pediatric hematology and oncology (scientific principles and clinical practice) || retinoblastoma

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Introduction and h istorical p erspective

Retinoblastoma is a rare childhood cancer of the retina, and may have been one of the fi rst diseases in which it was observed that excessive tissue growth led to death. Tumor growth in the ante-rior chamber of the eye and surrounding peri - ocular tissue could have been readily visualized in ancient times without specialized equipment, leading to the early understanding that this condition was virtually always fatal. Indeed, antiquities have been described that depict children with retinoblastoma [1] . Following the formal description of retinoblastoma in 1809 by James Wardrop [2] , clinical experience with the therapy of retinoblastoma has highlighted important advances in therapy and the understand-ing of cancer etiology: • Several of the earliest attempts to treat cancer were carried out on retinoblastoma patients, including the use of surgical enuclea-tion [3, 4] , radiation therapy [5] and chemotherapy with nitrogen mustard [6] . • The modern modalities of combination chemotherapy evolved from institutional experience of the treatment of this disease in the 1960s and 1970s [7,8] .

As retinoblastoma survivors grew up to have their own fami-lies, it became clear that some were transmitting retinoblastoma susceptibility to their children in a Mendelian - dominant fashion. Based on this pattern of inheritance, the age of onset, and pres-entation of the disease (multifocal bilateral versus focal unilat-eral), Alfred Knudson proposed the existence of a recessive tumor suppressor gene in 1971 [9] . Specifi cally, he hypothesized that both copies of a putative tumor suppressor gene must be mutated for retinoblastoma to form. Knudson reasoned that children who inherit a defective copy of the putative tumor suppressor gene are much more likely to sustain a second mutation in their normal allele during retinal development and develop multifocal bilateral retinoblastoma at an early age. In contrast, children who develop sporadic retinoblastoma must sustain two inactivating mutations

to affect each allele in the same cellular lineage. The lower statisti-cal probability of these combined events would result in fewer lesions (unilateral unifocal retinoblastoma) and a later age of onset that is precisely what is observed clinically.

Knudson ’ s hypothesis was verifi ed in 1986 with the identifi ca-tion and cloning of the fi rst tumor suppressor gene, RB1 , from families with retinoblastoma [10] . It was later discovered that the retinoblastoma (Rb) pathway is disrupted in virtually every human cancer [11] and this has led to a rapid increase in research on the RB1 gene (located on chromosome 13q1.4) and protein over the past three decades. Despite these advances in our under-standing of the Rb gene and pathway, only recently has laboratory research had an impact on the clinical management of retinoblas-toma. In this chapter we will discuss our current understanding of the epidemiology, presentation and treatment of retinoblast-oma along with recent developments for the modeling of retino-blastoma in mice and how those studies have led to novel therapeutic approaches. We will also discuss recent genetic studies to identify secondary genetic changes in retinoblastoma following RB1 gene inactivation and new drugs that specifi cally target those genetic perturbations.

Epidemiology (Box 18.1 )

Retinoblastoma is the most common pediatric cancer of the eye accounting for approximately 3% of all childhood cancers. There is no retinoblastoma predisposition by geographic origin or gender. Retinoblastoma affects very young children and is the third most common form of cancer in infants after neuroblast-oma and leukemia [12] . • Overall, retinoblastoma is a rare cancer with an incidence between 1:14 000 and 1:34 000 live births for a total of approxi-mately 300 cases per year in the United States, 115 cases per year in Western Europe and 5000 – 10 000 cases per year worldwide. • The majority of retinoblastomas (90%) are diagnosed by 5 years of age with a median age of diagnosis of 18 months. • There have been some reports of overlap of epidemiological risk factors for the development of retinoblastoma and papilloma

18 Retinoblastoma

Edward J. Estlin 5 , Fran ç ois Doz 3,4 and Michael Dyer 1,2 1 Department of Developmental Neurobiology, St Jude Children ’ s Research Hospital, Memphis, TN , 2 Department of Ophthalmology, Howard Hughes Medical Institute, University of Tennessee Health Science Center, Memphis, TN, USA , 3 Department of Pediatric Oncology, Institute Curie, Paris, France , 4 University Ren é Descartes, Paris, France and 5 Department of Paediatric Oncology, Royal Manchester Children ’ s Hospital, Manchester, UK

Pediatric Hematology and Oncology: Scientific Principles and Clinical Practice

Edited by Edward J. Estlin, Richard J. Gilbertson and Robert F. Wynn

© 2010 Blackwell Publishing Ltd. ISBN: 978-1-405-15350-8

Chapter 18 Retinoblastoma

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above, this refl ects the inheritance of one defective copy of the RB1 gene from an affected parent (15 – 25% of cases) or a new germ line mutation (75 – 85% of cases). • The remaining 60% of retinoblastoma cases typically present as unifocal unilateral disease. In these cases, both copies of the RB1 gene are mutated in a developing retinal cell. The lower statistical probability of inactivation of two alleles in a single cellular lineage is believed to account for the unifocal and unilateral disease pres-entation [9] . • The average age of diagnosis for bilateral retinoblastoma is around 12 months and for unilateral retinoblastoma it is 24 months. If there is a family history, the median age of diagnosis is younger because of more frequent genetic tests and monitoring to determine if the child inherited a defective copy of the RB1 gene. When the disease is familial, the penetrance exceeds 90% in subsequent generations. Clinically and histologically, the herit-able and sporadic forms of retinoblastoma are indistinguishable from one another. This is consistent with the common initiating genetic event in retinoblastoma, RB1 gene inactivation [16] .

