Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

Melanocyte

Conclusions

Figure 47.4  Schematic depiction of class 1 and class 2 uveal melanomas in relation to melanocyte development. Class 1 tumor cells closely resemble normal melanocytes in their gene expression profile, whereas class 2 tumor cells resemble more primitive, less differentiated cells.

2A (no 8p loss), and class 2B (8p loss). Although much attention has been focused on 8q gain as a prognostic factor, this change occurs so frequently in both class 1 and class 2 tumors that it does not appear to provide independent prog­ nostic information.42 Nevertheless, the frequent gain of 8q suggests that relevant genes on this chromosomal arm are targeted for upregulation during tumor progression. Several possible oncogenes on 8q that are upregulated in uveal melanoma have been identified, including MYC, DDEF1, and NBS1 (now called NBN).66–68

The identification of genes that are expressed differen­ tially in class 1 versus class 2 uveal melanomas has provided an opportunity to explore biological differences between these tumors that may provide insights into the metastatic process. The gene expression signature of class 1 tumors is very similar to normal melanocytes, including the expres­ sion of genes involved in melanocyte specification from neural crest (e.g., EDNRB, ERBB3, CTNNB1) and genes involved in melanocyte differentiation and pigment produc­ tion (e.g., MITF, DCT, TYR, TRP1). In contrast, class 2 tumors express genes involved in cell-cell adhesion (e.g., CDH1), basement membrane/extracellular matrix production (e.g., COL4A1, SPARC, LAMC1), and other epithelial-like func­ tions (e.g., EMP1, EMP3, CITED1).45 Recent work in our laboratory suggests that the class 2 tumors express a signa­ ture similar to neural/epithelial stem/progenitor cells, and it is these cells that are present in metastatic lesions (JW Harbour, unpublished data). Taken together, these findings suggest that class 1 tumors are composed mostly of melanoma cells that can traverse far down the melanocyte differentiation pathway, where they are resistant to meta­ static progression. Class 2 tumors, on the other hand, contain melanoma cells that are less differentiated and more stemlike, and perhaps it is this property that conveys metastatic potential (Figure 47.4).

The epithelial-like phenotype of class 2 tumors may promote metastasis by allowing them to survive after detach­ ment from surrounding extracellular matrix by substituting the survival signals emanating from cell–matrix interactions with ones from E-cadherin-mediated cell–cell interactions. Consistent with this possibility, E-cadherin expression is required for anchorage-independent growth of uveal melanoma cells in soft agar.45 The primitive nature of class 2 melanoma cells may also promote the dissemination and

colonization of distant sites, which is reminiscent of the behavior of neural crest cells, from which melanocytes are derived.

Unifying concepts

Many clinical and pathologic features of uveal melanoma have been shown to be associated with metastatic risk, including advanced patient age, ciliary body involvement, larger tumor size, epithelioid cell type, and extracellular matrix patterns.69 However, the pathogenetic relationships between these factors remain unclear. When viewed from the perspective of our current genetic understanding of uveal melanoma, many of these other features can be reconciled into a unifying concept of uveal melanoma pathobiology. For example, epithelioid cytology, looping extracellular matrix patterns, and monosomy 3 are all significantly associ­ ated with the class 2 gene expression signature.60,63,70,71 Even though none of these features predicts metastasis as accu­ rately as gene expression profiling, their association with the class 2 signature suggests that they may be biologically related. Perhaps epithelioid cytology and looping extracel­ lular matrix patterns represent manifestations of the epithe­ lial-like phenotype of class 2 melanoma cells.45 It is tempting to speculate that one or more genes on chromosome 3 are required for melanocytic differentiation, and the loss of these genes in tumors with monosomy 3 inhibits differentia­ tion and promotes primitive class 2 melanoma cells with stem/progenitor qualities.

