Diameter (mm)
Range (mean)
Range (mean)
3.0-3.3 3.1-3.6 3.2-4.2 3.0-4.6
3.2-6.0 3.8-6.0 7.0-7.6 5.1-6.8
(3.1) (3.2) (3.8) (3.4)
(4.9) (5.1) (7.3) (5.4)
Tumor Arrest
Tumor Growth
1 9 3 6 19
4 0 0 0 4
*Two animals were killed for histologic examination.
thermore, two received neither dye nor light. Two rabbits, one that received light alone and another that received BPD alone, were killed immediately for histologic stud ies. The remaining five control animals were followed up for 2 weeks and were killed because their tumors had filled the vitreous cavity. All animals treated with fluence of 80 J/cm2 or greater showed tumor arrest. Tumors with complete regression showed a typical clinical course. Immediately after treat ment, blanching of the tumor and closure of surrounding choroidal vessels were noted. Serous retinal detachment occurred in all animals treated with light and dye. This exudative process usually peaked within several hours of the treatment, and the absorption of subretinal fluid was spontaneous and complete within 14 days of treatment. As the subretinal fluid resolved gradually, the tumors were observed to be less pigmented, and reductions in the tu mor dimensions were evident. Overlying and adjacent choroid became avascular, leading to fibrotic chorioretinal scars surrounded by a ring of chorioretinal atrophy (Fig 1c). As shown in Figure 1d, a tumor of 4.2 mm in thick ness had shrunk to 1.7 mm only 4 weeks after treatment. Tumor growth occurred in four animals treated with a fluence of 60 J/cm2 • This was first evident by the persistent heavy pigmentation in the treated area followed by exten sion of pigmentation through the treated margins. As the subretinal fluid overlying the tumor cleared, the increase in thickness was evident with indirect ophthalmoscopy and documented by ultrasonography. Frequently, the growth manifested as a small pigmented nodule on the surface within the first week after treatment. The fluores cein angiograms from these tumors showed diffuse leak age. An example of fluorescein angiographies before and after treatment is shown in Figure 2. The untreated tumors showed delayed filling followed by hyperfluoresc'ence of the mass in the late phase of the angiograms (Figs 2a, b). Angiography immediately after treatment with BPD and light showed significant hypofluorescence of the tumor and surrounding choroidal vessels, corresponding to the areas of treatmenf(Fig 2c). Fluorescein angiographies of the control groups treated with either light or dye only showed no difference from that of the untreated animals. No apparent toxicity was encountered after the intrave-
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nous administration of 1 mg/kg of BPD in the rabbit. All the rabbits treated with 120 J/cm2 had subconjunctival chemosis. Serous retinal detachment was detected in all animals treated with light and dye, and the ones treated with higher fluences had more prominent serous detach ments compared to those treated with 60 J/cm2 , but all underwent spontaneous resolution. Histopathologic Examination Histopathologic examination results of tumors performed immediately after treatment with 100 J/cm2 plus dye showed markedly dilated vascular channels packed with erythrocytes throughout the full thickness of the tumor (Fig 3a). Tumors receiving dye alone or laser irradiation alone (Fig 3b) did not show any changes compared to untreated tumors. No vascular changes were noted in these eyes. Four to 6 weeks after treatment with light plus dye, no mitotic figures or viable tumor cells were seen at the tumor site (Fig 4a). The necrotic tumors were infil trated with pigment-laden macrophages and mononuclear cells. In a control eye, intact tumor cells with prominent mitotic figures were identified (Fig 4b).
Discussion A major problem with current conventional treatment methods for malignant tumors is the lack of tumor speci ficity and low therapeutic-toxic ratios. Photodynamic therapy is a relatively new and experimental means of treating tumors that are accessible to light. 8' 24 The princi ple of this method is based on the activation of photosensi tizing agents by light of the appropriate wavelength, which, in turn, causes tissue destruction. This approach offers dual selectivity for tumor tissue, which is accom plished by preferential retention of photosensitizer dye within the tumor and restriction of the illumination field to the tumor site. The potential of PDT to selectively target tumor cells is particularly attractive in anatomic regions where structural integrity is crucial for mainte nance of vital functions such as the larynx, bronchus, esophagus, urinary bladder, and brain. 4 •5•10•25 Ocular melanoma may be an ideal candidate for PDT
Kim et al · PDT of Pigmented Choroidal Melanomas Figure 2. a, a red-free pretreatment fundus photograph of a pigmented choroidal melanoma measuring 3.6 mm in thickness. b, pretreatment fluo rescein angiogram of the same tumor. The tumor vasculature shows delayed hyperfluorescence. c, fluorescein angiogram of the same tumor immediately after treatment. H ypofluorescence of the tumor and surrounding areas corresponding to the areas of treatment is noted.
