Various forms of chemotherapy

and targeted therapies have helped

improve rates of survival and

ocular salvage in retinoblastoma.

Photo courtesy of Lynn E. Harman, MD. Themes at the Shard.

Therapeutic Options for Retinoblastoma Pia R. Mendoza, MD, and Hans E. Grossniklaus, MD

Background: Retinoblastoma is the most common primary intraocular malignancy in children. The manage- ment of retinoblastoma is complex and depends on several factors. Methods: This review provides an update on current and emerging therapeutic options for retinoblastoma. The medical literature was searched for articles relevant to the management of retinoblastoma. The results of prospective and retrospective studies on chemotherapy and focal therapy for retinoblastoma are summarized. Animal models for novel therapeutic agents are also discussed. Results: Treatment strategies for retinoblastoma involve intravenous chemoreduction, local administration routes of chemotherapy (eg, intra-arterial, intravitreal), focal therapy for tumor consolidation (eg, photocoagulation, thermotherapy, cryotherapy, plaque brachytherapy), external beam radiotherapy, and surgical enucleation. Emerging therapies include alternative chemotherapeutic agents, molecularly targeted therapies, and novel drug-delivery systems. Conclusion: In the past 10 years, the management strategy for retinoblastoma has significantly changed, shifting toward local chemotherapy and away from systemic chemotherapy. Innovations in the field of molecular biology and the development of targeted therapies have led to improvements in survival rates and ocular salvage for this disease. However, the need still exists to further assess the long-term effects of such directional changes in therapy.

Introduction sor RB1. Major advances, such as our understand- Retinoblastoma is the most common primary intraoc- ing of the molecular biology of the tumor and the de- ular malignancy in children; its incidence rate is 1 in velopment of targeted therapy, have improved survival 14,000 to 20,000 livebirths.1 However, compared with rates in developed countries.3 In the United States, the other pediatric malignancies, retinoblastoma is rare, 5-year overall survival rate is 97%.4 Much of this suc- comprising 3% to 4% of all pediatric cancers.2 It is a cess is due to the benefits of effective treatments, in- tumor of the developing retina that arises from primi- cluding chemotherapy, focal or consolidation therapy, tive retinal stem cells or cone precursor cells associated external beam radiotherapy, and surgical enucleation with inactivation of both alleles of the tumor suppres- (Table 1).5 With early diagnosis and aggressive, multi- modal treatment strategies, near-complete cure rates are possible, and many patients retain functional vi- From the Department of Ophthalmology, Emory University School of Medicine, Atlanta, Georgia. sion in at least 1 eye. However, in developing nations, Address correspondence to Hans E. Grossniklaus, MD, 1365 where access to health care is limited, retinoblastoma Clifton Road, Northeast, BT428, Atlanta, GA 30322. E-mail: can cause blindness and death.6 [email protected] Among the mainstay treatments for retinoblastoma Submitted October 14, 2015; accepted January 5, 2016. are intravenous chemotherapy (involving agents asso- No significant relationships exist between the authors and the companies/organizations whose products or services may be ref- ciated with significant toxicities), surgical enucleation, erenced in this article. external beam radiotherapy, or all 3 options, poten-

April 2016, Vol. 23, No. 2 Cancer Control 99 tially result in disfigurement and sec- Concepts and Principles of ondary malignancies. However, in re- Table 1. — Therapeutic Options Treatment in Retinoblastoma cent years, an ongoing evolution has The goal of treatment in retinoblas- taken place, where treatment is being Chemotherapy toma is the elimination of the tumor geared toward more targeted therapy Intravenous while concurrently minimizing col- and local medication delivery. Local Chemoreduction lateral injury to other tissues. The delivery increases exposure to intra- Adjuvant therapy priorities in management are (1) pre- ocular target areas, reduces overall venting metastasis, (2) reducing the Metastatic retinoblastoma systemic exposure and harmful late risk of long-term secondary tumors Intra-arterial effects, and improves effectiveness (eg, osteosarcoma, soft-tissue sarco- and rates of tolerability. The intro- Intravitreal ma), (3) saving the eye, and (4) pre- duction of intra-arterial and intravit- Periocular serving vision.11 Caring for a patient real chemotherapy has resulted in a Focal Therapy with retinoblastoma must be a mul- change in treatment algorithms and Thermotherapy tidisciplinary effort involving pedi- improved outcomes, with their pos- Photocoagulation atric oncologists, ocular oncologists, sibility of ocular salvage and mainte- radiation oncologists, ocular pathol- Cryotherapy nance of vision.7 ogists, and geneticists to optimize Preclinical models that simulate Plaque brachytherapy treatment outcomes. human disease are needed for rare External Beam Radiotherapy Treatment depends on tumor childhood cancers such as retinoblas- Enucleation stage, laterality and number of tu- toma because not enough patients mor foci (unifocal, unilateral, mul- with the disease exist for large-scale, tifocal, or bilateral), the localization multicenter clinical trials. Despite the lack of clinical and size of tumors within the eye, presence of vit- trials, rates of survival and ocular preservation have reous seeding, age and health of the child, and the improved.7 In vitro studies and animal models remain family’s wishes.12 The type of management depends instrumental in understanding biological mechanisms on the tumor classification, which is based on 2 sys- and assessing safety and effectiveness of the treatment tems: the International Classification of Retinoblas- options for this disease.8-10 Examples of emerging ther- toma13 used to predict success of chemoreduction14 apies under investigation for retinoblastoma are alter- and the tumor, node, and metastasis classification native chemotherapeutic agents, molecularly targeted of the American Joint Committee on Cancer15 for tu- therapies, and novel drug-delivery systems. mor staging (Table 2). Location of the tumor in -