Investigation and s taging Typically, examination under anesthesia (EUA), ultrasound scans, magnetic resonance imaging (MRI) and histological exam-ination can all play a role in the diagnosis and staging of retino-blastoma [16] : • Ophthalmosocopy (and in particular indirect ophthalmoscopy for the visualization of anterior lesions of the eye) reveals retino-blastoma to have a white appearance with angiomatoid dilatation of the associated blood vessels. In order to visualize the entire retina, an examination under anaesthetic is required, and the retina must be examined after maximal pupil dilatation with scleral indentation needed to determine the number of tumors, their size, and situation in the retina whether or not there are tumor foci in the peripheral retina and the presence or not of any sub - retinal fl uid and vitreal seeds. In more advanced cases, hyphema, glaucoma, or infl ammation may also be present. • Ocular ultrasound demonstrates a tumor that has increased echogenicity in comparison to the vitreous, with fi ne calcifi cation and retinal detachment observed in exophytic tumors.

virus infection (HPV) [13] . Interestingly, human papillomavirus (HPV) genomic DNA has been detected in retinoblastomas from patients with these overlapping epidemiological risk factors in developing countries [13] . This has led to the hypothesis that HPV may contribute to retinoblastoma progression through the activity of the E7 and E6 viral oncoproteins. It is not known if this is due to the inactivation of the Rb pathway, the p53 pathway, or other pathways targeted by E7 and E6. Clearly, there may be some complex epidemiological factors that contribute to retino-blastoma progression and careful molecular, cellular, and bio-logical studies are required to follow up on the signifi cance of overlapping epidemiological risk factors. • Recently, an elevated risk of retinoblastoma in children born after in vitro fertilization (IVF) [14] was reported in the Netherlands. However, subsequent analysis of retinoblastoma incidence in children born after IVF throughout the world has shown no increase relative to children born without IVF [15] . The mechanism underlying the increased retinoblastoma inci-dence in the children born by IVF in the Netherlands is not known but retinoblastoma predisposition should be closely mon-itored in children born by IVF to determine if there may be an epigenetic component to this increase as a result of current labo-ratory procedures used for assisted reproduction.

Clinical p resentation and i nvestigation (Box 18.2 )

Presentation In many cases, retinoblastoma is fi rst detected by a parent or health professional as an abnormal pupillary refl ex (leukocoria) or squint (strabismus). One of the major challenges with the clinical presentation of retinoblastoma is delayed diagnosis, and other clinical signs at presentation include iris rubeosis and orbital cellulitis [16] . Other non - malignant differential diagnoses need to be excluded by an expert team. There are two clinical forms of retinoblastoma: • For approximately 40% of cases, the disease presents as a hered-itary form that tends to be bilateral and multifocal. As mentioned

Box 18.1 Epidemiology of retinoblastoma.

• Incidence is between 1 in 14 000 and 1 in 34 000 live births

• 90% of cases diagnosed by the age of 5 years

• Median age of diagnosis 12 months for bilateral disease and 18 months for unilateral cases.

• 40% of cases hereditary with bilateral and multifocal features

• RB1 tumor suppressor gene mutations as a paradigm for understanding of cancer predisposition

• Genetic counselling is always proposed and screening offered where appropriate

Box 18.2 Presentation, investigation and grouping of retinoblastoma.

• Leucocoria and squint predominate as clinical fi ndings

• Direct and indirect ophthalmoscopy under general anesthetic required for adequate examination

• Magnetic resonance imaging scans to defi ne involvement of the optic nerve, anterior chamber, ocular fat and central nervous system extension

• Clinical risk grouping systems (Reese - Ellsworth and ABC Classifi cation) help to predict success, in terms of ocular preservation, of conservative therapies

Part III Solid Tumors of Childhood

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• Computerized tomography describes an enhancing mass with a higher density than the vitreous. • MRI scans are the investigation of choice to defi ne the clinical extension to structures such as the optic nerve, anterior chamber of the eye or ocular fat, and also extension into the central nervous system (CNS) or the occurrence of the trilateral form of the disease as described below. Typically, retinoblastoma demon-strates higher signal intensity than the vitreous on T1 - weighted imaging, and a relatively lower signal of T2 - weighted sequences [17] .