because of the clear ocular media that permit precise alignment of the laser beam with the lesions, thus min imizing photodestruction of adjacent healthy tissues. Fur thermore, the presence of a rich and leaky tumor vascula-
ture and an intact blood-retina barrier in adjacent tissues may result in enhanced localization of photosensitizer dye within tumors? 6 During the 1980s, a few investigators applied PDT to treat ocular tumors using HPD as the photosensitizer. 27 - 31 However, efficacy of HPD was difficult to determine be cause the few small clinical trials were carried out on patients with tumors of different origins and sizes, in whom many had recurrent or advanced diseases. Complete tumor destruction was shown in some selected cases, but most tumors displayed superficial necrosis with viable cells in the deeper layers. In addition, HPD now is widely recog nized for its limitations. The recent resurgence of PDT in cancer therapy is attributed to the development of second-generation photo sensitizers with significantly less phototoxicity and mark edly improved tissue penetration and enhanced drug de livery systems. Preassociation of BPD with either lipopro teins or liposomes has been shown to result in increased tumor-cell killing in vivo as well as in vitro. 13 •31 The route of tumor uptake of BPD is thought to be via a low density lipoprotein receptor-mediated pathway? 2 Miller et al 17 have shown successful closure of experimental cho roidal neovascularization using low-density lipoprotein delivered BPD. In our study, we used liposomes as vehi cles for the delivery of BPD. A dehydrated liposomal preparation can be stored easily and reconstituted, re sulting in entrapment of a high percentage of BPD, which then is injected intravenously. Previous studies using second-generation photosensi tizers in treating choroidal melanoma have been favor able. Ozier et al 19 and Panagopoulos et al 18 have used chloroaluminum sulfonated phthalocyanine with Greene hamster melanomas transplanted into the rabbit eye. Al though both studies showed promising data, the tumors were amelanotic with obvious limitations to clinical appli cations for choroidal melanomas. A recent study from our laboratory 20 used heavily pig mented choroidal tumors in evaluating PDT with chloro aluminum sulfonated phthalocyanine. We were successful in treating tumors as high as 4.8 mm. However, because chloroaluminum sulfonated phthalocyanine is not ap proved for clinical use in the United States, we chose BPD to treat thick, pigmented choroidal melanomas. Pre liminary data with small pigmented23 and nonpigmented 14 choroidal melanomas have been encouraging. Although there has been general concern that pigmen tation may impede the effectiveness of PDT, studies from our laboratory have shown that PDT is effective in treat ing thick, pigmented choroidal melanomas with chloro aluminum sulfonated phthalocyanine?0 Our current data
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Figure 3. a, histopathologic examination results of a tumor immediately after treatment with 100 J/cm 2 plus dye. Dilated vessels packed with erythrocytes were identified (arrows). b, histopathologic studies of a tumor treated with light only (120 J/cm2 ). The findings are similar to those of untreated B16F10 melanomas.
also attest to the ability of PDT to destroy heavily pig mented choroidal tumors as thick as 4.6 mm. Some investigators33 actually have proposed a syner gistic effect between PDT and hyperthermia generated by the melanin granules. The latter converts light energy to thermal energy. Hyperthermia has been shown to be induced by light at the red spectrum, 33 - 37 and with irradi ances of >300 mW/cm2 , we would expect some thermal effects to be present. However, these effects alone were incapable of destroying choroidal tumors, as shown in our control animals treated with light only. Ideally, we would have preferred to use lower irradiances to minimize any contributing thermal effects, but higher irradiances were chosen with the purpose of maintaining short treat ment times to keep the therapy clinically practical. Miller et al 17 used similar irradiances to obliterate experimental choroidal neovascular membranes, and they showed no adverse angiographic or histologic changes in the overly ing retina when compared with eyes treated with lower irradiances.
Our histopathologic evaluation of tumors treated with both light and dye suggests that vascular occlusion lead ing to tumor necrosis is at least partially responsible for tumor arrest. Two studies by Nelson et al 38•39 suggest that the rapid necrosis of tumor tissue seen after PDT is not the result of direct killing of tumor cells but secondary to destruction of the tumor microvasculature. They observed that the first signs of destruction occurred in the subendo thelial zone of the tumor capillary wall. Other studies also have shown occlusion of choroidal vessels using BPD. 40.4 1 In summary, our study suggests that PDT using liposo mal BPD is effective in destroying heavily pigmented choroidal melanomas even more than 3 mm in height. This method may provide an alternative therapy for poste rior pole tumors in which conventional radiation therapy may result in poor visual outcome because of radiation maculopathy or optic neuropathy. 42 - 44
Acknowledgments. The authors thank QLT PhotoThera peutics, Inc, for providing BPD and Coherent Laser Medical Group for assistance with the development of the laser delivery system.
Figure 4. a, 6 weeks after treatment with 100 J/cm2 , the tumor is infiltrated with pigment-laden macrophages and mononuclear cells. No mitotic figures or viable tumor cells are seen at the tumor site. b, in the control eye, treated with benzoporphyrin derivative only (1 mg/kg), intact tumor cells with prominent mitotic figures (arrows) are identified (hematoxylin-eosin; original magnification, X200).