Table 2. — International Classification and General Therapeutic Options for Retinoblastoma Eye Group General Feature Specific Feature (Presence of ≥ 1) Therapy A Small tumor away from Tumor ≤ 3 mm Laser photocoagulation fovea and optic disc Thermotherapy Cryotherapy Plaque brachytherapy B Larger tumor Tumor > 3 mm Laser photocoagulation Macular Tumor located < 3 mm from fovea Thermotherapy Juxtapapillary Tumor located ≤ 1.5 mm from optic disc Cryotherapy Subretinal fluid Presence of subretinal fluid ≤ 3 mm from tumor margin Plaque brachytherapy Intravenous/intra-arterial chemoreduction C Focal seeds Presence of subretinal, vitreous seeds, or both located ≤ 3 mm Intra-arterial chemotherapy from main tumor Intravitreal chemotherapy D Diffuse seeds Presence of subretinal, vitreous seeds, or both located > 3 mm Intra-arterial chemotherapy from main tumor Intravitreal chemotherapy Enucleation E Extensive tumor Tumor occupying > 50% of the globe Intra-arterial chemotherapy Neovascular glaucoma Enucleation Opaque media from hemorrhage in anterior chamber, vitreous, Adjuvant intravenous chemotherapy or subretinal space if high-risk histopathological features Invasion of postlaminar optic nerve, choroid (> 2 mm), sclera, present orbit, anterior chamber

100 Cancer Control April 2016, Vol. 23, No. 2 tion to the optic disc and macula is important in the that systemic chemotherapy delivered prior to external clinical evaluation because it largely affects the vi- beam radiotherapy contributed to tumor control and sual prognosis. Involvement of certain tissues in and ocular salvage.19 A subsequent study on eyes treated around the eye, including the optic nerve, choroid, with chemoreduction combined with focal treatments iris, sclera, and orbit, increase the likelihood of tumor (cryotherapy, thermotherapy, or plaque radiotherapy) metastasis.16 demonstrated tumor regression and decreased need In children, unilateral retinoblastoma is differ- for additional external beam radiotherapy and enucle- ently managed than bilateral tumors. In general, ation.20 This was a major advancement in management unilateral cases are the result of sporadic mutations, because it was evident that satisfactory tumor control whereas bilateral cases are typically multifocal, he- could be attained with systemic chemotherapy while reditary, and occur in patients carrying a germline saving the eye and avoiding the adverse events of exter- RB1 mutation, thus placing them at risk for other ma- nal beam radiotherapy.20 Using the International Classi- lignancies (eg, intracranial neuroblastic tumors [pine- fication of Retinoblastoma, treatment success was found aloblastoma in trilateral retinoblastoma]). In general, in 100% of group A eyes, 93% of group B eyes, 90% of unilateral tumors can be managed with focal therapy group C eyes, and 48% group D eyes.14 This launched alone; chemoreduction, intra-arterial chemotherapy the so-called “systemic chemotherapy era” (1990s to with focal therapy, or both; or enucleation. If genetic 2006); however, with the advent of local routes of ad- testing is not performed, then patients with unilateral ministration (intra-arterial and intravitreal), use of sys- disease require close follow-up of the unaffected eye temic chemotherapy has decreased in recent years.14 to exclude bilateral involvement. Alternatively, treat- Intravenous chemotherapy is indicated as initial ment for bilateral tumors will depend on the extent of therapy for bilateral advanced disease in which at- tumor in each eye. In asymmetric disease, an attempt tempts are made to salvage both eyes. Many centers is made to salvage the eye with less severe disease. use systemic chemotherapy in bilateral (germline) ret- One may consider giving patients systemic intrave- inoblastoma for intraocular retinoblastoma control as nous chemoreduction as initial therapy to treat both well as to prevent metastasis, reduce the likelihood of eyes as well as to prevent metastasis and associated the development of pineoblastoma, and to reduce the secondary tumors or to use local (intra-arterial) che- long-term risk of secondary cancers.17,18 Systemic che- motherapy with focal treatment.7,17,18 motherapy is also utilized as adjuvant treatment to pre- Initial chemotherapy in retinoblastoma can be giv- vent metastasis following enucleation in patients with en either systemically (intravenous) or locally (directly optic nerve invasion posterior to the lamina cribrosa, through the ophthalmic artery/intra-arterial). Chemo- massive choroidal invasion, or any combination of op- reduction is then usually followed by consolidation, tic nerve and choroidal invasion.21 The treatment regi- which is achieved by focal therapy (eg, laser photo- mens used include vincristine/etoposide/carboplatin, coagulation, thermotherapy, cryotherapy, plaque vincristine/doxorubicin/idarubicin/cyclophosphamide, brachytherapy). Focal therapy may be employed as or hybrid regimens, typically delivered monthly for the only or initial therapy for small retinoblastoma 6 to 9 months.22 A major obstacle to use of intravenous tumors located away from the fovea and optic nerve chemotherapy in retinoblastoma is the blood–retina (group A); it is often used for tumor consolidation in barrier, which limits the entry of some chemotherapeu- retinoblastoma following chemoreduction for more tic agents into the eye, thereby reducing their efficacy.23 advanced (groups B–D) tumors. Surgical removal of Systemic chemotherapy for retinoblastoma is generally the eye or enucleation is reserved for very advanced safe and effective, but it is not without adverse events, (group E) cases. such as neurotoxicity, hyponatremia, nephrotoxicity, ototoxicity, and secondary leukemia. Treatment fail- Management ure in the form of persistent vitreous seeds, subretinal Chemotherapy seeds, and intraretinal tumors following therapy have Intravenous: Systemic chemotherapy generally in- been attributed to the inability of the treatment drugs volves a combination 2-, 3-, or 4-drug regimen deliv- to reach the tumor.24 Tumor resistance or unresponsive- ered through an intravenous catheter. Three classes ness to chemotherapy is thought to occur more often of agents are commonly employed: DNA-crosslink- in well-differentiated tumors, presumably because cells ing agents (carboplatin, cisplatin), DNA topoisomer- are not cycling, so they are less likely to respond to ase 2 inhibitors (etoposide, topotecan, teniposide) and treatment modalities affecting cell division.25 Vinca alkaloids (vincristine; Fig 1). The most common- Intra-Arterial: In the late 1980s, melphalan was ly used regimen is vincristine/etoposide/carboplatin.11 found to be effective for the management of retinoblas- Chemoreduction via systemic chemotherapy was toma in vitro.26 Yamane et al27 and Suzuki et al28 then introduced as a management option for retinoblastoma began to treat patients with intracarotid artery melpha- in the mid-1990s following preliminary observations lan, developing a technique to safely and effectively