Retinoblastoma tumor growth has been classifi ed into three patterns. • Endophytic retinoblastoma tumor growth is characterized by a disruption in the retinal inner limiting membrane and the tumor appears as a white mass with small and disorganized tumor - associated blood vessels. Vitreous seeding often accompanies endophytic retinoblastoma and it is believed that these small fragments of tumor can initiate new foci if they become juxta-posed to the normal retinal vasculature. • Exophytic retinoblastoma tumor growth is characterized by expansion in the sub - retinal space and is often associated with retinal detachment. In this type of retinoblastoma growth, tumor cells can invade the choroids through Bruch ’ s membrane and invade the associated vasculature. The vasculature in the overly-ing retina appears abnormally patterned and may become larger. However, it is not uncommon for tumors to exhibit features of both endo - and exophytic growth. • The third pattern of retinoblastoma growth is the diffuse infi l-trating type. This is a rare form of retinoblastoma growth making up only 1 – 3% of tumors and is characterized by tumor infi ltra-tion of the retina without a discrete mass, often grows more slowly than exophytic or endophytic retinoblastoma growth, and the differential diagnosis of this condition is diffi cult also [18] .

Classifi cation, h istopathological v ariants, and m olecular o ncology

Clinical r isk g rouping There are currently two main grouping systems in use for retino-blastoma. The Reese - Ellsworth (Table 18.1 ) was developed in the 1960s at a time when most children received external beam radio-therapy as their conservative treatment [19] , and is used to deter-mine the likelihood of the success of preserving vision for children with intra - ocular disease. There are fi ve groups in the Reese - Ellsworth system based on the number of lesions, the size of the lesions, and their location including the presence of vitreal seeds and the probability of success of conservative treatment through external beam radiotherapy.

Similarly, the ABC grouping system for Intraocular Retinoblastoma (Table 18.2 ) has been developed to predict the success of conservative approaches to modern treatments, in terms of the chances of ocular salvage and vision preservation in accordance with age, tumor location, vitreous seeding, and retinal

Table 18.1 Reese - Ellsworth Retinoblastoma Grouping System.

Group I (very favorable) Ia Single tumor mass smaller than 4 dd, at or behind the equator Ib Multiple independent tumors smaller than 4 dd, all at or behind equator

Group II (favorable) IIa Single tumor 4 – 10 dd, at or behind equator IIb Multiple tumors 4 – 10 dd, at or behind equator

Group III (doubtful) IIIa Any tumor anterior to equator IIIb Single tumor larger than 10 dd, behind equator

Group IV (unfavorable) IVa Multiple tumors with some larger than 10 dd IVb Any tumor extending anterior to the ora serrata

Group V (very unfavorable) Va Massive tumors involving more than half the retina Vb Vitreous seeding

dd, optic disc diameter.

Table 18.2 The ABC Grouping System.

Group A: small tumors away from the fovea and disc • Tumors < 3 mm in greatest dimension and confi ned to the retina and • Located at least 3 mm from the foveola and 1.5 mm from the optic disc

Group B: all remaining tumors confi ned to the retina • All other tumors confi ned to the retina and not in group A • Subretinal fl uid (without subretinal seeding) < 3 mm from the base of the

tumor

Group C: local subretinal fl uid or vitreous seeding • Subretinal fl uid alone > 3 mm and < 6 mm from the tumor • Vitreous or subretinal seeding > 3 mm from the tumor

Group D: diffuse subretinal fl uid or seeding • Subretinal fl uid > 6 mm from the tumor • Vitreous or subretinal seeding > 3 mm from the tumor

Group E: presence of any one or more of these poor prognosis features • More than two - thirds of the globe fi lled with tumor • Tumor in the anterior segment or anterior to the vitreous • Tumor in or on the ciliary body • Iris neovascularization • Neovascular glaucoma • Opaque media from hemorrhage • Tumor necrosis with aseptic orbital cellulitis • Phthisis bulbi


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detachment [20] . Plate 18.1 illustrates examples of localized and extensive intraocular disease. Another recent classifi cation (Table 18.3 ), as proposed by Chantada [21] relates directly to survival but not to ocular preservation.

Histopathology Only a small number of histological features have been described in retinoblastoma including the Flexner - Wintersteiner and Homer - Wright rosettes [22] . Despite the classically better prog-nosis of the differentiated form of retinoblastoma, histological subtype has not been reproducibly found to relate to prognosis. More importantly, retinoblastoma is one of the few cancers for which there is a defi nitive diagnosis without histopathological review. This is because surgery is not performed for retinoblast-oma due to the high risk of dissemination of the tumor outside of the eye as a result of surgical biopsy. Therefore, it is impossible to classify tumors by histopathological criteria and predict response to therapy. However, the following features can be discussed: • For the largest tumors that require enucleation, the most important features of retinoblastomas that infl uence adjuvant treatment are the extent of optic nerve, choroidal/scleral and anterior segment invasion. • When tumor size and location allow conservative approaches, the most important features that infl uence the success of these treatments are the presence of vitreal and sub - retinal seeds.

Vitreous s eeding Vitreal seeds are small clusters of retinoblastoma cells ranging from tens to hundreds of cell that are free - fl oating in the vitreous. The presence of extensive vitreal seeding in retinoblastoma is an important risk factor for conservative treatment outcome and is refl ected in the retinoblastoma staging systems discussed above. Vitreal seeds are believed to be largely quiescent because they do

Table 18.3 International Classifi cation of Retinoblastoma (staging system).