Conclusions

We are entering a new era in the management of uveal melanoma. It will remain important to manage the primary ocular tumor with the most effective techniques available, but it is clear that this alone will not have an impact on future improvements in patient survival. Rather, such improvements will require the identification and preventa­ tive treatment of high-risk patients. As discussed herein, we now have highly accurate molecular tests for identifying patients at high risk of metastasis. The next step is to develop adjuvant treatments for high-risk patients that will delay or prevent the development of metastatic disease, such as by

inducing sustained dormancy in micrometastatic lesions. Such progress will require a greater understanding of tumor dormancy and the mechanisms governing successful coloni­ zation of secondary sites by metastatic melanoma cells. A recent study has shed important light on this issue by com­ paring the gene expression profiles of primary and metastatic uveal melanoma cells.72 This study identified the NF-κB pathway as potentially important in regulating the develop­ ment of metastatic tumors. Such studies will be critically important for identifying “druggable” molecular targets for treating high-risk patients.

Acknowledgments

This work was supported by grants to ZMC from Quest for Vision, Research to Prevent Blindness, Inc., Mary Knight Asbury Chair of Ocular Pathology and E Vernon and Louise Smith Fund, and to JWH from the National Cancer Institute (R01 CA125970), Research to Prevent Blindness, Inc., Barnes-Jewish Hospital Foundation, the Kling Family Foundation, the Horncrest Foundation, and the Tumori Foundation.

2.Ramaiya KJ, Harbour JW. Current management of uveal melanoma. Exp Rev Ophthalmol 2007;2:

939–946.

7.Gamel JW, McLean IW, Foster WD, et al. Uveal melanomas: correlation of cytologic features with prognosis. Cancer 1978;41:1897–1901.

9.Harbour JW, Brantley MA Jr, Hollingsworth H, et al. Association between posterior uveal melanoma and iris freckles, iris naevi, and choroidal naevi. Br J Ophthalmol 2004;88: 36–38.

13.Egan KM, Seddon JM, Glynn RJ, et al. Epidemiologic aspects of uveal melanoma. Surv Ophthalmol 1988;32: 239–251.

17.Girnita A, All-Ericsson C, Economou MA, et al. The insulin-like growth factor-I receptor inhibitor picropodophyllin causes tumor regression and attenuates mechanisms involved in invasion of uveal melanoma cells. Clin Cancer Res 2006;12:1383–1391.

22.Rimoldi D, Salvi S, Lienard D, et al. Lack of BRAF mutations in uveal melanoma. Cancer Res 2003;63:5712–5715.

25.Zuidervaart W, van Nieuwpoort F, Stark M, et al. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS. Br J Cancer 2005;92:2032–2038.

42.Ehlers JP, Worley L, Onken MD, et al. Integrative genomic analysis of aneuploidy in uveal melanoma. Clin Cancer Res 2008;14:115–122.

45.Onken MD, Ehlers JP, Worley LA, et al. Functional gene expression analysis uncovers phenotypic switch in aggressive uveal melanomas. Cancer Res 2006;66: 4602–4609.

54.Gamel JW, McLean IW. Quantitative analysis of the Callender classification of uveal melanoma cells. Arch Ophthalmol 1977;95:686–691.

59.Onken MD, Worley LA, Person E, et al. Loss of heterozygosity of chromosome 3 detected with single nucleotide

polymorphisms is superior to monosomy 3 for predicting metastasis in uveal melanoma. Clin Cancer Res 2007;13: 2923–2927.

60.Onken MD, Worley LA, Ehlers JP, et al. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res 2004;64:7205–7209.

61.Tschentscher F, Husing J, Holter T, et al. Tumor classification based on gene expression profiling shows that uveal melanomas with and without monosomy 3 represent two distinct entities. Cancer Res 2003;63:2578–2584.

63.Worley LA, Onken MD, Person E, et al. Transcriptomic versus chromosomal prognostic markers and clinical outcome in uveal melanoma. Clin Cancer Res 2007;13:1466–1471.

72.Meir T, Dror R, Yu X, et al. Molecular characteristics of liver metastases from uveal melanoma. Invest Ophthalmol Vis Sci 2007;48:4890–4896.