April 2016, Vol. 23, No. 2 Cancer Control 101 Fig 1. — Chemotherapeutic agents used in retinoblastoma. Alkylating agents such as melphalan cause DNA intra–strand-linking and cross-linking, resulting in DNA damage and disruption of transcription. Topoisomerase inhibitors such as etoposide and topotecan cap free double-strand breaks in DNA, preventing DNA repair and . Platinum-based agents such as carboplatin bind DNA bases, resulting in kinked DNA. Vinca alkaloids such as vincristine bind tubulin dimers and block mitotic spindle formation. Reprinted from Grossniklaus HE. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson Memorial Lecture. Am J Ophthalmol. 2014;158(5):875-891, with permission from Elsevier. cannulate the ophthalmic artery for chemotherapy in- years, globe-salvage results (85%–94% globe salvage fusion. In the United States, intra-arterial chemothera- rate in group D tumors with primary IAC) have been py (IAC) was initially used by Abramson et al.29 IAC in- reported in study patients with advanced retinoblas- volves transfemoral catheterization with advancement toma.30,31 In some centers, most group D eyes are pri- into the ophthalmic artery, which is where melphalan, marily treated with IAC, and, on occasion, group D topotecan, or carboplatin is infused. The medication and E eyes are managed with combined intravenous is slowly delivered for 30 minutes in a pulsatile fash- chemotherapy followed by intra-arterial chemotherapy ion with care to not occlude the artery and to minimize — particularly if the patient has advanced bilateral dis- reflux into the internal carotid artery. Most patients ease or disease in a single eye.7 Fair success has been receive 3 monthly sessions. Melphalan is the most fre- reported in group E tumors with total retinal detach- quently used agent, with topotecan added if extensive ment cautiously managed with initial IAC.32,33 Treat- vitreous seeding is present. ment of more advanced disease with initial IAC has Generally, IAC is employed for unilateral or non- also not been shown to compromise survival rates.7 germline retinoblastoma, recurrent tumors following After adequate reduction is visualized and subretinal previous intravenous chemotherapy or plaque radio- fluid is resolved, complete consolidation with either therapy, recurrent subretinal seeds involving at least thermotherapy or cryotherapy and supplemental in- 2 quadrants, recurrent vitreous seeds, or, if the patient travitreal chemotherapy after IAC are commonly per- wishes to avoid systemic therapy, enucleation can be formed. Consolidation is generally performed during considered if consent is obtained. In a 5-year experi- the second or third cycle of chemoreduction. ence with IAC, complete regression was achieved for In infants younger than 3 months who weigh less solid tumors in 94%, for subretinal seeds in 95%, and than 7 kg, IAC is initially deferred to allow the femo- for vitreous seeds in 87% of study patients.30 In recent ral artery to grow before attempting catheterization.