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Kim et al · PDT of Pigmented Choroidal Melanomas
References 1. Dougherty TJ. Photosensitizers: therapy and detection of malignant tumors. Photochem Photobiol 1987;45:879-89. 2. Mannyak MJ, Russo A, Smith P, Glastein E. Photodynamic therapy. J Clin Oncol 1988;6:380-91. 3. Marcus SL. Current status of photodynamic therapy for human cancer. Society of Photo-Optical Instrumentation Engineers 1991;1426:302-10. 4. Prout GR, Jr, Lin CW, Benson R Jr, et al. Photodynamic therapy with hematoporphyrin derivative in the treatment of superficial transitional cell carcinoma of the bladder. N Engl J Med 1987;317:1251-5. 5. Karanov S, Kostadinov D, Shopova M, Durtev P. Photody namic therapy in lung and gastrointestinal cancers. J Pho tochem Photobiol B 1990;6:175-81. 6. Moan J, Peng Q, Evenson JF, et al. Photosensitivity effi ciencies: tumors and cellular intake of different photosensi tizing drugs relevant for photodynamic therapy of cancer. Photochem Photobiol 1987;46:713-21. 7. Krohn DL, Jacobs R, Morris DA. Diagnosis of model cho roidal malignant melanoma by hematoporphyrin derivative fluorescence in rabbits. Invest Ophthalmol Vis Sci 1974; 13:244-55. 8. Spikes JD. Photobiology of porphyrins. In: Doiron DR, Gomer CJ, eds. Porphyrin Localization and Treatment of Tumors. New York: Alan R. Liss Inc, 1984; 19-39. 9. Tralau CJ, Young AR, Wolkern PJ, et al. Mouse skin photo sensitivity with dihaematoporphyrin ether and aluminum sulphonated phthalocyanine: a comparative study. Pho tochem Photobiol 1988;49:305-12. 10. Marcon NE. Photodynamic therapy and cancer of the esophagus. Semin Oncol1994;21:20-3. 11. Lui H, Hruza L, Kollias N, et al. Photodynamic therapy of malignant skin tumors with benzoporphyrin derivative monoacid ring A (BPD-MA): preliminary observations. In: Proceedings of the Lasers in Otolaryngology, Dermatology, and Tissue Welding. Bellingham, WA: Society of Photo Optical Instrumentation Engineers, 1993; 1876:147-51. 12. Richter AM, Cerruti-Sola S, Sternberg ED, et al. Biodistri bution of tritiated benzoporphyrin derivative eH-BPD MA), a new potent photosensitizer in normal and tumor bearing mice. J Photochem Photobiol B 1990;5:231-44. 13. Richter AM, Waterfield E, Jain AK, et al. Liposomal deliv ery of a photosensitizer, benzoporphyrin derivative mono acid ring A (BPD), to tumor tissue in a mouse tumor model. Photochem Photobiol 1993;57:1000-6. 14. Schmidt-Erfurth U, Bauman W, Gragoudas E, et al. Photo dynamic therapy of experimental choroidal melanoma us ing lipoprotein-delivered benzoporphyrin. Ophthalmology 1994; 101:89-99. 15. Jamieson CH, McDonald WN, Levy JG. Preferential uptake of benzoporphyrin derivative by leukemic versus normal cells. Leuk Res 1990;14:209-19. 16. Neyndorff HD, Bartel DL, Tufaro F, Levy JG. Develop ment of a model to demonstrate photosensitizer-mediated viral inactivation in blood. Transfusion 1990;30:485-90. 17. Miller JW, Walsh AW, Kramer M, et al. Photodynamic therapy of experimental choroidal neovascularization using lipoprotein-delivered benzoporphyrin. Arch Ophthalmol 1995; 113:810-8. 18. Panagopoulos JA, Svitra PP, Puliafito CA, Gragoudas ES. Photodynamic therapy for experimental intraocular mela noma using chloroaluminum sulfonated phthalocyanine. Arch Ophthalmol 1989; 107:886-90.
19. Ozier SA, Nelson SJ, Liggett PE, et al. Photodynamic ther apy of experimental subchoroidal melanoma using chloro aluminum sulfonated phthalocyanine. Arch Ophthalmol 1992; 110:555-61. 20. Gonzales VH, Hu LK, Theodossiadis PG, et al. Photody namic therapy of pigmented choroidal melanoma. Invest Ophthalmol Vis Sci 1995;36:871-8. 21. Hill RA, Reddi S, Kenney ME, et al. Photodynamic therapy of ocular melanoma with bis silicon 2,3-naphthlocyanine in a rabbit model. Invest Ophthalmol Vis Sci 1995; 36:2476-81. 22. Hu LK, Huh K, Gragoudas ES, Young LHY. Establishment of pigmented choroidal melanomas in a rabbit model. Ret ina 1994; 14:264-9. 23. Young LHY, Howard MA, Hu LK, et al. Photodynamic therapy of pigmented choroidal melanoma using a liposo mal preparation of benzoporphyrin derivative. Arch Oph thalmol 1996; 114:186-92. 24. Dougherty TJ. Photodynamic therapy of malignant tumors. Crit Rev Oncol Hematol 1984; 2:83-116. 25. Noske DP, Wolbers JG, Sterenborg HJ. Photodynamic ther apy of malignant glioma: a review of literature. Clin Neurol Neurosurg 1991;93:293-307. 26. Tse DT, Dutton JJ, Weingeis TA, et al. Hematoporphyrin photoradiation therapy for intraocular and orbital malignant melanoma. Arch Ophthalmol 1984; 102:833-8. 27. Ohnishi Y, Yamana Y, Minei M. Photoradiation therapy using argon laser and a hematoporphyrin derivative for retinoblastoma: a preliminary report. Jpn J Ophthalmol 1986;30:409-19. 28. Sery TW, Shield JA, Augsburger JJ, Shah HG. Photody namic therapy of human ocular cancer. Ophthalmic Surg Lasers 1987; 18:413-8. 29. Murphree AL, Cote M, Gomer CJ. The evolution of photo dynamic therapy techniques in the treatment of intraocular tumors. Photochem Photobiol 1987;46:919-23. 30. Bruce RA, Jr. Evaluation of hematoporphyrin photoradia tion therapy to treat choroidal melanoma. Lasers Surg Med 1984;4:59-64. 31. Allison BA, Waterfield E, Richter AM, Levy JG. The ef fects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative. Photochem Photobiol 1991;54:709-15. 32. Rutledge JC, Curry F-R, Lenz JF, Davis PA. Low density lipoprotein transport across a microvascular endothelial barrier after perfl\eability is increased. Circ Res 1990; 66:486-95. 33. Berns MW, Coffey J, Wile AG. Laser photoradiation ther apy of cancer. Possible role of hyperthermia. Lasers Surg Med 1984;4:87-92. 34. Milanesi C, Biolo R, Reddi E, et al. Ultrastructural studies on the mechanism of the photodynamic therapy of tumors. Photochem Photobiol 1987;46:675-81. 35. Svaasand LO. Photodynamic and photohyperthermic re sponse of malignant tumors. Med Phys 1985; 12:455-61. 36. Kinsey JH, Cortese DA, Neel HB. Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983;43:1562-7. 37. Mattiello, Hetzel F, Vandenheede L. Intratumor tempera ture measurements during photodynamic therapy. Pho tochem Photobiol 1987;46:873-9. 38. Nelson JS, Liaw LH, Berns MW. Mechanism of tumor destruction in photodynamic therapy. Photochem Photobiol 1987;46:829-36. 39. Nelson JS, Liaw LH, Orenstein A, et al. Mechanism of tumor destruction following photodynamic therapy with