Clinical background

Epidemiology

Retinoblastoma is a tumor of the developing retina. It is the most common malignant ocular tumor in childhood, affecting approximately 1 in 20 000 live births.1–3 In the USA retinoblastoma is the 10th most common pediatric cancer,4 with an incidence of 10.6 per million children under the age of 4, 1.53 per million in children between the ages of 5 and 9 years and only 0.27 per million in children over the age of 10.5 Worldwide, retinoblastoma is responsible for 1% of childhood cancer deaths and 5% of childhood blindness.6 No gender or race predilection and no significant environmental risk factors have been identified. However there may be an association between retinoblastoma and low socioeconomic status worldwide.7

Historical development

Knudson in 1971 provided critical insight into the genetic understanding of retinoblastoma by postulating the “twohit” hypothesis.8 He proposed that two mutational events were necessary for retinoblastoma tumorigenesis. Comings later expanded the theory by proposing that the mutations were in both alleles of a gene with a tumor-suppressive function.9 The retinoblastoma gene (RB1) was later localized10–12 and cloned13–15 on chromosome 13q14, becoming the first tumor suppressor gene identified and laying the groundwork for great advances in the understanding of oncogenesis.

In this chapter we will briefly discuss the clinical presentation and management of retinoblastoma. We will also summarize the current understanding of the molecular pathophysiology and genetics of hereditary retinoblastoma.

Signs and symptoms

Patients usually present in the first year of life (average 7 months) for bilateral cases and at approximately 24 months for unilateral cases.16 In patients without a family history, when retinoblastoma is not suspected, the most common

Genetics of hereditary retinoblastoma

Alejandra G de Alba Campomanes and Joan M O’Brien

clinical presentation is leukocoria or white pupil (Figure 48.1). The second most common presenting sign is strabismus, which is usually constant and unilateral and can manifest as an esoor exodeviation. This is the result of macular involvement and represents an early sign of the disease, with higher survival rates and higher chances of globe preservation. In contrast leukocoria is a late sign and is associated with lower rates of globe salvage. A small proportion of patients have a more atypical presentation with consequent poor prognosis.16 If the tumor spreads into the anterior chamber, the patient can develop hypopyon, rubeosis, or glaucoma or present with an apparent orbital cellulitis secondary to extensive tumor necrosis. Other presenting signs include unilateral mydriasis, heterochromia, hyphema, uveitis, and nystagmus. Extraocular extension of the disease may present with proptosis. Systemically, patients can present with signs of increased intracranial pressure secondary to an intracranial mass when affected by trilateral retinoblastoma.

Diagnostic workup

All patients with any clinical suspicion of retinoblastoma should have a complete ophthalmic exam and a careful family history. In the presence of hypopyon or orbital cellulitis, retinoblastoma should be ruled out before performing any surgical intervention such as a needle tap or biopsy. Exam under anesthesia is required for a thorough examination of the posterior pole and peripheral retina with scleral depression in infants and young children. A wide-angle camera (Ret-Cam) is commonly used and provides 130° imaging of the retina and the anterior segment.

On dilated fundus exam a round creamy yellow-white mass can be identified projecting into the vitreous cavity with large irregular blood vessels on the surface and penetrating the tumor (Figure 48.2). Vitreous or subretinal seeding of tumor cells can be observed. Calcification within the tumor mass is common. The localization of the tumor is variable but related to the age at presentation. Posterior pole masses tend to present at an earlier age. Patients can also present with a retinal detachment covering an underlying tumor mass. An irregular gray plaque on the retinal surface can be seen in the diffuse infiltrating form of this

Figure 48.1  Leukocoria is the presenting sign of retinoblastoma in

60% of patients, as demonstrated here in a patient with unilateral disease. Strabismus is a presenting sign in 20% of patients with retinoblastoma. The remaining 20% of patients present with atypical clinical presentations like orbital cellulitis.

Figure 48.2  Fundus photograph showing multiple round elevated white masses projecting into the vitreous cavity.

disease. This is an uncommon presentation that is more difficult to diagnose; the presence of a hypopyon can sometimes alert the clinician to this unusual presentation.

In the presence of vitreous opacity or a retinal detachment, when visualization of the tumor mass is difficult, B-scan ultrasonography or computed tomography (CT) can identify calcifications (Figure 48.3). These tests should always be performed to exclude retinoblastoma before any surgical intervention is performed in a child with a restricted fundus exam.

B-scan ultrasonography characteristically demonstrates a high reflectivity mass with shadowing behind the tumor. Magnetic resonance imaging may be preferred to CT to reduce the risk of radiation-associated cancer in these pediatric patients.