102 Cancer Control April 2016, Vol. 23, No. 2 In such cases, patients are treated with 1 to 2 doses of nique does not increase the risk of tumor spread.46 single-agent carboplatin as bridge therapy before de- Treatment of retinoblastoma with recurrent vitre- finitive IAC.34 ous seeds has been reported to have a success rate of Reports of high rates of efficacy of IAC abound, 83%.47 The number of injections depends on the re- but studies also exist on its associated ocular toxici- sponse, and 6 injections delivered weekly or biweek- ties.35-37 Use of intra-arterial infusion of melphalan ly is recommended. Reported adverse events include and carboplatin compromise the ocular vasculature transient vitreous hemorrhage, chorioretinal atrophy, in nonhuman primates and induces inflammation and extraocular tumor spread.42,48,49 and leukostasis in human retinal endothelial cells.35,36 Melphalan is the most commonly used drug for in- Other complications include vitreous hemorrhage, travitreal injections. Although it caused no systemic tox- microemboli to the retina and choroid, myositis, icity in humans or rabbits, melphalan did result in sig- eyelid edema, orbital congestion with resulting dys- nificant retinal toxicity at high doses and lower but safer motility, choroidal atrophy, optic atrophy, ophthalmic doses only achieved incomplete tumor control.42,50 artery stenosis, and branch-retinal artery occlusion Periocular: Chemotherapeutic agents may be resulting in blindnesss.37 periocularly injected, either as subconjunctival or sub- A major drawback to this therapy is that only tenon. Periocular injection enables transcleral drug skilled neurosurgeons and/or interventional radi- delivery, using the large surface area of the sclera ologists at major cancer centers can treat patients us- and its high permeability to small molecules without ing IAC.38 The therapy is also expensive.39 In devel- the danger of puncturing the globe.51 Periocular che- oping countries, IAC is not available or is too costly, motherapy achieves levels within the vitreous rapidly so enucleation as initial therapy is still a mainstay of and at levels 6 to 10 times higher than the intravenous treatment for advanced retinoblastoma.40 In addition, route.52 In general, periocular injection of chemothera- a study has shown that initial chemotherapy prior to peutic agents has been used for retinoblastoma control enucleation for advanced retinoblastoma can mask as an adjunct to systemic chemotherapy and, on occa- pathological evidence of tumor extension, thus lead- sion, to treat tumor recurrence. Either carboplatin or ing to reduced surveillance and the undertreatment of topotecan can be employed. The first-line indications high-risk disease — thereby increasing the risk of met- for periocular chemotherapy are bilateral advanced astatic death.41 group D or E eyes in which a higher local dose of che- Intravitreal: The major question encountered motherapy is needed, treatment of vitreous seeds, and in the management of retinoblastoma is how to treat recurrent localized tumor. Subtenon chemotherapy us- subretinal and vitreous seeds of tumor unresponsive ing carboplatin can increase tumor control, especially to laser treatment, conventional chemoreduction ther- if it is coupled with intravenous chemoreduction. Sub- apy, or IAC.24 Treatment of vitreous seeds is challeng- tenon carboplatin showed initial favorable results as ing due to lack of vasculature in the vitreous. Another single therapy, but long-term follow-up revealed a high route of administration developed to address this prob- failure rate as initial treatment; therefore, it should be lem is the intravitreal method, which involves injecting combined with other therapeutic approaches.53 the therapeutic agent into the vitreous cavity of the eye Complications of periocular chemotherapy include through the pars plana under aseptic precautions. orbital and eyelid edema, ecchymosis, orbital fat atro- Although health care professionals were initially phy, muscle fibrosis leading to strabismus, and optic skeptical of injecting drugs through the sclera into the atrophy.53-55 Because of its significant toxic effects, peri- vitreous for fear of tumor spread along needle tracks, ocular chemotherapy is rarely used in current practice. techniques for safe and effective intravitreal injections have been developed. Intraocular pressure is first low- Focal Therapy ered prior to the injection. A small-volume dose of mel- Thermotherapy/Transpupillary Thermotherapy: phalan, topotecan, or a combination of both is injected This type of focal therapy uses an 810-nm diode laser into the eye using a fine needle (30- or 32-gauge) and directly on the tumor to heat it up to subcoagulation the needle is frozen with a cryoprobe as it is with- temperatures of 42 to 60 °C to induce a cytotoxic ef- drawn from the eye to prevent tumor seeding.42 The fect. It is used for local control and for small (< 4.5-mm first-line indication for intravitreal chemotherapy in- base and 2.5-mm thickness) tumors posteriorly located cludes vitreous seeds refractory to standard therapy to the equator of the globe. In cases of macular or jux- and recurrent vitreous seeds after previous therapy.43,44 tapapillary tumors, consolidation should be performed In an analysis of 57 eyes with viable recurrent vit- with caution so as to protect the optic disc and neuro- reous seeds, intravitreal melphalan led to eye salvage sensory papillomacular bundle. The tumor may be ob- in 51% of eyes after 10 years of follow-up; no cases served while on chemoreduction and, if unresponsive, of extraocular extension were reported during that foveal-sparing thermotherapy may be administered. In time.45 A systematic review showed that proper tech- an analysis of 68 macular retinoblastomas treated with