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hematoporphyrin derivative, chlorine and phthalocyanine. J Nat! Cancer Inst 1988;80:1599-1605. 40. Lin SC, Lin CP, Feld JR, eta!. The photodynamic occlusion of choroidal vessels using benzoporphyrin derivative. Curr Eye Res 1994; 13:513-22. 41. Schmidt-Erfurth U, Hasan T, Gragoudas ES, eta!. Vascular targeting in photodynamic occlusion of subretinal vessels. Ophthalmology 1994; 101:1953-61. 42. Seddon JM, Gragoudas ES, Polivogianis L, et a!. Visual
outcome after proton beam irradiation of uveal melanoma. Ophthalmology 1986;93:666-74. 43. Seddon JM, Gragoudas ES, Egan KM, eta!. Uveal mel anomas near optic disc or fovea: visual results after proton beam irradiation. Ophthalmology 1987;94:354 61. 44. Cruess AF, Augsburger JJ, Shields JA, et a!. Visual results following cobalt plaque radiotherapy for posterior uveal melanomas. Ophthalmology 1984;91:131-6.
Centennial Advertisement From the Sur une nouvelle methode de querir la cataracte par ['extraction du cristalin, a report by Jacques Daviel (1693 or 1696-1762), showing his method for cataract extraction. Daviel is best remembered for developing the modem methods of cataract extraction. Instead of couching the cataract with a needle, Daviel developed and used a knife needle for the incision, two curved scissors, a gold spatula to remove the lens, a needle to open the anterior capsule, and a spoon and forceps to remove the lens matter.*
* Centennial advertisement provided courtesy of the Museum of Ophthalmology, Foundation of the American Academy of Ophthalmology, San Francisco, California
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Photodynamic therapy (PDT) involves the systemic ad ministration of photosensitizers in combination with light of appropriate wavelength for the treatment of abnormal Originally received: March 22, 1996. Revision accepted: August 2, 1996. From Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston. Presented in part at the ARVO Annual Meeting, Ft. Lauderdale, May 1995. Supported in part by NIH EY10975-0l, Cancer Research Institute, Re search to Prevent Blindness, Inc, New York, New York, and Massachu setts Lions Eye Research Fund, Inc, Northboro, Massachusetts. The Massachusetts Eye and Ear Infirmary has a proprietary interest in this technology under a research agreement with Coherent Inc, Palo Alto, California, and as part of a patent application. Reprint requests to Lucy H. Y. Young, MD, PhD, Massachusetts Eye and Ear Infirmary, Department of Ophthalmology, 243 Charles St, Bos ton, MA 02114.
tissue. It is one of the newest strategies for the local treatment of malignant tumors. 1' 2 Phase III clinical trials for cancer of the lung and phase II trials for cancer of the bladder with porfimer sodium (photofrin, QLT Photo therapeutics, Inc, Vancouver, BC), a purified preparation of hematoporphyrin derivative (HPD), have been com pleted.3-5 Photofrin has been accepted recently by the U.S. Food and Drug Administration for the treatment of advanced esophageal cancer. Porphyrins have been used in PDT because of their ability to generate oxygen radi -cals wl;J.en irradiated with light. 6 - 8 However, PDT using these compounds has been limited because of skin photo sensitivity lasting up to 2 months and poor tissue penetra tion of light at 630 nm, the wavelength used to activate HPD. 9 Recently, PDT has gained much interest with the de velopment of second-generation photosensitizers. 10' 11 Benzoporphyrin derivative (BPD) is one such photosensi
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tizer whose peak absorption occurs at 692 nm, allowing improved tissue penetration and significantly decreased skin phototoxicity. In addition, BPD exhibits 10 to 70 times more cytotoxicity than does HPD in vitro. 12 Because of its absorption and emission peaks in the infrared region, BPD has been effective for PDT in experimental murine tumors 13 and choroidal melanomas 14 and currently is in phase II clinical trials for the treatment of cutaneous le sions. Furthermore, it also holds some promise as a bone marrow purging agent in the treatment of leukemia, 15 as 16 an antiviral agent for the decontamination of blood, and as a treatment for nonmalignant ocular diseases (e.g., subretinal neovascularization). 17 Previous studies using various second-generation pho tosensitizers have treated experimental choroidal melano mas. 18 - 2 1 In these studies, the Greene hamster amelanotic melanoma cell line was used. Even though these results are encouraging, data obtained with amelanotic melano mas are not applicable to clinical practice because more than 90% of human ocular melanomas are pigmented. Recently, we developed an animal model of pigmented choroidal melanoma in our laboratory,22 and earlier expe rience in treating small tumors less than 2.5 mm in height was quite successful. 23 In this study, we set out to evaluate the effects of PDT in the destruction of tumors ~ 3 mm in thickness using a liposomal preparation of BPD.