Ultrasound biomicroscopy is useful to detect disease anterior to the ora serrata: this is an indication for immediate enucleation due to the increased risk for systemic metastasis. A biopsy of the tumor or vitreous is contraindicated due to the associated risk for tumor spread outside the eye. Bone

A

B

Figure 48.3  (A) B-scan ultrasonography demonstrating a high-reflectivity mass with characteristic shadowing. (B) Computed tomography can identify calcified masses.

marrow aspiration and lumbar puncture should be performed to screen for metastasis in children who present late or with high-risk features of the disease.

Screening examinations of babies with a family history allow early detection of tumors even before they are clinically evident. Currrent recommendations for screening include: initial fundoscopic exam under anesthesia at birth and subsequently every 2–4 weeks for the first several months. The oldest age that a patient with a family history of retinoblastoma has presented is 48 months.17

Genetic testing for retinoblastoma

Genetic testing can characterize the specific mutation affecting an individual patient as well as identify the presence of a nonpenetrant mutation in a carrier parent. Karyotypic studies are less useful for clinical diagnosis because only 3–5% of retinoblastoma patients carry large deletions detectable by these methods.18 Occasionally 13q deletions or translocations are evident through application of these techniques. In these cases, other systemic abnormalities, including severe developmental delay and dysmorphic features (13q deletion syndrome), can be clinically observed.

More sophisticated direct and indirect DNA analysis techniques are needed to detect smaller mutations. These techniques identify the initial germline mutation in approximately 85% of patients.19 Direct methods involve extracting DNA from a fresh unfixed fragment of the tumor after enucleation. If this is not available, testing can be performed with leukocytes from peripheral blood. These

techniques include single-strand conformation polymorphism (SSCP) analysis, gel electrophoretic analysis of synthetically amplified exons, and fluorescent in situ hybridization (FISH). Indirect methods can also be used in cases where the initial mutation cannot be found. These methods involve restriction fragment length polymorphism (RFLP) or variable number of tandem repeats (VNTR) analysis of parental and tumor DNA to detect the presence of a genetic marker that segregates along the retinoblastoma gene (RB1). Indirect techniques require the presence of two or more affected family members20 and are in general less sensitive than direct techniques.

A recently described multistep testing strategy combines multiplex polymerase chain reaction (PCR) with double exon sequencing and promoter-targeted methylationsensitive PCR to achieve a sensitivity of 89% in detecting the RB1 mutation.21 Protein truncation testing has also been shown to be effective in screening for germline mutations.22 New methodologies using microarray chips and robotic sequencing are currently on the horizon. In the future, knowing the specific gene mutation in a particular patient could be useful to predict disease severity and to provide prognostic and therapeutic guidance.

Genetic testing of affected patients and their families is extremely important, not only because patients with a germline mutation are at risk of developing secondary tumors, but also for genetic counseling. Occasionally, lowpenetrance pedigrees can be identified where there is unilateral or even no detectable disease or family history. It is possible to perform preimplantation genetic diagnosis during in vitro fertilization. Therefore, screening for constitutional RB1 mutations should become an integral part of the management of patients with retinoblastoma, irrespective of tumor laterality or family background.

Differential diagnosis

The differential diagnosis of retinoblastoma includes lesions that simulate retinal tumors like Toxocara canis and astrocytic hamartoma, lesions that can cause retinal detachments such as retinopathy of prematurity, Coats disease, and persistent hyperplastic primary vitreous and other conditions like retinal dysplasia and medulloepithelioma (dikytoma).

Retinoma is a benign growth of the retina that is also produced by a mutation in the RB1 gene. It presents as a nonprogressive, elevated gray retinal mass that can have calcification and pigmentation. It may develop when the second mutation occurs in a nearly developed retinal cell and does not acquire the additional necessary mutations for full malignancy.

Treatment (Box 48.1)

Management algorithms for retinoblastoma have changed rapidly over the past few decades and continue to evolve. The goals of treatment are cure of the disease, globe salvage, preservation of vision, and early detection and treatment of secondary malignancies. A wide array of systemic and local treatments exists (Table 48.1).