April 2016, Vol. 23, No. 2 Cancer Control 103 chemoreduction, tumor recurrence occurred in 17% of Consolidation alone may be used in group A and B those consolidated with foveal-sparing thermotherapy tumors. Bilateral cases with small, extrafoveal tumors compared with 35% of those observed without consoli- can be managed with focal treatments alone. In asym- dation.56 Complications of transpupillary thermothera- metric disease, a single eye can be managed with focal py include iris atrophy, focal cataracts, tumor seeding techniques and the other with enucleation or intra-ar- into the vitreous, and retinal fibrosis, transition, and terial chemotherapy with or without systemic chemo- vascular occlusion. therapy.7 The desired end point is tumor regression in Laser Photocoagulation: This type of therapy the form of a completely calcified tumor (type 1 regres- uses an 520-nm argon laser to coagulate the blood sion) or a flat chorioretinal scar (type 4 regression). supply of the tumor by generating heat with tempera- tures in excess of 65 °C within the treatment spot. Di- Enucleation rect photocoagulation to the tumor must be avoided. Enucleation remains a favored approach in cases of ex- A second mechanism occurring around the ring of tis- tensive retinoblastoma with buphthalmos, neovascular sue beyond the laser spot is the increase in tempera- glaucoma, aqueous seeding, and transcleral extension ture to a thermotherapeutic range (45–60 oC). In this (advanced group E eyes) — particularly for unilateral region, synergism exists with carboplatin, so this laser cases with no likelihood of functional vision.6 Surgery treatment is typically performed within 24 hours of involves careful removal of the eye with minimal globe the intravenous chemotherapy cycle. Small posterior trauma to prevent tumor seeding into the orbit, and tumors without seeding respond well to laser photo- then excising a long section of optic nerve for proper coagulation. It is not used for tumors impinging on the histopathological evaluation.57 Following enucleation, fovea, because of the risk of compromising a patient’s an orbital implant is placed to provide the socket with central vision. In such cases, some form of chemother- enough volume and allow the 4 rectus muscles to at- apy can be used to shrink the tumor prior to the start tach for motility. Immediately after enucleation, fresh of definitive laser treatment. Photocoagulation is also tissue should be harvested on a separate tray for ge- used to treat tumor-associated retinal neovasculariza- netic studies. tion. Complications include vitreous seeding if the la- A corollary to enucleation is the adequate histo- ser power is too high, and retinal fibrosis, traction, and pathological evaluation of globe pathology, because vascular occlusion. retinoblastoma with histological features signifying a Cryotherapy: Cryotherapy involves a cryoprobe high risk of metastasis requires adjuvant intravenous by which liquid nitrogen is delivered and directly chemotherapy.58 High-risk features include choroidal applied to the outer surface of the sclera adjacent invasion, retrolaminar optic nerve invasion, scleral and to the tumor. It is suitable for small (< 3.5-mm base orbital invasion, and anterior chamber invasion. An and < 2-mm thickness) tumors anteriorly located to the analysis of 1,020 study patients with high-risk features equator of the globe. Complications of cryotherapy in- after enucleation showed that 24% of study volunteers clude retinal tears and detachment, proliferative vitreo- at high risk developed metastasis if not treated with ad- retinopathy, and chorioretinal atrophy. juvant chemotherapy, whereas 4% of these study pa- Plaque Brachytherapy: This type of treatment tients had metastasis if treated with chemotherapy.59 refers to implantation of radioactive material on the The regimen for adjuvant chemotherapy is vincris- sclera over the base of the tumor. Tissue absorption of tine/etoposide/carboplatin for 4 to 6 cycles. ionizing radiation causes DNA damage and cell death. Because retinoblastoma has a high rate of proliferat- External Beam Radiotherapy ing cells, it is quite radiosensitive. Brachytherapy is External beam radiotherapy can be used to treat eyes used for solitary, medium-sized tumors (6–15 mm and unresponsive to other treatments, those with a tumor > 3 mm from the optic disc or fovea) and, if little sub- located at the optic nerve resection margin (36 Gy to retinal fluid is present, plaque radiotherapy can gen- orbit and 10 Gy boost to chiasm), and as treatment of erally be used to achieve tumor control. Iodine-125 extraocular retinoblastoma such as those extending and ruthenium-106 isotopes are the most common through the sclera, orbit, or intracranially (40–45 Gy).18 source of radiation currently used in brachytherapy External beam radiotherapy is rarely used and em- for retinoblastoma to deliver 40 to 45 Gy to the apex ployed only when necessary because of its subsequent of the tumor. In addition to treatment for retinoblasto- risks. It is known to cause midfacial hypoplasia in ma, ocular brachytherapy is commonly used for uveal young patients and increase the risk of development of melanoma, in which a clinician uses a similar surgi- soft-tissue sarcomas, brain tumors, osteosarcomas, and cal technique of plaque application of suturing to the other cancers.60 In patients with bilateral disease, the episcleral surface but uses a lower dose of radiation. incidence of secondary malignancy at 50 years from di- Complications of plaque brachytherapy include radia- agnosis of retinoblastoma is 21% in patients who were tion retinopathy and optic neuropathy.18 not treated vs 38% for those who were treated with ra-