Materials and Methods Cell Line B16F10 mouse melanoma cells (obtained from Dr. R. Haming) were passaged in vitro in Dulbecco modified Eagle medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (Hyclone Laboratories, Logan, UT). Two weeks before subchoroidal implantation, 106 B16F10 cells in 0.1-m\ phosphate-buffered saline were inoculated subcutaneously into the flank area of a C57B 1/6 mouse. With this procedure, a palpable tumor usually was estab lished within 10 to 14 days of implantation. Tumor Model All animals were treated in accordance with the Associa tion for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Re search. The establishment of choroidal tumors was carried out as described. 22 Briefly, female New Zealand albino rabbits weighing 1.8 to 2.5 kg were immunosuppressed with daily injections of cyclosporine (Sandimmune) at a concentration of 20 mg/kg for the first week and 'r5 mg/ kg for the remainder of the experiment. Animals were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg) before tumor implantation. The donor mouse was killed immediately before implantation, and the tumor was dissected free and stored in cold normal saline solu tion. Two limbal 7-0 polygalactin traction sutures were
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placed to rotate the globe of the rabbit superiorly. An inferior conjunctival peritomy was performed, and a 2 mm circumferential scleral incision was made with a number 64 blade (Beaver Surgical Products, Waltham, MA) 6-mm posterior to the limbus to expose the choroid. A 0.5-mm3 tumor fragment was aspirated into the blunted tip of a 22-gauge spinal needle and deposited into the subchoroidal space approximately 2 disc diameters infe rior to the optic nerve. The scleral incision was closed with 8-0 nylon sutures. Indirect ophthalmoscopy was used to verify tumor position through a pupil dilated with a mixture of 2.5% phenylephrine hydrochloride and 1o/o tropicamide. Animals were examined every 3 days by indirect ophthalmoscopy starting 1 week after implanta tion. Tumor heights were determined by B-scan ultraso nography (Quantum 200; Siemens, Issaquah, W A). The range of tumor heights at the time of entry into the treat ment groups was 3.1 to 4.6 mm (mean height, 3.3 mm). Fundus photographs were taken with a fundus camera (CF-60ZA; Canon, Lake Success, NY) before and after treatment and at 1-week intervals after treatment. Fluores cein angiography was performed immediately after treat ment using 10% sodium fluorescein (0.1 mg/kg) injected into the marginal ear vein. Photosensitizer Freeze-dried lyophilized liposomal BPD (QLT Photo Therapeutics, Inc, Vancouver, British Columbia) was re constituted with sterile water to an isotonic solution of 1.47 mg/ml immediately before use and administered in travenously at a dose of 1 mg/kg through an ear vein 30 minutes before light exposure. The animals were housed in the dark for 24 hours to protect them against skin sensitivity and to ensure that all observed effects were caused by laser light alone. Photodynamic Therapy Laser radiation was produced by an argon-pumped dye laser (Coherent Medical Laser, Coherent, Inc, Palo Alto, CA) tuned to emit light at 692 nm. The laser fluence delivered was determined by a power meter (Field Master, Coherent, Inc) before and after each treatment. The light was delivered via a 200-p,m fiberoptic cable coupled to a slit lamp (Zeiss, Carl Zeiss, Oberkochen, Germany). The beam diameter on the rabbit retina was 2 mm, and the desired irradiance was achieved by calculating the power output in relation to the area covered by the light after correction for the optics of the rabbit eye and the fundus contact lens (Ogfa-FA, Ocular Instruments, Belleview, WA). Estimated fluences ranging between 60 and 120 J/cm 2 were provided by the application of over lapping spots until the tumor was completely treated; a half-spot size margin was given around the tumor. Irradi ances of 300, 600, and 1000 mW/cm 2 were used. Thirty-two tumor-bearing animals were included in the study. Twenty-five animals were treated with light and dye. Control animals were treated with light only (three animals), dye only (two animals), or observation (two
Kim et al · PDT of Pigmented Choroidal Melanomas
Figure 1. a, pretreatment fundus photograph of a pigmented choroidal melanoma. The height of this tumor is 4.2 min and the diameter is 7.6 mm. b, pretreatment ultrasonogram of the tumor shown in a. c, post-treatment fundus photograph of the same tumor 4 weeks after photodynamic therapy using 100 J/cm2• The tumor is less heavily pigmented, and adjacent areas of atrophy are seen. d, post-treatment ultrasonogram shows the height of the same tumor at this stage to be 1. 7 mm.
animals). All animals treated with light and dye were followed for 4 to 6 weeks, except for the two rabbits killed immediately after treatment for histologic examination. Follow-up for the control animals was limited to 2 weeks because of rapid tumor growth. Histopathologic Studies After the animals were killed, the eyes were enucleated, fixed in 10% buffered formaldehyde solution (formalin, Sigma Chemical Co, St. Louis, MO), and processed for paraffin sectioning. Seven micron-thick sections were stained with hematoxylin-eosin and examined by light microscopy (BHS System, Olympus Optical Co Ltd, Tokyo, Japan).