Stage 0 Conservative treatment subject to pre - surgical ophthalmic classifi cation

Stage I Eye enucleated and resected histologically Stage II Eye enucleated with microscopic residual tumor (extrascleral disease

and/or optic nerve margin and/or subarachnoid space involvement) Stage III Regional extension of retinoblastoma

IIIa Overt orbital disease IIIb Pre - auricular and/or cervical lymph node extension

Stage IV Metastatic disease IVa Hematogenous metastasis

(1) Single lesion (2) Multiple lesion

IVb Central nervous system metastasis (1) Pre - chiasmatic lesion (2) Central nervous system mass (3) Leptomeningeal disease

not tend to get noticeably larger over time as visualized with the ophthalmoscope. As the seeds settle near the retinal vasculature it is believed that they re - enter the cell cycle and begin to form new tumor foci. Tumor cell seeds which grow in the sub - retinal space, when there is a localized or total retinal detachment in the exophytic forms of retinoblastoma, raise the same kind of chal-lenges as vitreous seeds because of slower cell division and the lower access of the drugs.

Origin of r etinoblastoma A recent study has described some of the morphological changes that occur in mouse and human retinoblastomas as they progress and some hypotheses regarding the cell - of - origin for retinoblas-toma have been discussed [23] .

It is believed that most human retinoblastomas are made up of undifferentiated tumor cells that have high mitotic indices and resemble retinal progenitor cells [23 – 25] . There is emerging con-sensus in the fi eld that retinoblastomas arise from retinal pro-genitor cells during development of the retina [26] . There are several lines of evidence to support this hypothesis. • Firstly, tumors clearly initiate in utero and premature babies have been delivered that have retinoblastoma already established [27] . • Secondly, the likely mechanism for mutations in the RB1 gene is through errors in DNA replication that only occurs in retinal progenitor cells during fetal development [28] . • Thirdly, the retinoblastoma cells express markers of retinal pro-genitor cells and have many ultrastructural features of these cells [28] . • Finally, whereas tumors in the very young tend to affect the posterior pole of the retina, those found with follow up or for older children affect mainly the anterior part of the retina, a fi nding that may have a basis in the ontogeny of this structure [1] .

However, even if retinoblastoma arises from a retinal progeni-tor cell, it does not preclude the possibility that the tumors may exhibit some morphological features of more differentiated retinal cells [28] . Thus, it is likely that Verhoeff ’ s original pro-posal in the 1920s that retinoblastoma arose from embryonic retinal cells was correct and that any differentiated features of retinoblastoma cells are the consequence of the competence state of these progenitor cells, and the development of the fi rst knock-out mouse model of retinoblastoma and subsequent studies on retinoblastoma differentiation have provided valuable insight into this process [29, 30] .

Contemporary molecular biological techniques have allowed the characterization of the gross deletions and insertions in the RB1 gene for patients with retinoblastoma. For example, carriers of cytogenetic and submicroscopic whole RB1 gene deletions mostly have unilateral tumors, and almost all patients with gross deletions with one breakpoint in RB1 have bilateral disease [31] . Furthermore, gain of chromosome 1q, 2p, 6p and 13q, and loss of 16q may play a part in retinoblastoma oncogenesis, and these observations are leading to further work to investigate candidate genes with oncogenic and tumor - suppressor function, and also


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The most important features of retinoblastomas that predict metastatic progression are the extent of optic nerve, and invasion of the choroid and sclera, and anterior segment invasion is a less classic histological criteria, but also seems to be of value. At this time little is known about the genetic, molecular, or cellular changes that accompany metastatic retinoblastoma progression, although deletion of 1p and MYCN amplifi cation is found more frequently in intraocular retinoblastoma [16] . Preventing meta-static retinoblastoma is highly important. This may be facilitated both by avoiding inappropriate and prolonged conservative attempts in the case of treatment failure, and by taking into account the histopathological risk factors that determine the need for adjuvant treatment. When metastatic disease occurs, the most successful approach for treating metastatic retinoblastoma cur-rently involves intensifi ed chemotherapy, and this will be dis-cussed in more detail later in this chapter.

General p rinciples of t reatment for r etinoblastoma (Box 18.3 )

Many factors, in relation to tumor size, stage/grouping, age, and visual status need to be taken into account when deciding upon treatment for retinoblastoma, but: • The fi rst goal of retinoblastoma treatment is to preserve life, including performing enucleation when the intraocular tumors are not amenable to conservative approaches, and adjuvant treat-ment when this is indicated according to risk grouping and histology. • If possible, the second aim is to preserve the eye, since enuclea-tion is a radical procedure and should be avoided every time it is possible, even if no vision can be salvaged. • Finally, the third aim is to preserve as much vision as possible, depending on the tumor site and size.

their biological signifi cance in terms of epigenetic phenomenon such as gene promoter methylation [32] .