104 Cancer Control April 2016, Vol. 23, No. 2 diation.61-63 If possible, any radiation (including radi- preservation have improved.7 Animal models remain ography, computed tomography, and external beam) instrumental in understanding biological mechanisms should be avoided in individuals with heritable retino- and designing targeted therapies for this disease.8-10 blastoma to minimize their lifetime risk of developing Novel treatment options being explored in vitro and secondary cancers. in vivo include alternative chemotherapeutic agents, molecularly targeted therapies, gene therapy, localized Extraocular Therapy delivery, and the sustained-release of broad-spectrum Although their incidence is rare in developed coun- chemotherapy agents. tries, cases of extraocular disease are occasionally seen in neglected or inadequately treated patients.64,65 Alternative Chemotherapeutic Agents Tumors may directly extend through emissary vessels Topotecan, a topoisomerase inhibitor that works of the sclera into the orbital soft tissue surrounding in the G0 phase of the cell cycle, may be used as a the eye. Treatment of orbital retinoblastoma includes single agent or in combination with other agents for systemic chemotherapy and external beam radiother- the treatment of retinoblastoma. Subconjunctival, sus- apy (40–45 Gy) with a rate of cure between 60% and tained-release topotecan has been shown to decrease 85%.18 Treatment may also extend to the optic nerve tumor burden in a transgenic mouse model of retino- into the brain and meninges with subsequent seeding blastoma.69 In a similar fashion, periocular injections of spinal fluid. Most recurrences occur in the central of topotecan in fibrin sealant in humans have been nervous system and are associated with a low cure shown to achieve retinoblastoma tumor volume re- rate; regimens proven to be effective include vincris- duction.70 Topotecan can also reduce retinoblastoma tine, cyclophosphamide, doxorubicin, and platinum as tumor burden when given systemically,71 via IAC,72 well as epipodophyllotoxin-based drugs.66 No preclin- or via intravitreal injections.43 When compared with ical or clinical evidence supports the use of intrathe- standard chemotherapeutic agents used to treat reti- cal chemotherapy. Craniospinal irradiation using 25 to noblastoma, topotecan avoids the retinal toxicity of 35 Gy is sometimes provided. Retinoblastoma may also melphalan, the nephrotoxicity of carboplatin, and the hematogenously spread to the bone marrow, bones, hematotoxicity of etoposide.73 Topotecan is a good lungs, or liver. For metastatic disease, multiple-agent, candidate drug for intravitreal injection because it has high-dose chemotherapy and autologous hematopoi- minimal retinal toxicity and achieves maximum con- etic stem cell transplantation can be administered as centration levels 50 times higher than systemic expo- rescue therapy.67 The Children’s Oncology Group de- sure.73,74 Our laboratory has shown that 20-µg topo- veloped a prospective, multi-institutional, internation- tecan can provide sustained retinoblastoma cell-line al trial for metastatic retinoblastoma, and data will be killing in vitro,75 similar to the clinically demonstrated available in early 2016; that study hopes to define dif- retinoblastoma cell-line killing ability with 20-µg in- ferences in site of metastasis and outcome.68 travitreal topotecan.43 Anthracyclines are a class of chemotherapeu- Emerging Therapies tic drugs that inhibit topoisomerase and intercalate New developments in the treatment of retinoblastoma DNA. They are used to treat several cancers and are are geared toward an ideal system that is affordable active against retinoblastoma; however, because of and can easily be used by ophthalmologists or ocular their limited blood–brain barrier penetration, their oncologists — even those in developing nations — and use is typically limited to extraocular and metastat- is locally effective to the main tumor as well as tumor ic retinoblastoma. The anthracycline idarubicin has seeds, causes minimal complications, and can achieve been shown to be effective against retinoblastoma tu- a sustained treatment effect to retinoblastoma without mor cells in bone marrow (response rate, 60%), but spreading tumor cells. A sustained treatment effect is sufficient concentrations have not been achieved at desired because, even when compounds do reach the target sites (eg, central nervous system).76 A study to target site, rapid clearance from the eye often results improve intraocular exposure of the anthracycline in short intraocular residence times. In addition, many doxorubicin while also reducing systemic exposure patients with retinoblastoma are young children who to the drug was successful in maintaining sustained oftentimes require sedation to receive intraocular ther- levels of doxorubicin after the intravitreal injection of apy — sustained drug-release mechanisms will enable the drug encapsulated in polyhydroxybutyrate micro- continuous medication dosing. spheres in rabbits.77 Preclinical models that accurately reflect human disease are needed for rare childhood cancers such as Molecularly Targeted Therapy retinoblastoma because not enough patients exist for RB1 was the first tumor suppressor gene identified and large-scale multicenter clinical trials.8 Despite this lack cloned, yet no effective, molecularly targeted cure cur- of clinical trials, rates of patient survival and ocular rently exists for retinoblastoma.78 This could perhaps