Systemic chemotherapy protocols have replaced en­ ucleation and radiation as the primary treatment for retino­blastoma. Chemotherapy reduces tumor volume

Clinical background

Box 48.1  Treatment key points

Table 48.1  Treatment of retinoblastoma

(chemoreduction) to permit application of focal techniques such as laser photocoagulation or cryotherapy to ablate remaining tumor mass. The choice of agents, combination, and dosage varies among treatment centers. The most commonly used chemotherapeutic agents include vincristine, carboplatin, etoposide, and teniposide. Ciclosporin A is sometimes used to combat multidrug resistance. Subtenon injections of carboplatin can be used as an adjunct to systemic chemotherapy. Intravenous carboplatin can also be used in conjunction with infrared diode laser radiation applied directly to the tumor through the pupil (transpupillary thermotherapy) because heat increases the permeability of the cellular membrane to antimitotics, reinforcing their cytotoxic effect. For small tumors (diameter less than 3 mm), transpupillary thermotherapy can be used alone, relying on the cytotoxic effect of heat by raising the temperature of the tumor above 45°C. Complete tumor control can be achieved in 85% of appropriately selected patients.23 Complications include iris atrophy, lens opacities, retinal traction, retinal detachment, and disk edema. Chemotherapy alone does not achieve permanent tumor control. When local therapy is applied in conjunction with chemotherapy, success rates approach 85%.24 Systemic chemotherapy carries potentially serious systemic adverse effects, including hearing loss, cytopenia, neutropenia, infections, gastrointestinal toxicity, and neurotoxicity.

External-beam radiation is indicated in advanced bilateral cases or in cases of disease relapse. It may also be considered for small tumors located within the macula, because it offers a better chance for useful vision when compared to other focal treatments. Radiation increases the risk of secondary nonocular malignancies. Side-effects include cataract formation, dry eye, retinopathy, vitreous hemorrhage, growth retardation of the orbit, and resultant midface hypoplasia.

Brachytherapy uses radioactive plaques, like iodine-125 or ruthenium-106, on the sclera over the base of the tumor. The total dose of radiation is ~4000 cGy delivered at a rate of 1000 cGy daily. It can be used for medium-sized tumors (4–10 disk diameters) for consolidation or as a secondary method after treatment failure with localized relapse. Tumors involving the macula or optic disk are not optimal candidates for this treatment modality. The recurrence rate for this treatment is 12% at 1 year if used as primary treatment and 8–34% if used as salvage therapy after failure of other methods.25 Radiation retinopathy, cataract, and neovascular glaucoma are reported complications of brachytherapy. Accelerated proton beam irradiation uses tantalum rings sutured to the sclera to mark the tumor edges in order to deliver an accelerated particle beam to active intraocular tumor. It can also be applied as an adjunct to enucleation with positive tumor margins in the optic nerve stump.

Focal treatments include transpupillary argon laser photocoagulation and transscleral cryotherapy. Small tumors (less than 3 mm in diameter and 2 mm in thickness) without vitreous seeding are good candidates for photocoagulation. Complications include retinal detachment, fibrosis, and vascular occlusions. Alternatively, cryotherapy can be used if a small tumor is located peripherally. Cryotherapy causes intracellular ice crystal formation, protein denaturation, pH changes, and cell membrane rupture. Circulation to the tumor is also disrupted. Cryotherapy can also be applied as a secondary treatment for tumor previously treated with laser, transpupillary thermotherapy, or external-beam radiation. Tumor destruction is usually achieved after one or two sessions of triple-freeze therapy at 1-month intervals. Complete destruction is achieved in 90% of tumors.26 Complications include pain, intraocular inflammation, chemosis, lid edema, vitreous hemorrhage, and retinal detachment.

In the USA, enucleation is reserved for advanced cases of retinoblastoma with massive involvement of the retina and vitreous, rubeosis, glaucoma, or tumor invasion into the anterior segment or optic nerve. It remains the most common form of treatment worldwide.16 Fortunately, enucleation is effective, achieving total cure in 99% of patients with no extraocular involvement.7 During enucleation it is of utmost importance to avoid inadvertent perforation of the globe, since this carries a very high risk for extraocular tumor seeding. A long section of the optic nerve should be obtained for histopathological analysis. Enucleation is the only treatment modality that allows genetic analysis of fresh tissue. High-risk characteristics of enucleated specimens include massive choroidal infiltration, anterior-chamber seeding, tumor invasion beyond the lamina cribrosa, and scleral invasion. Children with these high-risk features are recommended to have adjunctive chemotherapy (Children’s Oncology Group, Group E Prospective Trial).27 Tumor at the surgical margin of the optic nerve has an associated mortality rate of 50–81%.28 High-dose chemotherapy with autologous marrow transplantation is therefore indicated to prevent metastases in patients with tumor extending beyond the cut end of the optic nerve (Children’s Oncology Group, Group F Concept Proposal).27

