Retinoblastoma, a malignant tumor of the embryonic neural retina, is the most common intraocular malignancy in children. Although usually not recognized at birth, retinoblastoma predom-inantly affects young children. The tumor has a variable growth rate, can originate from single or multiple foci in one or both eyes, and, in bilateral cases, may be manifest in one eye many months before it is evident in the other. Retinoblastoma is caused by a mutation in a gene that expresses a protein central to the control of the cell cycle and may occur sporadically or be inherited. Children with the hereditary type of retinoblastoma have a particular susceptibility to developing other malignant tumors. This disease serves as a model for understanding the genetics and heredity of child-hood cancer.

The term retinoblastoma was first adopted by the American Ophthalmological Society in 1926.¹ The cellular origin of retinoblastoma had been a topic of debate since 1809, when, based only on gross pathological findings, the Scottish surgeon Wardrop first recognized that retinoblastoma is a discrete tumor arising from the retina.^2,3 After this was published, other pathologists, including Robin and Langenbeck, confirmed the observations at a microscopic level. Virchow, however, thought that the cell of origin was glial and named it “glioma of the retina.” In the late 1800s, the term neuroepithelioma was proposed by Flexner and supported later by Wintersteiner because they believed that the tumor originated from the neuroepithelium and that the typical rosettes that now bear their names were attempts to form photoreceptors. In the early 1900s, Verhoeff concluded that the tumor was derived from undifferentiated embryonic retinal cells called retinoblasts and proposed the term retinoblastoma.¹ Zimmerman proposed the term retinocytoma for the well-differentiated tumor that displays benign features.⁴ For this same tumor, Gallie et al.⁵ described the clinical features and proposed the term retinoma. The histopathological, ultrastructural, immunohistochemical and molecular characteristics of retinoblastoma support the concept that this tumor originates from a multipotent precursor cell. This cell could develop into almost any type of inner or outer retina cell including photoreceptors.^{6,7,8,9,10,11,12,13,14,15} Alternatively, investigations have supported the concept that a differentiated cell could be the precursor cell by de-differentiating into a malignant cell.^16,17 That the benign retinoma is a precursor to the malignant retinoblastoma has also been proposed.¹⁸

The Third National Cancer Survey predicts an average incidence of 11 new cases of retinoblastoma per million population less than 5 years of age, or 1 in 18,000 live births in the United States,¹⁹ and this rate has proven stable over time.²⁰ Although data from developing countries are less complete, oncologists in Central and South America, the Middle East, and India generally feel that the incidence may be greater in these regions.^21,22 A multicenter report from Mexico concluded that retinoblastoma is the second most frequent solid malignancy after CNS tumors in children.²³ The estimated frequency of bilateral retinoblastoma ranges from 25% to 35% of reported cases.^20,24 Thus, in the United States, an estimated 300 children per year develop retinoblastoma; of these 300, 60 to 90 cases are bilateral. There are no racial or gender predilections.

Retinoblastoma is often present at birth and is almost entirely restricted to early childhood. About 80% of cases are diagnosed before 3 to 4 years of age, with a median age at diagnosis of 2 years.^20,21,24,25 The discovery of retinoblastoma beyond the age of 6 years is rare. Bilateral disease is diagnosed earlier than unilateral disease. Sporadic bilateral retinoblastoma has been associated with advanced parental age, but a recent analysis shows no increase in incidence of retinoblastoma with advancing maternal age.²⁶

Multiple congenital anomalies associated with retinoblastoma have been reported in approximately 0.05% of US patients with retinoblastoma.²⁷ The reported anomalies include congenital cardiovascular defects, cleft palate, infantile cortical hyperostosis, dentinogenesis imperfecta, familial congenital cataracts, and incontinentia pigmenti or Bloch-Sulzberger syndrome (an X-linked inherited disease that is lethal in males but affects females with pigmentary retinopathy, corneal opacities, cataracts, nystagmus, blue sclerae, myopia, pseudoglioma, dental abnormalities, abnormal skin pigmentation, and mental deficiency).²⁵ An association with mental retardation has been suggested in children with the D-deletion syndrome; however, most patients with retinoblastoma have no intellectual impairment.

The majority of retinoblastomas appear sporadically; however, an inherited form of the disease has been documented²⁸ and is transmitted with few exceptions as a typical Mendelian autosomal dominant trait with high but incomplete penetrance. Of all cases, about 60% are nonhereditary and unilateral, 15% are hereditary and unilateral, and 25% are hereditary and bilateral.^29,30

A two-hit model has been proposed to explain the observations that familial cases are generally multifocal and bilateral, whereas sporadic cases typically present with unilateral unifocal disease at a later age.^29,31 According to the model, as few as two stochastic mutational events are required for tumor initiation, the first of which can be inherited through the germ line (in heritable cases) or can occur somatically in individual retinal cells (in nonheritable cases). The second event occurs somatically in either case and leads to tumor formation from each doubly defective retinal cell.