April 2016, Vol. 23, No. 2 Cancer Control 105 be attributed to the variety of unknown secondary mu- Gene Therapy tations and differential expression of other aside Suicide gene therapy is based on the introduction of a from RB1 involved in tumorigenesis, as well as ques- viral or bacterial gene into tumor cells, thus allowing tions on the cellular origin of retinoblastoma.79,80 Sig- the conversion of a nontoxic compound into a lethal nificant progress has been made in our understanding drug to kill the tumor cells. In a phase 1 study, intra- of tumor biology, leading to the discovery and devel- vitreal injections of an adenovirus vector containing opment of small molecules for the treatment of retino- HSVtk followed by treatment with ganciclovir were blastoma, including novel, targeted therapeutics such shown to be safe and effective against vitreous seeds.90 as inhibitors of the MDMX– response (nutlin-3a), Although these results were promising, it may be un- histone deacetylase (HDAC) inhibitors, and spleen ty- likely that this type of gene therapy could be used as rosine kinase (SYK) inhibitors.9,81 first-line treatment for retinoblastoma but rather its use Nutlin-3 is an imidazole analogue that plays a role may be better suited as an adjunct to standard therapy in the activation of p53, a tumor suppressor . for the treatment of refractory vitreous seeds.90 It is a small molecule inhibitor of MDM2 and MDMX and prevents the association of both these Local Drug-Delivery Systems with p53. Studies have shown that nutlin-3 restores Nanotechnology-based drug-delivery systems for the the p53 pathway in retinoblastoma cells lacking both treatment of cancer have significantly evolved during and p53 activity, thus inducing the past decade by enabling options for site-specific p53-mediated apoptosis.82 Subconjunctival injection of delivery and increased bioavailability.91 Investigations nutlin-3 in mouse models of retinoblastoma resulted have been conducted on the potential use of different in reduced tumor burden when used in combination materials as intraocular drug carriers, such as biode- with topotecan, demonstrating a synergistic effect.83 gradable polyesters, dendrimers, liposomes, mesopo- Nutlin-3 is being studied in phase 1 clinical trials for rous silica, and gold nanoparticles.91 These particles the treatment of retinoblastoma.82,83 can be modified to target specific cells, and they can be Although it is not expressed in normal human reti- designed to ensure sustained drug release to increase nas, SYK was found to be upregulated in retinoblasto- the therapeutic effectiveness of the drug molecules ma tumor samples and has been shown to be required they contain.92 Nanoparticle-based systems designed for retinoblastoma cell survival.84 Inhibition of SYK for the treatment of retinoblastoma have improved with BAY-61-3606 and R406 caused cell death in reti- rates of drug delivery to the posterior segment of the noblastoma, and an in vivo study of orthotopic, xeno- eye and have increased the intravitreal half-life of che- graft mice showed that subconjunctival BAY-61-3606 motherapy agents, thus highlighting their potential was effective at blocking retinoblastoma cell prolifera- in treatment of this cancer.93 The intravitreal delivery tion.84 In addition, studies of SYK inhibition have also of doxorubicin-loaded, polyester-based microspheres implicated several downstream signaling molecules as in the ocular tissue from rabbits exhibited reduced mediators of SYK survival, including the B-cell chronic rates of toxicity to surrounding normal structures.77 lymphocytic leukemia/lymphoma 2 (BCL2) family of Dendrimer nanoparticles containing carboplatin sig- proteins.85 Myeloid cell leukemia 1, a member of the nificantly reduced tumor volume compared with free BCL2 family, is a potential target because it is upreg- carboplatin in a retinoblastoma mouse model.94 Poly- ulated in retinoblastoma and is being developed as a lactic glycolic acid nanoparticles developed to deliver therapy for other cancers.86 doxorubicin95 and etoposide96 were tested on Y79 reti- Another class of targeted therapies in phase 1 noblastoma cell lines and may be potential candidates clinical trials are the inhibitors of HDAC.87 Cells with for sustained drug-release models. elevated activity are uniquely sensitive to inhibi- Gold-based nanoparticles have unique physi- tors of HDAC through the overexpression of proapop- cal properties, including a strong ability to absorb totic factors. Cells lacking retinoblastoma protein near-infrared light, which enables the photothermal have increased E2F1 activity, and retinoblastoma-de- destruction of cancer cells.97 Light-activated drug re- rived cell lines have demonstrated particular sensitiv- lease can be attained using gold nanoparticles conju- ity to apoptosis induced from inhibitors of HDAC.88 gated with chemotherapeutic agents.98 For example, In a preclinical trial, inhibitors of HDAC slowed the gold nanocages are encapsulated with a smart poly- growth of retinoblastoma-derived tumors in both mer, wherein the nanocages absorb light converted transgenic and xenograft murine models of retino- to heat, thus causing the collapse of the polymer and blastoma, with minimal off-target effects, suggesting the subsequent release of doxorubicin.99 The light-re- that inhibitors of HDAC may specifically inhibit the sponsiveness of gold and other photosensitive nano- proliferation of retinoblastoma tumor cells and, thus, carriers infers considerable potential for ophthalmic have lower systemic toxicities relative to chemothera- conditions such as retinoblastoma because of the reg- peutic agents.89 ular use of lasers in the treatment of retinal disease

