machine dependent. Optimal immobilization allows minimum planning target volume and increases the possibility of lens sparing. Techniques have been described to immobilize the eye so that the beam orientation can be optimized for treatment delivery.

Proton beam radiation therapy, electron beam therapy, and intensity-modulated radiation therapy are EBRT modalities that have been used to reduce the dose of radiation to the brain, orbital and facial bones, and contralateral eye and lens. Dosimetric studies have been reported; however, long-term benefits and reduction of possible secondary malignancies have not been established yet. Proton radiation therapy is particularly interesting in these vulnerable patients because of the reduction of the integral dose to the patient’s head. The fractionation schemes remain the same as those used for other forms of EBRT, with a typical dose of 40–45 Gy delivered over 20–25 treatments over 4–5 weeks.

Brachytherapy is another effective radiation therapy delivery system. A plaque is sutured on the sclera directly over the tumor using either ruthenium 106 (Ru-106) plaque or a plaque with radioactive seeds (iodine 125 [I-125]). Ru-106 is a beta-emitting isotope that is well suited for patients with retinoblastoma; however, availability is a problem in the United States. I-125 is a gamma-emitting isotope that can be customized to the size and shape of the tumor. Brachytherapy is ideally used in patients who have small, accessible, discrete single tumors.

14.8 Multi-institutional Clinical Trials

For many decades, pediatric oncologists in North America were involved in multiinstitutional clinical trials through the establishment of cooperative groups like the Pediatric Oncology Group, Children’s Cancer Group, National Wilms Tumor Study Group, and Intergroup Rhabdomyosarcoma Study Group. In 2001, these groups were brought together to form the COG. The formation of COG gave fresh impetus to the study of retinoblastoma as a disease and the establishment of clinical trials to develop a uniform approach to the management of retinoblastoma in children in order to improve outcomes.

A retinoblastoma committee was formed within the COG, which included ophthalmologists, oncologists, pathologists, statisticians, radiation oncologists, epidemiologists, and researchers interested in the study of the biology of the tumor. The goal of this committee is to investigate all aspects of the disease. For the first time, there is an opportunity to enroll most of the approximately 300 patients diagnosed each year with retinoblastoma in North America in clinical trials. The new international retinoblastoma classification and staging systems for intraocular and extraocular disease are used in the staging and assignment of patients to specific protocols.

A study of patients with unilateral disease was the first protocol to open; the study’s main objective was estimating the incidence of “high-risk” histopathologic features following enucleation. There is consensus about the definitions of high-risk

features that predict recurrence in patients who undergo enucleation for unilateral advanced disease. Involvement of the choroid by the tumor and extension of the tumor into the optic nerve posterior to the lamina cribrosa are features that are generally considered to be predictive of recurrence. However, this is not widely accepted, as the previous studies were mostly retrospective and had different definitions of choroid and optic nerve involvement. In an effort to prospectively evaluate all enucleated eyes from patients with unilateral disease for specific histopathologic features, a committee of three pathologists was formed as part of this study to develop a consensus pathology report based on definite criteria for choroidal involvement and other features. In addition, patients who meet the criteria for choroidal involvement and postlamina optic nerve involvement receive chemotherapy consisting of three agents as per the protocol. This protocol is currently accruing.

With the wide acceptance of the ABC international classification system for intraocular retinoblastoma [2], the role of chemotherapy can be more definitively evaluated in multi-institutional studies. Extensive intraocular disease that corresponds to group B disease requires chemotherapy in addition to local therapies to salvage the vision and eye. Chemotherapy regimens include carboplatin, vincristine, and etoposide. However, data from a single institution suggest that carboplatin and vincristine without the use of etoposide can be effective in this stage of the disease. This would decrease toxicity as well as the number of visits to the hospital. Therefore, a study was initiated by the COG to evaluate the efficacy of two drugs— carboplatin and vincristine—in conjunction with local treatments to salvage eyes with group B intraocular disease. This study also evaluates the response to the initial course of chemotherapy that is not accompanied by local therapies.

A third COG study addresses group C and D intraocular disease and evaluates the efficacy of high-dose carboplatin given over 2 days along with etoposide and vincristine in saving the globe from enucleation. This study is based on pilot data from Children’s Hospital Los Angeles who received high doses of carboplatin along with subtenon carboplatin. The recommended number of subtenon injections is 3. When six injections were given, there was evidence of orbital retraction and cosmetic defects. With this regimen, clinicians from the Children’s Hospital Los Angeles were able to save 2 of 3 group C eyes and 11 of 19 group D eyes without EBRT or enucleation. This study is open only to a limited number of institutions. The goal of this study is to salvage the globes and the residual vision of patients with group C or D disease.