Trilateral r etinoblastoma The term trilateral retinoblastoma refers to a patient who has bilateral retinoblastoma and a second intracranial malignancy generally several months or years later, a phenomenon that has the following characteristics [33] . • The average time of diagnosis between retinoblastoma and the intracranial malignancy is 35 months. • In most cases, the second tumor is a pinealoblastoma. This is not surprising because many of the genes and pathways that regu-late retinal development also regulate pineal development. • The pinealoblastomas have some histopathological features in common with retinoblastoma but detailed studies of human or mouse pinealoblastoma have not been carried out. • Pinealoblastomas are highly invasive and usually fatal. Prior to the widespread use of systemic chemotherapy for the treatment of retinoblastoma, 3 – 6% of bilateral retinoblastoma patients went on to develop pinealoblastoma.

However, with the use of systemic broad - spectrum chemo-therapy, pinealoblastoma and trilateral retinoblastoma incidence has decreased signifi cantly. This is an important consideration as we move forward with locally delivered chemotherapy. It is likely that trilateral retinoblastoma may increase in bilateral retinoblas-toma patients and will require at least some form of systemic chemotherapy. It is also likely that another factor that may have contributed to the reduction in incidence of pinealoblastoma in bilateral retinoblastoma patients is the reduction in external beam radiotherapy that coincided with the increased use of sys-temic chemotherapy. Long - term follow up in patients who receive ocular chemotherapy without radiotherapy will allow us to determine the relative contribution of these two mechanisms underlying the induction of trilateral retinoblastoma.

Metastatic r etinoblastoma The challenges, in terms of ocular salvage and patient survival for this poor prognosis disease, are well described in the face of metastatic retinoblastoma [16, 34] . Retinoblastoma can metasta-size via several different routes depending on its pattern of growth in the eye, and extra - retinal involvement may increase the risk of extra - ocular disease. • If the tumor invades the choroid, it often invades the extensive choroidal vasculature that may provide a route for distant metastases. • In more advanced cases, the tumor can penetrate directly through the sclera into the peri - orbital tissue (as in localized disease also). • Tumor cells that have invaded the anterior segment often metastasize to the regional lymph nodes. • Optic nerve invasion can also lead to intracranial metastases, especially in cases undergoing enucleation where tumor involve-ment of the resection margin of the optic nerve and involvement of the subarachnoid space is demonstrated.

Box 18.3 Treatment and prognosis of retinoblastoma.

• Overall aim is to preserve vision and avoid enucleation where possible

• Chemoreduction with vincristine, carboplatin, and etoposide mainstay of initial therapy, with a variety of local techniques such as cryotherapy, photocoagulation and brachytherapy successful in international experience

• External beam radiotherapy utilized for more extensive disease

• Improving local delivery with subconjunctival and intra - arterial delivery is being evaluated

• Metastatic disease still carries a very poor prognosis

• Late effects relate to disease, genetic background and to therapy and include poor vision and eye damage (such as cataract) and second cancers


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as attempts to spare the anterior retina have met with a higher risk of anterior retinal relapse, and a dose of 45 Gy external beam radiotherapy appears to be suffi cient for local control for tumors smaller than 10 disc diameters in size [43] or for Reese - Ellsworth Group I – II tumors [44] , and larger or more extensive tumors may require higher doses of irradiation.

Focal t herapies Brachytherapy with ruthenium 106 or iodine125 plaque therapy has a long - established role as a focal therapy for anterior tumors with vitreous seeding [16] , has been evaluated in the context of disease following failure of chemoreduction, and also for the control of extrascleral disease following enucleation. Although not entirely free of late sequelae, this modality may be expected to be associated with fewer late effects in terms of irradiation to surrounding tissues than external beam irradiation [45] .

Chemotherapy and focal techniques such as cryotherapy can allow the successful treatment of retinoblastoma without the requirement for enucleation or external beam radiotherapy [46] . Cryotherapy is employed for small localized tumors without vit-reous seeding and that are not in the vicinity of the macula and are anterior to the equator [47] . This approach can be repeated as necessary when the patient is undergoing an examination under anesthesia. However, for multiply recurrent tumors, other focal treatments should be envisaged (such as laser or brachy-therapy); the key limiting factors for cryotherapy are the size and location of the tumor [16] .

Laser photocoagulation is used to both kill tumor cells and to interrupt the blood supply to the tumor. This approach is only useful for smaller tumors, but allows repetitive application to tumors, including the foveal area of the retina, and alongside chemotherapy, can result in durable control of localized disease [48] . A further approach that is used in combination with carbo-platin chemotherapy is chemo - thermotherapy. This technique relies upon transpupillary thermotherapy shortly after adminis-tration of systemic carboplatin and is most effective for small - or medium - sized tumors that are less than 12 mm in diameter [49] .

Treatment of l ocalized u niocular d isease The general approach to the treatment of localized retinoblast-oma has changed over the past 10 years, with individual centers advocating an approach to therapy that has the dual aims of avoidance of enucleation and preservation of vision [16, 35, 46] . Current treatment strategies generally employ combinations of local therapies and systemic chemotherapy for this purpose, with the general principles of: • Every effort is made to preserve sight, with initial chemoreduc-tion to reduce tumor volume and enable the local therapies described above to consolidate the effects of chemotherapy. • Radiotherapy for advanced disease.