Results Treatment Response Thirty-two rabbits bearing heavily pigmented tumors of heights 2:: 3 mm were used in our study. Figures 1a and 1b show an example of a tumor 4.2 mm in thickness. Twenty~five tumor-bearing animals were treated with light and dye, and their dimensions and responses are summarized in Table 1. Two of these animals were killed immediately for histologic examination, and the re maining 23 animals were followed-up for 4 to 6 weeks. Seven control animals were included in this study. Two animals were treated with BPD alone, and three animals were treated with light alone (120 J/cm2 , 150 J/cm 2). Fur
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Table 1. Comparison of Fluence to Treatment Outcome for Animals Treated with Dye and Light
Fluence (J/cm 2 )
No. of Animals
60 80 100 120
5 9 5* 6 25
Total
Height (mm)
Diameter (mm)
Range (mean)
Range (mean)
3.0-3.3 3.1-3.6 3.2-4.2 3.0-4.6
3.2-6.0 3.8-6.0 7.0-7.6 5.1-6.8
(3.1) (3.2) (3.8) (3.4)
(4.9) (5.1) (7.3) (5.4)
Tumor Arrest
Tumor Growth
1 9 3 6 19
4 0 0 0 4
*Two animals were killed for histologic examination.
thermore, two received neither dye nor light. Two rabbits, one that received light alone and another that received BPD alone, were killed immediately for histologic stud ies. The remaining five control animals were followed up for 2 weeks and were killed because their tumors had filled the vitreous cavity. All animals treated with fluence of 80 J/cm2 or greater showed tumor arrest. Tumors with complete regression showed a typical clinical course. Immediately after treat ment, blanching of the tumor and closure of surrounding choroidal vessels were noted. Serous retinal detachment occurred in all animals treated with light and dye. This exudative process usually peaked within several hours of the treatment, and the absorption of subretinal fluid was spontaneous and complete within 14 days of treatment. As the subretinal fluid resolved gradually, the tumors were observed to be less pigmented, and reductions in the tu mor dimensions were evident. Overlying and adjacent choroid became avascular, leading to fibrotic chorioretinal scars surrounded by a ring of chorioretinal atrophy (Fig 1c). As shown in Figure 1d, a tumor of 4.2 mm in thick ness had shrunk to 1.7 mm only 4 weeks after treatment. Tumor growth occurred in four animals treated with a fluence of 60 J/cm2 • This was first evident by the persistent heavy pigmentation in the treated area followed by exten sion of pigmentation through the treated margins. As the subretinal fluid overlying the tumor cleared, the increase in thickness was evident with indirect ophthalmoscopy and documented by ultrasonography. Frequently, the growth manifested as a small pigmented nodule on the surface within the first week after treatment. The fluores cein angiograms from these tumors showed diffuse leak age. An example of fluorescein angiographies before and after treatment is shown in Figure 2. The untreated tumors showed delayed filling followed by hyperfluoresc'ence of the mass in the late phase of the angiograms (Figs 2a, b). Angiography immediately after treatment with BPD and light showed significant hypofluorescence of the tumor and surrounding choroidal vessels, corresponding to the areas of treatmenf(Fig 2c). Fluorescein angiographies of the control groups treated with either light or dye only showed no difference from that of the untreated animals. No apparent toxicity was encountered after the intrave-
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nous administration of 1 mg/kg of BPD in the rabbit. All the rabbits treated with 120 J/cm2 had subconjunctival chemosis. Serous retinal detachment was detected in all animals treated with light and dye, and the ones treated with higher fluences had more prominent serous detach ments compared to those treated with 60 J/cm2 , but all underwent spontaneous resolution. Histopathologic Examination Histopathologic examination results of tumors performed immediately after treatment with 100 J/cm2 plus dye showed markedly dilated vascular channels packed with erythrocytes throughout the full thickness of the tumor (Fig 3a). Tumors receiving dye alone or laser irradiation alone (Fig 3b) did not show any changes compared to untreated tumors. No vascular changes were noted in these eyes. Four to 6 weeks after treatment with light plus dye, no mitotic figures or viable tumor cells were seen at the tumor site (Fig 4a). The necrotic tumors were infil trated with pigment-laden macrophages and mononuclear cells. In a control eye, intact tumor cells with prominent mitotic figures were identified (Fig 4b).
Discussion A major problem with current conventional treatment methods for malignant tumors is the lack of tumor speci ficity and low therapeutic-toxic ratios. Photodynamic therapy is a relatively new and experimental means of treating tumors that are accessible to light. 8' 24 The princi ple of this method is based on the activation of photosensi tizing agents by light of the appropriate wavelength, which, in turn, causes tissue destruction. This approach offers dual selectivity for tumor tissue, which is accom plished by preferential retention of photosensitizer dye within the tumor and restriction of the illumination field to the tumor site. The potential of PDT to selectively target tumor cells is particularly attractive in anatomic regions where structural integrity is crucial for mainte nance of vital functions such as the larynx, bronchus, esophagus, urinary bladder, and brain. 4 •5•10•25 Ocular melanoma may be an ideal candidate for PDT
Kim et al · PDT of Pigmented Choroidal Melanomas Figure 2. a, a red-free pretreatment fundus photograph of a pigmented choroidal melanoma measuring 3.6 mm in thickness. b, pretreatment fluo rescein angiogram of the same tumor. The tumor vasculature shows delayed hyperfluorescence. c, fluorescein angiogram of the same tumor immediately after treatment. H ypofluorescence of the tumor and surrounding areas corresponding to the areas of treatment is noted.