Novel treatment modalities under investigation include subconjunctival (Figure 48.4) and intravitreal delivery of chemotherapeutic agents,29 injection of photosensitizing agents followed by selective laser treatment, gene therapy,

and ophthalmic artery injection of chemotherapeutic agents. New understanding in the genetic alterations in retinoblastoma tumorigenesis will likely produce novel molecular targets to treat the tumors directly with reduced systemic side-effects.30

Prognosis and complications

When diagnosis is timely, local control is excellent and survival exceeds 85% in patients who present with disease confined to the globe. Survival has improved dramatically in the past century, approaching 96% in specialized centers where patients have access to modern therapeutic strategies. However in developing countries mortality is still as high as 50%.31 Extraocular disease carries a significantly worse prognosis and is fatal in 50–85% of cases.16 Visual prognosis depends on the size of the tumor, location, and multifocality, and the presence of retinal detachment, vitreous seeding, or subretinal seeding.

The most common route for metastasis is direct extension into the optic nerve with subsequent intracranial or meningeal involvement. Systemic metastasis can also occur through extensive involvement of the choroidal circulation. The bone marrow is the most common site for metastasis. Other less frequent sites for metastases include bone, lymph nodes, liver, and lungs. The greatest risk factor for metastasis is extensive invasion down the optic nerve or into the orbit.

Patients who carry an RB1 germline mutation are at risk of developing other nonocular malignancies, such as midline intracranial tumors (also known as pinealoma, pinealoblastoma, ectopic intracranial retinoblastoma, trilateral retinoblastoma, or primitive neuroectodermal tumor), osteosarcoma, other soft-tissue sarcomas, and cutaneous melanoma. The cumulative incidence of second tumor development is 1% per year; after 50 years, 50% of patients would have developed another primary malignant tumor. The risk of developing osteosarcoma is increased by radiation, especially when administered within the first year of life.32 Secondary primary tumors, rather than retinoblastoma itself, are the most common cause of death in retinoblastoma patients.33

Pathology

Different growth patterns can be identified on gross pathology: the endophytic form of retinoblastoma grows into the vitreous cavity, whereas the exophytic form grows into the subretinal space, producing an overlying retinal detachment (Figure 48.5). A mixed-growth pattern is a combination of these two endophytic and exophytic forms. Diffuse infiltrating or plaque-like retinoblastoma presents with no obvious mass or calcification.34

Histopathologically, retinoblastoma consists of poorly differentiated neuroblastic cells with large hyperchromatic nuclei and scant cytoplasm. Mitotic figures are common. More differentiated cells can form Flexner–Wintersteiner rosettes, a spherical structure of columnar cells arranged around a lumen composed of internal limiting membrane. Flexner–Wintersteiner rosettes are a unique histopathologic feature of retinoblastoma.

Etiology

Genetics

The retinoblastoma tumor arises from loss or mutation of both alleles of the RB1 on the q14 band of chromosome 13.13,14 Approximately 60% of affected children have the sporadic or nonheritable form of this disease.1 Characteristically unilateral and unifocal, this form of the disease arises when both of the RB1 alleles are inactivated somatically in a single developing retinal cell. The remaining 40% of patients have heritable or familial retinoblastoma (Table 48.2), where affected individuals carrying a predisposing germline mutation suffer an additional mutational event in the normal allele of the developing retinal cell (Figure 48.6). In general, heritable retinoblastoma is characterized by high

Figure 48.5  Gross histopathology section of advanced retinoblastoma demonstrating an exophytic tumor with overlying retinal detachment.

Table 48.2  In heritable retinoblastoma a germline mutation is present 100% of the time1

penetrance (nearly 90% of carriers develop the disease) and high expressivity (bilateral and multifocal tumors). However least common mutations can result in a different pattern with reduced penetrance and expressivity (“low-penetrance retinoblastoma”). Approximately 11% of patients with unilateral tumors harbor a germline mutation (i.e., have the heritable form).35 Heritable retinoblastoma is considered a genetic cancer predisposition syndrome since the presence of a germline mutation imposes an elevated risk for the development of cancer in other organ systems.