Treatment of e xtensive u niocular d isease Even with contemporary treatment strategies, enucleation is still often required for extensive (e.g. Reese Ellworth Group V

The recently published experience of conservative treatments for intraocular retinoblastoma from the Institute Curie in Paris highlights the contemporary approaches to treatment with chem-oreduction, chemothermotherapy, cryotherapy, brachytherapy, and local therapy that are now achieving satisfactory rates of tumor control and a low need for external beam radiotherapy [35] . The general principles that guide the relative contribution of each of these treatment modalities is described below.

Chemotherapy for r eduction of l ocal d isease and s ystemic c ontrol Chemotherapy with combinations of the agents vincristine, car-boplatin, and etoposide are the most commonly employed in contemporary treatment protocols for retinoblastoma, and in particular the chemoreduction of unilateral retinoblastoma in order to salvage the affected eyes and vision alongside local therapies. Chemotherapy has been found to lower the need for enucleation or external beam radiotherapy in institutional series, but this effect is greater for those patients with more localized disease with ocular salvage rates of 70 – 80% with localized com-pared with only 30 – 50% for more extensive intraocular disease [35 – 37] .

Experience is also being reported for the use of subcon-junctival carboplatin [16, 38] as a means for ocular salvage. In the setting of systemic administration, over 90% of tumors will respond to single agent carboplatin [39] , and subconjunc-tival administration affords high vitreal concentrations of this agent than that achieved by systemic administration [38] . The utility of this subconjunctival therapy is currently being evalu-ated in the setting of therapy with systemic chemotherapy and local treatments for patients with vitreous seeding. In addition increased local delivery of chemotherapy with melphalan by the intra - arterial route is also being evaluated in an effort to reduce enucleation rates [40] .

Surgery For patients with advanced unilateral disease, if there is no chance of preserving vision in an eye with retinoblastoma, enucleation is performed, provided that there is no rupture of the ocular globe and the optic nerve is of suffi cient length. Also, surgical proce-dures must take into account the requirement for careful patho-logical sampling for genetic analyses. Following enucleation, prosthetic implants, such as polymer - coated hydroxyapatite globes, are generally employed for cosmetic purposes, and also to promote bony growth of the residual socket [41] .

External b eam i rradiation Although the mainstay of conservative therapy for localized retin-oblastoma before the advent of chemoreduction and other con-temporary local treatments, external beam radiotherapy is now mainly employed for the therapy of advanced disease, especially when there is diffuse vitreous or subretinal seeding after failure of other methods of treatment and when preservation of vision is still a priority [16, 42] . Radiation is given to the entire retina


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• The same factors plus vitreous seeding are risk factors for the eventual use of external beam irradiation [35] ; female gender and greater number of chemoreduction treatments have also been reported [53] . • The risk factors for eventual enucleation (20 – 30% of cases) include advanced disease and sub - retinal seeds [35] , tumor thick-ness, and tumor recurrence and older age at presentation have also been reported [53] . • The risk factors for extraocular relapse follow enucleation after failure of chemoreduction for retinoblastoma include scleral invasion and bilateral enucleation [54] .

Although chemoreduction with agents such as vincristine, etoposide and carboplatin is an established practice for the treatment of retinoblastoma, there is a paucity of studies to relate patient outcomes with known cellular and molecular determi-nants of chemosensitivity for retinoblastoma. However, although in vitro drug sensitivity, as measured by means of the MTT assay, does not relate to tumor characteristics such as invasion and seeding, the relative chemosensitivity of undifferentiated tumors and lack of effi cacy of cytosine arabinoside has been identifi ed [55] . The effi cacy of the chemotherapy agents vincristine, etopo-side and anthracyclines could be impaired by the multi - drug resistance - associated protein MDR1, and although MDR1 posi-tivity is more prevalent post - chemotherapy for patients with retinoblastoma, which may indicate MDR1 as a mechanism for resistance [56] , MDR1 has not been found to relate to response [57] or histological invasiveness [58] . Therefore, the 60% response rate of retinoblastoma tumors to single agent idarubicin results may not relate to cellular factors such as MDR1, but to down-stream determinants of cellular engagement of apoptosis [59] .

Strategies for f ollow u p and o verview of i mportant l ate e ffects

EUA is routinely performed at different time intervals after treat-ment is complete. The timing of the examinations depends on the age and particular presentation at diagnosis, the genetic coun-seling and the response to therapy. For children at risk of devel-oping retinoblastoma, screening is typically carried out every month in the very young, and then at gradually increasing inter-vals of time thereafter, typically throughout childhood and increasingly, early adult life. However, it is recognized that insti-tutional surveillance programs carry with them a signifi cant impact on the patient, family, and hospital resources [60] , and the late - effects burden for children with retinoblastoma is well characterized as follows: • Chemoreduction and local control strategies result in over 80% vision salvage rates for Reese - Ellsworth Groups I – IV tumors, but only a 20% success for group V retinoblastoma [61] . The quality of preserved vision remains a function of tumor location and absence of subretinal fl uid, with tumor margins at least 3 mm from the foveola predictive of eventual visual acuities of 20/40 or better [62] . Indeed, visual acuity for almost one - half of patients

tumors), but the role of adjuvant chemotherapy with agents such as carboplatin - etoposide or vincristine - cyclophosphamide - doxo-rubicin is uncertain, although benefi t is clearer for patients with stage II disease. External beam or interstitial radiotherapy can be employed in the case of microscopically - incomplete resection of tumor [16, 35] .