because of the clear ocular media that permit precise alignment of the laser beam with the lesions, thus min imizing photodestruction of adjacent healthy tissues. Fur thermore, the presence of a rich and leaky tumor vascula-
ture and an intact blood-retina barrier in adjacent tissues may result in enhanced localization of photosensitizer dye within tumors? 6 During the 1980s, a few investigators applied PDT to treat ocular tumors using HPD as the photosensitizer. 27 - 31 However, efficacy of HPD was difficult to determine be cause the few small clinical trials were carried out on patients with tumors of different origins and sizes, in whom many had recurrent or advanced diseases. Complete tumor destruction was shown in some selected cases, but most tumors displayed superficial necrosis with viable cells in the deeper layers. In addition, HPD now is widely recog nized for its limitations. The recent resurgence of PDT in cancer therapy is attributed to the development of second-generation photo sensitizers with significantly less phototoxicity and mark edly improved tissue penetration and enhanced drug de livery systems. Preassociation of BPD with either lipopro teins or liposomes has been shown to result in increased tumor-cell killing in vivo as well as in vitro. 13 •31 The route of tumor uptake of BPD is thought to be via a low density lipoprotein receptor-mediated pathway? 2 Miller et al 17 have shown successful closure of experimental cho roidal neovascularization using low-density lipoprotein delivered BPD. In our study, we used liposomes as vehi cles for the delivery of BPD. A dehydrated liposomal preparation can be stored easily and reconstituted, re sulting in entrapment of a high percentage of BPD, which then is injected intravenously. Previous studies using second-generation photosensi tizers in treating choroidal melanoma have been favor able. Ozier et al 19 and Panagopoulos et al 18 have used chloroaluminum sulfonated phthalocyanine with Greene hamster melanomas transplanted into the rabbit eye. Al though both studies showed promising data, the tumors were amelanotic with obvious limitations to clinical appli cations for choroidal melanomas. A recent study from our laboratory 20 used heavily pig mented choroidal tumors in evaluating PDT with chloro aluminum sulfonated phthalocyanine. We were successful in treating tumors as high as 4.8 mm. However, because chloroaluminum sulfonated phthalocyanine is not ap proved for clinical use in the United States, we chose BPD to treat thick, pigmented choroidal melanomas. Pre liminary data with small pigmented23 and nonpigmented 14 choroidal melanomas have been encouraging. Although there has been general concern that pigmen tation may impede the effectiveness of PDT, studies from our laboratory have shown that PDT is effective in treat ing thick, pigmented choroidal melanomas with chloro aluminum sulfonated phthalocyanine?0 Our current data
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Figure 3. a, histopathologic examination results of a tumor immediately after treatment with 100 J/cm 2 plus dye. Dilated vessels packed with erythrocytes were identified (arrows). b, histopathologic studies of a tumor treated with light only (120 J/cm2 ). The findings are similar to those of untreated B16F10 melanomas.
also attest to the ability of PDT to destroy heavily pig mented choroidal tumors as thick as 4.6 mm. Some investigators33 actually have proposed a syner gistic effect between PDT and hyperthermia generated by the melanin granules. The latter converts light energy to thermal energy. Hyperthermia has been shown to be induced by light at the red spectrum, 33 - 37 and with irradi ances of >300 mW/cm2 , we would expect some thermal effects to be present. However, these effects alone were incapable of destroying choroidal tumors, as shown in our control animals treated with light only. Ideally, we would have preferred to use lower irradiances to minimize any contributing thermal effects, but higher irradiances were chosen with the purpose of maintaining short treat ment times to keep the therapy clinically practical. Miller et al 17 used similar irradiances to obliterate experimental choroidal neovascular membranes, and they showed no adverse angiographic or histologic changes in the overly ing retina when compared with eyes treated with lower irradiances.
Our histopathologic evaluation of tumors treated with both light and dye suggests that vascular occlusion lead ing to tumor necrosis is at least partially responsible for tumor arrest. Two studies by Nelson et al 38•39 suggest that the rapid necrosis of tumor tissue seen after PDT is not the result of direct killing of tumor cells but secondary to destruction of the tumor microvasculature. They observed that the first signs of destruction occurred in the subendo thelial zone of the tumor capillary wall. Other studies also have shown occlusion of choroidal vessels using BPD. 40.4 1 In summary, our study suggests that PDT using liposo mal BPD is effective in destroying heavily pigmented choroidal melanomas even more than 3 mm in height. This method may provide an alternative therapy for poste rior pole tumors in which conventional radiation therapy may result in poor visual outcome because of radiation maculopathy or optic neuropathy. 42 - 44
Acknowledgments. The authors thank QLT PhotoThera peutics, Inc, for providing BPD and Coherent Laser Medical Group for assistance with the development of the laser delivery system.
Figure 4. a, 6 weeks after treatment with 100 J/cm2 , the tumor is infiltrated with pigment-laden macrophages and mononuclear cells. No mitotic figures or viable tumor cells are seen at the tumor site. b, in the control eye, treated with benzoporphyrin derivative only (1 mg/kg), intact tumor cells with prominent mitotic figures (arrows) are identified (hematoxylin-eosin; original magnification, X200).