106 Cancer Control April 2016, Vol. 23, No. 2 and the added benefit of facilitating the controlled roidal and retinal concentrations targeted toward the timing and location of delivery of therapeutic agents. elimination of subretinal seeds and the prevention of Gold nanoparticles can also cross the blood–retina choroidal invasion, with minimal biodistribution and barrier without significant rates of cytotoxicity.100 The toxicity to surrounding tissue. Our laboratory is in- intravitreal injection of gold-tethered liposomes and vestigating the suprachoroidal and intravitreal injec- viral-like nanoparticles containing topotecan is being tion of topotecan using microneedles in a rabbit mod- investigated in a rabbit model of retinoblastoma that el of retinoblastoma (Fig 2).5 develops vitreous seeds.55,101 Another injectable agent being studied as a deliv- Conclusions ery system of chemotherapeutic agents for the treat- Management of retinoblastoma is individualized ac- ment of retinoblastoma is the biodegradable carrier cording to patient characteristics and preferences, with fibrin glue.102 Carboplatin103 and topotecan69 released the main goals being to save the patient’s life and to from fibrin depots have both been shown to retain preserve useful vision, if possible. Therapeutic op- their bioactivity against retinoblastoma cells in cul- tions for retinoblastoma have rapidly evolved in recent ture. Carboplatin released from a fibrin depot was also years, with a paradigm shift in standard treatment pro- shown to decrease tumor burden in a transgenic mu- tocols toward the targeted delivery of chemotherapeu- rine model of retinoblastoma cells.104 In another report, tic agents. Increased use of intra-arterial and intravit- fibrin sealants sustained delivery of carboplatin in ocu- real types of chemotherapy with focal consolidation, lar tissues compared with carboplatin in plain solution, along with the reduced use of systemic chemotherapy, which clears rapidly in vivo.105 Promising results have enucleation, and external beam radiotherapy, has con- been reported for topotecan combined with fibrin in tributed to improved rates of survival and globe sal- clinical studies.71,106 vage while also minimizing unwanted treatment-relat- ed adverse events. However, these approaches are not Suprachoroidal Injection without complications, and a need still exists to weigh Suprachoroidal injection with microneedles is a route the risks and benefits of treatment and to evaluate their of targeted drug delivery to the choroid and retina that long-term effects. New therapies being investigated in- poses minimal risk of extraocular tumoral spread, be- clude novel carriers and routes for local drug delivery cause the vitreous and subretinal spaces are not en- as well as molecularly targeted therapies. Our under- tered. In a rabbit model, beva- cizumab was determined to be safe and effective when injected A B into the suprachoroidal space; high concentrations were found in the posterior segment tis- sues.107 Microneedles are 0.8- to 1.0-mm long, 32-gauge needles with a short bevel that penetrate through the sclera into the su- praciliary space. When the mi- croneedle injects compounds into the supraciliary space, these compounds posteriorly spread C D into the suprachoroidal space.108 In phase 1 clinical trials, mi- croneedles for suprachoroidal injections have been used to in- ject triamcinolone into the supra- choroidal space in patients with uveitis and macular edema fol- lowing retinal venous occlusion (NCT02303184, NCT01789320). The suprachoroidal delivery of Fig 2A–D. — Intravitreal injection of topotecan in a rabbit model of retinoblastoma. (A) Vitreous chemotherapeutic agents is a seeds (asterisks) form in this retinoblastoma rabbit model. (B) A 32-gauge needle is inserted into the pars plana. (C) The needle (arrows) is inserted into its hub and topotecan is injected. (D) Vitreous promising approach for appli- seeds have disappeared after 3 weekly injections of 20 µg topotecan. cations in retinoblastoma, and, Reprinted from Grossniklaus HE. Retinoblastoma. Fifty years of progress. The LXXI Edward Jackson ideally, will result in high cho- Memorial Lecture. Am J Ophthalmol. 2014;158(5):875-891, with permission from Elsevier.

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