Treatment of b ilateral r etinoblastoma

For children with bilateral retinoblastoma, conservative appro-aches aimed to preserve vision in at least one eye have been developed. The relative use of conservative measures depends on the number of tumors, their situation in relation to the optic disc and macula, the degree of retinal detachment, the extent of inva-sion of the vitreous and pre - retinal space, age at diagnosis, and family history of retinoblastoma [16] .

Factors that r elate to p rognosis

The survival from a diagnosis of retinoblastoma in Western industrialized countries is excellent with more than 95% of chil-dren surviving their disease [50, 51] . Therefore, outcome meas-ures for children with retinoblastoma are more related to considerations such as preservation of vision, ocular salvage, and the avoidance of enucleation and external beam irradiation than survival. The relative contribution of the various treatment modalities to eventual success or failure is obviously complex due to the different modalities of therapy employed, but certain inferences may be drawn from the literature with respect to prognosis: • Treatments should be given in specialized onco - ophthalmolog-ical centres. • For retinoblastoma treated with primary chemotherapy alone, the 72% of patients with disease that included the features of location in the macula, measuring greater than 2 mm in diameter or patient age older than 2 months was more likely not to require other therapies for control [52] . • For retinoblastoma treated with radiotherapy alone, the likeli-hood of control relates to the extent of disease as defi ned by the Reece - Ellsworth Group classifi cation, with 79% of Group I – II eyes and 20% of Group III – V eyes being controlled by 45 Gy of external beam irradiation. A similar effect was also found for tumor size, with lesions larger than 15 mm less likely to be con-trolled than smaller lesions [44] .

Contemporary institutional studies also highlight the factors that relate to ocular relapse, the need for external beam irradia-tion, and enucleation following treatment with chemoreduction and local therapies. • The risk factors for ocular relapse include Reese - Ellsworth Group V and ABC Classifi cation Group D eyes and the presence of subretinal seeds [35] .


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Novel t herapeutic a pproaches

In this section, we will discuss the role of experimental therapeu-tic approaches in helping to defi ne novel combinations of chemo-therapy agents for the treatment of retinoblastoma. For example, preclinical studies have revealed that the combination of topote-can and carboplatin is more effective than carboplatin and vinc-ristine, or carboplatin, vincristine and etoposide in the xenograft setting for retinoblastoma [72] , and this information may help promote the study of topotecan now that phase I evaluation of this agent in children is complete [73] . Other novel approaches to therapy has seen the encouraging response rates for refractory retinoblastoma found with the adenovirus vector/herpes simplex thymidine kinase gene activation of systemic acyclovir [74] , and strategies to inhibit the VEGF - mediated growth and angiogenesis of retinoblastoma are being evaluated in the pre - clinical setting [75] . These strategies may eventually compliment therapeutic approaches based on the knowledge of cellular - adhesion mole-cules in the pathogenesis of retinoblastoma [76] . However, study of the functions of the retinoblastoma gene pathway interactions with p53 are the subject of preclinical developments that may soon translate into clinical practice.

Inactivation of the p 53 p athway in r etinoblastoma Tumorigenesis involves sequential genetic lesions in pathways that regulate cell proliferation, cell survival, and other biological processes [11] . It has been proposed that both the p16 Ink4a - CycD/Cdk4 - pRb and Arf - MDM2/MDMX - p53 pathways must be inac-tivated during cancer progression [77] . The primary role of the Rb pathway is to regulate cell division [77] , and that of the p53 pathway is to regulate responses to cellular insults such as DNA damage or oncogenic stress [78] . The Rb and p53 pathways may be inactivated by mutations in the RB1 and p53 tumor suppres-sor genes themselves or through genetic alterations of other genes in the pathway. For example, some cancers have MDM2 gene amplifi cations that functionally suppress the p53 pathway by reducing the steady - state levels of the p53 protein [79] . In relation to retinoblastoma, the following observations are emphasized: • The fi rst evidence that the p53 pathway may be important for retinoblastoma tumor progression came from studies on mouse models of retinoblastoma. In mouse models of retinoblastoma, tumor development is greatly enhanced when p53 is inactivated [80] ; mice develop 100% penetrant bilateral retinoblastoma that invades the anterior chamber and surrounding tissue [81] . To determine if any previously overlooked mutations within the p53 pathway effectively block p53 activity and lead to a growth advan-tage for retinoblastoma cells, the Arf - MDM2/MDMX - p53 onco-genic stress response pathway was analyzed in detail. • MDMX is amplifi ed in 65% of human retinoblastomas, and MDM2 is amplifi ed in 10% of human retinoblastomas [82] . MDMX and MDM2 are similar in structure, but they inhibit p53 by distinct mechanisms [83] . MDM2 is a ubiquitin ligase that

with orbital preservation is near - normal, i.e. better than or equal to 6/12 [63] . • Complications of external beam radiotherapy include cataract and retinopathy, which can affect one - quarter of patients, and orbital deformities, although the later complication is less common [64] . Rarer complications of radiotherapy include retinal tears and detachment, sub - retinal fi brosis, vitreous trac-tion bands, and pre - retinal fi brosis [65] . However, the evolution of techniques continues to allow a more accurate delivery of the 45 – 50 Gy treatment doses for retinoblastoma, and this may result in fewer ocular late effects and also reduce the risk of secondary cancers [66] .