Box 48.2  Mutational mechanisms

Germline mutations → first mutational event

onset with few intraocular recurrences, and shows an early and complete response to therapy.37

Mutations (Box 48.2)

In the majority of cases the first RB1 mutation is a single nucleotide change (point mutation) or small deletion. In 60% of cases the second mutational event occurs following loss of heterozygosity (LOH) by nondisjunction followed by reduplication or mitotic recombination, resulting in homozygosity of the initial mutation in the susceptible retinal cell. Mutations occur throughout the RB1 gene with no single mutational “hotspot.” Ninety percent of clinically significant mutations are characterized by frameshift, nonsense, or splice mutations, which produce a premature stop codon and a truncated transcript.36 These mutations involve the large pocket domain of the retinoblastoma protein (pRb) 98% of patients.36 The high frequency of truncating mutations in retinoblastoma suggests that tumorigenesis is most favored by mutations that globally inactivate pRb. In contrast truncating mutations are absent in low-penetrance retinoblastoma. The mutations reported in these kindreds minimally alter the expression of the pRb, permitting residual protein function. Retinoblastoma in these low-penetrance kindreds often skips generations, is unilateral, has a delayed

Pathophysiology

The human RB1 gene spans 180 kb of DNA39 and contains 27 exons. It encodes a 4.8-kb mRNA.40 Its protein product (pRb) is a 110-kDa phosphoprotein composed of 928 amino acids. This protein functions as a regulator at the cell cycle checkpoint between the G1 and S-phase. When inactivated, transcription of downstream genes that promote progression through the cell cycle occurs. Along with p107 and p130, pRb belongs to the pocket protein family.

Retinoblastoma likely arises from either a precursor cone photoreceptor or a multipotent retinoblast. The retinal cells in these patients develop and mature between the third month postconception and the age of 4 years, being susceptible to mutations at any point during this time.

Function of pRb

pRb inhibits cellular proliferation by altering the expression of genes that promote cellular division through an interaction with the E2F transcription factors. Of the six E2F members (E2F1–E2F6), pRb interacts preferentially with

Transcriptional repression G1 arrest

Proliferative signals

Cyclin D

Cyclin D -cdk4/6 P

E2F

Promoter E2F target genes

Transcriptional activation progression into S phase

Pathophysiology

Figure 48.7  The role of the retinoblastoma protein (pRb) in the cell cycle. pRb is hyperphosphorylated (inactivated) by cyclin-dependent kinases in response to mitogenic signals, leading to its dissociation from E2F and enabling the cell to progress into the DNA synthesis phase.

E2F1–E2F4, whereas p107 and p103 interact with E2F4 and E2F5.41 The activation of E2F1–E2F341 is required for cell proliferation, since these transcription factors activate the expression of genes required for G1/S-phase progression and for DNA replication.42

In its hypophosphorylated state, the active form of pRb binds to E2F in early G1,43 blocking its transactivating function and inhibiting cell cycle progression.44 This interaction may result in permanent or reversible cell cycle arrest (G0). Continuous mitogenic signaling is required to induce the cell out of G0 (quiescence) and allow it to progress through the cell cycle. pRb is hyperphosphorylated by cyc- lin-dependent kinases in response to mitogenic signals. Mitogenic stimuli induce the expression of cyclin D which in turn binds to cdk4 and cdk6 to form complexes. These complexes phosphorylate and inactivate pRb, leading to its dissociation from E2F and enabling the cell to progress into the DNA synthesis phase of the cell cycle (S) (Figure 48.7). The conformational changes that inactivate pRb can be mimicked by mutations in the RB1 gene or by binding to viral proteins (adenovirus E1A, simian virus 40, large T- antigen, and human papillomavirus E7).45,46 On the other hand, antiproliferative signals, such as DNA damage and senescence,47 induce the expression of cyclin-dependent kinase inhibitors, which positively regulate pRb by inactivating the cyclin–cdk complexes through the upregulation of Ink4 and Cip/Kip family proteins. Data suggest that pRb is only partially inactivated at G1. During later phases of the cell cycle, pRb has a significant function, such as inducing cell cycle arrest at G2/M.48

Loss of pRb does not necessarily lead to tumor formation. In some tissues, the response to pRb loss is apoptosis.49 This is because pRb plays a critical role in the terminal differentiation of many cell types, including neurons, muscle, bone, erythrocytes, and fibers of the crystalline lens.50 In these cell types, loss of the pRb results in massive apoptosis or developmental defects.51 In other tissues like the retina and bone, the loss of pRb initiates tumorigenesis.