The presence of a microscopically visible deletion in one of the chromosome 13 homologues in constitutional cells of a small number of retinoblastoma patients was the first evidence that supported an inherited mechanism for retinoblastoma development. ^32,33,34,35 Although the deletions varied between families, each deletion minimally encompassed chromosome 13, band q14.^36,37 This chromosomal locus contains the RB1 retinoblastoma gene. In the two-hit model, such deletions in the germ line could act as the first hit and confer the risk of tumor formation as an autosomal dominant trait. The increasing resolution of cytogenetic
technology and the development of DNA probes for loci in the immediate vicinity of the RB1 gene locus have allowed the detection of more subtle genomic rearrangements. These techniques can be used to identify people who carry nonpenetrant mutations in the retinoblastoma susceptibility locus.³⁸

Patients without a gross 13th chromosomal deletion but who have bilateral or familial retinoblastoma have submicroscopic mutations at the RB1 locus similar to mutations that have been found in the tumor cells of patients with nonhereditary retinoblastoma. The second step in tumorigenesis in both heritable and nonhereditary retinoblastoma involves somatic alteration of the normal allele at the RB1 locus in such a way that the mutant allele is unmasked. Thus, the first mutation in this process, although it may be inherited as an autosomal dominant trait in the child, is in fact a recessive defect in the individual retinal cell. Elimination of the chromosome containing the wild-type allele followed by reduplication of the remaining mutant chromosome may be one mechanism by which the affected RB1 locus becomes homozygous within the cell.^39,40 Other mechanisms of mutagenesis including the generation of point mutations, deletions and silencing of the promoter by methylation are frequently encountered in the second allele. The result is that the potential tumor cell becomes functionally recessive for the mutant allele.

Although the unmasking of predisposing mutations at the RB1 locus occurs in mechanistically similar ways in sporadic and heritable retinoblastoma, only the latter carries the initial mutation in each cell. Patients with heritable disease also seem to be at greatly increased risk for the development of second primary tumors, particularly osteosarcoma.⁴¹ This high propensity is genetically determined by the predisposing RB1 mutation. The notion of a pathogenetic association between these two rare tumor types was tested by determining the constitutional and osteosarcoma genotypes at restriction fragment length polymorphism (RFLP) loci on chromosome 13. The data indicated that osteosarcomas arising in patients with retinoblastoma had become homozygous specifically around the chromosomal region carrying the RB1 locus. Furthermore, these same chromosomal mechanisms eliciting losses of constitutional heterozygosity were observed in sporadic osteosarcomas, suggesting a genetic similarity in pathogenetic causality.

These studies provided data useful for the molecular isolation of the RB1 gene.⁴² The 200-Kb RB1 locus contains 27 exons that are transcribed into a 4.7-Kb transcript in normal human and rat tissues including brain, kidney, ovary, spleen, liver, placenta, and retina.⁴³ Although the number of different types of tumors that occur as a result of inherited mutations in the RB1 locus is small, the broad tissue expression and species conservation of this gene suggest a common and potentially important role in the growth or differentiation of many cell types.^44,45,46,47 Introduction of the wild-type gene into retinoblastoma and osteosarcoma cell lines using recombinant retroviral vector transfer resulted in a partial reversal of the tumorigenic phenotype.^48,49 Further characterization of the complete RB1 genomic sequence⁵⁰ allowed a rigorous cataloging of the different mutations affecting the gene in retinoblastoma tumors. Over 200 disease-causing mutations have been identified in the retinoblastoma genes of patients.^{38,51,52,53,54,55}

The examination of sporadic cases of bilateral retinoblastoma showed that disease frequently arises subsequent to a new germ line mutation in the paternal allele followed by somatic alteration or loss of the maternally derived wild-type allele.^56,57 This finding suggests either that mutations in the RB1 locus occur more commonly during spermatogenesis or that the paternal chromosome in the early embryo is at a higher risk of mutation. Analyses of sporadic osteosarcomas also showed preferential mutation of the paternal allele.⁵⁸

Investigations of RB1 gene alterations at both the DNA and the RNA levels cumulatively reveal a strong correlative relationship between the lack of RB1 gene product RB protein and the appearance of retinoblastoma tumors. In addition to osteosarcomas, other tumor types contain mutations involving the retinoblastoma gene. Molecular analyses of small cell lung carcinomas have revealed RB1 structural abnormalities in approximately 15% of cases.⁵⁹ Loss of heterozygosity for chromosome 13 has been detected in about 25% of breast cancers and related breast cancer-derived cell lines.^60,61 However, a more detailed analysis of the effects of chromosome 13 mutations in tumors has been compiled and clearly shows that not all tumors are either a direct or an indirect result of loss of heterozygosity of the RB1 locus.⁶² The cumulative data suggest that only subsets of tumors may share a common pathogenetic mechanism that results from unmasking mutations affecting the tumor suppressing function of the RB protein.