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Kim et al · PDT of Pigmented Choroidal Melanomas
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19. Ozier SA, Nelson SJ, Liggett PE, et al. Photodynamic ther apy of experimental subchoroidal melanoma using chloro aluminum sulfonated phthalocyanine. Arch Ophthalmol 1992; 110:555-61. 20. Gonzales VH, Hu LK, Theodossiadis PG, et al. Photody namic therapy of pigmented choroidal melanoma. Invest Ophthalmol Vis Sci 1995;36:871-8. 21. Hill RA, Reddi S, Kenney ME, et al. Photodynamic therapy of ocular melanoma with bis silicon 2,3-naphthlocyanine in a rabbit model. Invest Ophthalmol Vis Sci 1995; 36:2476-81. 22. Hu LK, Huh K, Gragoudas ES, Young LHY. Establishment of pigmented choroidal melanomas in a rabbit model. Ret ina 1994; 14:264-9. 23. Young LHY, Howard MA, Hu LK, et al. Photodynamic therapy of pigmented choroidal melanoma using a liposo mal preparation of benzoporphyrin derivative. Arch Oph thalmol 1996; 114:186-92. 24. Dougherty TJ. Photodynamic therapy of malignant tumors. Crit Rev Oncol Hematol 1984; 2:83-116. 25. Noske DP, Wolbers JG, Sterenborg HJ. Photodynamic ther apy of malignant glioma: a review of literature. Clin Neurol Neurosurg 1991;93:293-307. 26. Tse DT, Dutton JJ, Weingeis TA, et al. Hematoporphyrin photoradiation therapy for intraocular and orbital malignant melanoma. Arch Ophthalmol 1984; 102:833-8. 27. Ohnishi Y, Yamana Y, Minei M. Photoradiation therapy using argon laser and a hematoporphyrin derivative for retinoblastoma: a preliminary report. Jpn J Ophthalmol 1986;30:409-19. 28. Sery TW, Shield JA, Augsburger JJ, Shah HG. Photody namic therapy of human ocular cancer. Ophthalmic Surg Lasers 1987; 18:413-8. 29. Murphree AL, Cote M, Gomer CJ. The evolution of photo dynamic therapy techniques in the treatment of intraocular tumors. Photochem Photobiol 1987;46:919-23. 30. Bruce RA, Jr. Evaluation of hematoporphyrin photoradia tion therapy to treat choroidal melanoma. Lasers Surg Med 1984;4:59-64. 31. Allison BA, Waterfield E, Richter AM, Levy JG. The ef fects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative. Photochem Photobiol 1991;54:709-15. 32. Rutledge JC, Curry F-R, Lenz JF, Davis PA. Low density lipoprotein transport across a microvascular endothelial barrier after perfl\eability is increased. Circ Res 1990; 66:486-95. 33. Berns MW, Coffey J, Wile AG. Laser photoradiation ther apy of cancer. Possible role of hyperthermia. Lasers Surg Med 1984;4:87-92. 34. Milanesi C, Biolo R, Reddi E, et al. Ultrastructural studies on the mechanism of the photodynamic therapy of tumors. Photochem Photobiol 1987;46:675-81. 35. Svaasand LO. Photodynamic and photohyperthermic re sponse of malignant tumors. Med Phys 1985; 12:455-61. 36. Kinsey JH, Cortese DA, Neel HB. Thermal considerations in murine tumor killing using hematoporphyrin derivative phototherapy. Cancer Res 1983;43:1562-7. 37. Mattiello, Hetzel F, Vandenheede L. Intratumor tempera ture measurements during photodynamic therapy. Pho tochem Photobiol 1987;46:873-9. 38. Nelson JS, Liaw LH, Berns MW. Mechanism of tumor destruction in photodynamic therapy. Photochem Photobiol 1987;46:829-36. 39. Nelson JS, Liaw LH, Orenstein A, et al. Mechanism of tumor destruction following photodynamic therapy with
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hematoporphyrin derivative, chlorine and phthalocyanine. J Nat! Cancer Inst 1988;80:1599-1605. 40. Lin SC, Lin CP, Feld JR, eta!. The photodynamic occlusion of choroidal vessels using benzoporphyrin derivative. Curr Eye Res 1994; 13:513-22. 41. Schmidt-Erfurth U, Hasan T, Gragoudas ES, eta!. Vascular targeting in photodynamic occlusion of subretinal vessels. Ophthalmology 1994; 101:1953-61. 42. Seddon JM, Gragoudas ES, Polivogianis L, et a!. Visual
outcome after proton beam irradiation of uveal melanoma. Ophthalmology 1986;93:666-74. 43. Seddon JM, Gragoudas ES, Egan KM, eta!. Uveal mel anomas near optic disc or fovea: visual results after proton beam irradiation. Ophthalmology 1987;94:354 61. 44. Cruess AF, Augsburger JJ, Shields JA, et a!. Visual results following cobalt plaque radiotherapy for posterior uveal melanomas. Ophthalmology 1984;91:131-6.
Centennial Advertisement From the Sur une nouvelle methode de querir la cataracte par ['extraction du cristalin, a report by Jacques Daviel (1693 or 1696-1762), showing his method for cataract extraction. Daviel is best remembered for developing the modem methods of cataract extraction. Instead of couching the cataract with a needle, Daviel developed and used a knife needle for the incision, two curved scissors, a gold spatula to remove the lens, a needle to open the anterior capsule, and a spoon and forceps to remove the lens matter.*
* Centennial advertisement provided courtesy of the Museum of Ophthalmology, Foundation of the American Academy of Ophthalmology, San Francisco, California
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