Role of the pRb in the development of retinoblastoma

The precise manner in which loss of the retinoblastoma protein results in retinoblastoma tumor development is still unclear. Retinoblastoma presumably arises from the dependence of retinal cells on pRb for terminal differentiation

through a complex process involving the regulation of cell cycle progression, apoptosis, and the expression of differentiation genes. During organogenesis the retina begins as a single layer of neuronal stem cells (neuroblasts). Murine studies suggest that pRb plays an important role in the differentiation of these neuroblasts into ganglion cells, amacrine and horizontal cells, and cone photoreceptors. RB1 expression is absent in bipolar cells and rod photoreceptors.52 Knockout mice models (RB1 -/-) suggest that in the absence of RB1 there is ectopic proliferation and enhanced apoptosis in this inner retinal progenitor layer, indicating that pRb is required for terminal cell cycle arrest and for suppression of apoptosis.53

The biallelic loss of the RB1 gene is a necessary and ratelimiting event in the development of retinoblastoma, but it is not sufficient. In humans, it has been proposed that a third mutational event is required to allow retinoblastoma cells to escape apoptosis and to proliferate.52 However, studies from knockout and chimeric mouse models have shown intact apoptotic mechanisms.53 In murine models, retinoblastoma only results when RB1 loss is accompanied by at least another cell cycle regulatory gene alteration. The susceptibility in humans with RB1 mutations to develop retinoblastoma is unexplained by these models. The inactivation of other pocket proteins and also of the tumor suppressor p53 has been extensively studied without conclusive evidence. In the mouse, combined loss of p107 and pRb appears to be a requirement for the development of retinoblastoma54; however, in humans, p130 appears to be more important as a tumor suppressor protein acting in conjunction with pRb.55

Human retinoblastoma tumor analysis has identified other chromosomal alterations and additional mutations. Frequently found genetic alterations include + 1q, + 2p, + 6p, – 16, –16q, – 17, and –17p. p53 is located on 17p13; however, the potential involvement of p53 in human retinoblastoma has been controversial. As mentioned earlier, p53 inactivation may be required for murine retinoblastoma, but no p53 mutation has ever been characterized in primary retinoblastoma and studies in retinoblastoma tumor specimens suggest the presence of normal p53 function.56 MDM2 (mouse double minute 2 homolog) encodes a protein that inhibits transactivation by tumor protein p53. Overexpression of this gene can result in excessive inactivation of p53, diminishing its tumor suppressor function. MDMX is a gene related to MDM2, with the same p53 antagonistic properties.

p14 Arf (cyclin-dependent kinase inhibitor 2A) expression has been found to be increased in human retinoblastoma cells when compared to normal retina.57 p14 Arf inactivates MDM2/MDMX, leading to apoptosis mediated by p53. However, a recent study found MDMX to be amplified in human retinoblastoma samples. In vitro, MDMX suppressed cell death in RB1 deficient human retina and led to rosette formation similar to human retinoblastoma.57 This recent evidence suggests that retinoblastoma does not bypass tumor surveillance mechanisms and that there is inactivation of the p53 pathway after loss of RB1, probably by subsequent amplification of the MDMX gene in the preneoplastic retinoblastoma cells.57

Conclusion

Retinoblastoma is a rare disease, but understanding of retinoblastoma gene derangements which underlie this condition has provided major insights in cancer research. The retinoblastoma gene was the first tumor suppressor gene to be identified, representing a new category of genes which have been found to be etiologic in a spectrum of tumor predisposition syndromes. As knowledge of the RB1 gene and the pathway it regulates continues to emerge, so too will a greater understanding of mechanisms for carcinogenesis.

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