The RB protein is comprised of 928 amino acids and has an estimated molecular mass of 110 kDa. The expressed protein has been shown to be primarily localized in the cell nucleus.⁶³ Posttranslational phosphorylation of the RB protein in quiescent cells overrides growth suppression and allows cell division to occur.⁶⁴ The RB protein also has a role in the regulation of the cell cycle of actively dividing cells. The unphosphorylated RB protein (p110RB) has been shown to bind E2F1, a transcription factor and a cell cycle regulator during the G1 stage of the cell cycle. The RB/E2F1 complex masks the E2F1 transactivation domain and inhibits surrounding enhancer elements, thereby causing transcription of E2F1-regulated genes to cease. The RB protein accomplishes this by physically associating with a histone deacetylase (HDAC1). This recruitment of the deacetylase to the E2F1 regulating domain by RB allows deactylation of histone, thereby modulating the local structure of the chromatin.^65,66 Phosphorylation of the RB protein at the G1/S boundary results in the release of these transcriptional factors, allowing them to become positive transcriptional elements. Additional cell cycle-specific kinases become activated and facilitate the progression of the cells through G2 and M. At the completion of the cell cycle, phosphatases dephosphorylate the RB protein, allowing the protein to again sequester E2F1 and form an inactive complex. Thus, positive and negative regulation of transcription and, therefore, cell proliferation are linked to the phosphorylation cycle of the RB protein. In tumors in which RB protein is mutated or absent, these intracellular transcriptional elements are dissociated and free to promote consistent and uncontrolled progression through the cell cycle. Such behavior results in unchecked cell proliferation consistent with a malignant phenotype.

The viral oncoproteins of polyomaviruses (SV40), adenoviruses (Ad-2 and Ad-5), and papillomaviruses (HPV-16) have also been shown to complex with the RB protein.^67,68,69 Because one function of these viral oncoproteins appears to be the creation of a cellular environment that is permissive for DNA synthesis, one of their modes of action may involve sequestration of the antiproliferative unphosphorylated RB protein. Releasing the cell from its negative regulation by RB might allow the cell to enter the S phase and synthesize DNA. Taken together, the data support a model in which the unphosphorylated form of RB is the species active in growth suppression.

Mutations in the RB1 gene have been associated with greater than 95% of all retinoblastomas; however, there are a small number of tumors where RB1 mutations fail to be found. In at least some of these tumors, the MYCN gene is amplified and appears to be associated with the malignant phenotype.⁷⁰

The understanding of the complex interrelationships of the RB proteins, mutations in the RB proteins that lead to disease, and the metabolic consequences of these events in the cell have led to the investigation of novel therapies to treat a variety of cancers including retinoblastoma.^71,72

Most cases of retinoblastoma in the United States are diagnosed while the tumor remains contained within the eye (intraocular) without local invasion or distant metastases. In developing countries, however, the diagnosis is frequently made only after an enlarged eye (buphthalmos) or gross orbital extension is apparent. These patients more commonly present with local invasion.

The signs and symptoms of an intraocular tumor depend on its size and position. The most common presenting sign is leukocoria of one or both eyes (Fig. 27.1). Leukocoria, white pupillary reflex instead of the normal red reflex, is manifest when the tumor is large or has caused a total retinal detachment leading to a retrolental mass visible through the pupil. In some cases, a small tumor centered in the macula (central portion of the retina) can produce leukocoria. If vitreous hemorrhage occurs because of bleeding from the retinoblastoma vessels, the pupil can appear to have a dark red reflex (hemocoria) or black reflex (nigrocoria) instead of the white reflex typically seen in retinoblastoma^73,74 (Fig. 27.2). The second most common presenting sign is strabismus. Loss of central vision from a tumor in the macula can result in a disruption of fusional ability and cause the affected eye to drift.

Other ophthalmic features accompany some cases of retinoblastoma and may indicate the necessity for immediate enucleation. Heterochromia (different color for each iris) can present as an initial sign of retinoblastoma secondary to iris neovascularization. The diagnosis of retinoblastoma should be excluded in children that present with this condition.¹⁵ Rubeosis iridis (neovascularization of the surface of the iris) occurs in approximately 17% of patients with retinoblastoma and in more than 50% of patients with advanced retinoblastoma requiring enucleation¹⁵ (Fig. 27.2). Extensive necrosis of the tumor and liberated angiogenic factors may be responsible for this neovascularization of the iris.

Spontaneous bleeding from rubeosis iridis can also cause hyphema (blood in the anterior chamber), and the potential diagnosis of retinoblastoma should be investigated in a child presenting with spontaneous hyphema without history of trauma.^15,73,74 Glaucoma can be secondary to neovascularization of the anterior chamber angle and/or anterior synechia as a result of rubeosis iridis. Closed-angle glaucoma can also be secondary to mechanical obstruction of the anterior chamber angle by the iris and lens that has been pushed forward by a large intravitreal tumor. Most children with these presentations undergo enucleation.^73,74 Anterior chamber seeding from endophytic tumors or diffuse infiltrating tumors may produce pseudohypopyon (cells in the anterior chamber)⁷⁵ (Fig. 27.2). Intraocular tumors are not associated with pain unless secondary glaucoma or inflammation is present.