The fourth COG study that is currently accruing patients addresses patients with extraocular disease. This involves patients with orbital disease, extracranial metastatic disease, and central nervous system disease. Staging for this protocol is based on the international staging system for extraocular disease. High-dose chemotherapy and stem cell rescue are part of the treatment for patients with extensive disease beyond the orbit. Radiation therapy is also part of the treatment, and the dose and the volume irradiated depend on response to systemic chemotherapy. This study is actively recruiting patients from institutions from countries all over the world and is an international collaboration.

14.9 Animal Models of Retinoblastoma

Retinoblastoma is uniquely a disease of human children. Rb1 mutations do not result in the development of retinal tumors in other animals. This has been hypothesized to be due to redundancy in the expression of retinoblastoma gene family members during fetal eye development in animals other than humans. Two approaches have been used to create animal models of retinoblastoma: the xenografting of human tissue in animals and the development of transgenic mice.

Establishing xenografts of human retinoblastoma cell lines necessitates the use of immune-compromised animals to prevent rejection. Such xenografts have been successfully established in the eyes of mice, rats, and rabbits, using either transgenic immune-compromised animals or drug-induced immune suppression. Because the animals receive intravitreous injections of tumor cells, these models mimic retinoblastoma with vitreous seeds rather than disease originating from primary retinal tumors. A murine model that exhibits extensive optic nerve and choroidal invasive disease that can result in central nervous system metastasis has been described [47]. These models have been successfully used to model gene transfer and oncolytic virus strategies to treat human disease [48, 49].

Another approach to animal models of retinoblastoma has been the development of murine transgenic models. The oncogenic viral protein SV40 T-antigen binds to and sequesters all retinoblastoma family members and p53. Introduction of the gene encoding this SV40 protein driven by appropriate promoters into embryonic mice has resulted in primary retinal tumors that are similar to retinoblastoma in appearance and, to varying degrees, biology. Another approach has been to knock out varying combinations of retinoblastoma gene family members in mice. Again, retinal tumors develop in these animals. Transgenic models have been used to study intraocular tumor response to chemotherapy and tumor biology.

While both types of animal models have proven useful to study preclinical response to potential novel therapeutic interventions, both have drawbacks. Xenograft models use immune-deficient animals, and therefore the host immune response to the therapies cannot be monitored. Transgenic tumors are not the result of the same genetic mutations as human tumors, and nonhuman cells do not respond to therapeutic modalities in the same way as do human cells. Therefore, the animal tumors might respond differently than the human counterparts, especially to therapies using delivery systems derived from viral vectors. These caveats must be kept in mind when interpreting therapeutic results of preclinical trials.

14.10Gene Transfer Technology for Treatment of Retinoblastoma

Adenoviral vectors have been shown to transfer transgenes to human retinoblastoma both in vitro and in vivo [48, 50]. While the introduction of the normal Rb1 gene can reduce the proliferation of retinoblastoma, the transfer of the herpes simplex thymidine kinase gene using adenoviral vectors into retinoblastoma tumors and

then treating the animal with the anti-herpes drug ganciclovir (suicide gene therapy) is superior [48]. The cytotoxic phosphorylated ganciclovir can be transported to adjacent tumor cells and result in the killing of cells that have not been transduced by the viral vector. This is termed a “bystander effect” and can greatly amplify the therapeutic potential of therapeutic gene transfer.

The successful validation of this preclinical strategy in xenograft models of retinoblastoma led to the development of a phase I trial to test the use of intravitreous adenoviral vector delivery of the herpes simplex thymidine kinase gene followed by ganciclovir for bilateral retinoblastoma complicated by vitreous seeds in children in whom all standard therapies for the disease had failed and who were facing a second enucleation. Nine children were treated and all had a complete clinical response of their vitreous seeds to this potential therapy. However, recurrent retinal disease and local toxic effects resulted in the subsequent enucleation of these eyes. Toxic reactions consisted of a transient inflammatory response that could be controlled with anti-inflammatory therapy. The inflammation appeared to be more severe in children with more extensive tumor seeding. Importantly, the intraocular injections did not result in the spread of the tumor through the needle track, suggesting that other therapies could be delivered by intraocular injection [51].

Use of an oncolytic picornavirus for the treatment of metastatic retinoblastoma has also been studied in a murine xenograft model of retinoblastoma [49]. In contrast to gene transfer using a viral vector, for which the virus is made replication incompetent, this oncolytic virus is a naturally occurring replication-competent virus. All of the treated animals had at least a partial response and most of the animals had a complete extraocular response to a single intravenous injection of this virus. None of the treated animals developed central nervous system metastases. Intravenous delivery of the virus did not control intraocular disease. A phase I trial to determine the toxicity of this virus in adults is ongoing, and a phase I trial to treat neuroendocrine tumors in children is under development.

14.11 Future Development

As translational research progresses from the laboratory to the clinic, it is likely that new approaches to retinoblastoma will be focused on localizing drug delivery directly to the eye, thereby avoiding systemic toxicity associated with current modalities. Intra-arterial chemotherapy and transscleral depot reservoirs are two such approaches that hold promise for the future.

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