Genetic c ounseling and s creening

One of the most important outcomes of the cloning of the RB1 tumor suppressor gene was the ability to provide retinoblastoma survivors with genetic counseling. Point mutations and small deletions represent the vast majority of RB1 germline mutations. Chromosomal aberrations have also been reported and these can be detected by cytogenetic analysis and/or molecular analyses [67 – 69] . The following features of genetic counseling for retino-blastoma include: • It is essential to perform RB1 gene analysis for every retinoblas-toma patient because the absence of multiple bilateral tumors does not exclude the possibility of germline RB1 mutations. • The hereditary type of retinoblastoma shows an autosomal dominant pattern of inheritance with at least 90% penetrance on average. Long - term follow up is essential to educate retinoblast-oma survivors of the risks of passing retinoblastoma susceptibility on to their children. • The risk of developing retinoblastoma to survivors of inherited retinoblastoma is approximately 45% and it is 2.5% for survivors of unilateral retinoblastoma. • The risk for passing on retinoblastoma susceptibility for unilat-eral retinoblastoma survivors (2.5%) is higher than in the general population (0.003%) because of the possibility of a low - pene-trance RB1 mutation or an individual with mosaic inactivation of RB1 . • Similarly, the risk of developing retinoblastoma for siblings of retinoblastoma patients with bilateral disease and a family history is 45%, and for the siblings of retinoblastoma patients with no family history and unilateral disease is 1%.

In the context of familial risk for retinoblastoma, surveillance investigations allow the detection of disease before the clinical sign of leucocoria is present, and this is associated with a higher ocular salvage rate [70] . However, it is also recognized that in patients in which retinoblastoma is detected early, at the time when tumors are smaller, actually do worse than in patients who are diagnosed later at a more advanced stage [71] , a fi nding that may result in the posterior retinal location, often involving the macula, in very young children. However, screening is of benefi t in terms of reduction of treatment burden and risk of enucleation.


314

Targeted c hemotherapy for r etinoblastoma Currently, no small - molecule inhibitors that bind MDMX and block its ability to bind p53 are available. It has been previously shown that the p53 - binding domains of MDM2 and MDMX are highly conserved [87, 88] . Therefore, an inhibitor of the MDM2 - p53 interaction may also inhibit the MDMX - p53 interaction [89] . A number of small molecule inhibitors of the MDM2 - p53 interac-tion have been constructed [90] . However, the most widely utilized small molecule inhibitor of the MDM2 - p53 interaction is nutlin - 3. • Nutlin - 3 interacts with the p53 - binding domain of MDM2, which is a hydrophobic pocket. The effects of nutlin - 3A on retin-oblastoma cells have been recently explored [90] . • Following treatment with nutlin - 3A, a p53 response is induced inWeri1 and Y79 human retinoblastoma cell lines as indicated by an increase in p53 protein levels, and an increase in the protein levels of its downstream targets, MDM2 and p21. The authors also observed an induction of apoptosis following nutlin - 3A treatment. • In addition, the sensitivity of retinoblastoma cells to nutlin - 3A was shown to be p53 - dependent, because Y79 cells expressing a siRNA to p53 were less sensitive to nutlin - 3A [90] .

Furthermore, binding studies have shown that racemic nutlin - 3 binds MDM2 and competes with fl uorescently labeled p53 with an inhibition constant (K i ) of 0.7 μ M; racemic nutlin - 3 binds MDMX and competes with a K i of 28 μ M [82] . Racemic nutlin - 3 also blocks MDMX - p53 binding in C33A cells, a cell line that expresses a mutant form of p53 (Cys273) [82] . Co - immunoprecipitation assays demonstrated that in cells treated with 10 μ M racemic nutlin - 3, the binding of MDM2 and MDMX to p53 was reduced. In Mdm2 - defi cient mouse embryonic fi brob-lasts (MEFs), we demonstrated that racemic nutlin - 3 can block MDMX in the absence of MDM2. MEFs expressing MDMX only were sensitive to racemic nutlin - 3, as demonstrated by reduced cell viability [82] . These studies demonstrate that racemic nutlin - 3 can inhibit p53 binding by both MDM2 and MDMX.

Local d rug d elivery for r etinoblastoma t reatment Systemic administration of nutlin - 3 to treat tumors with MDMX amplifi cation is not feasible due to its pharmacokinetics and tox-icity in multiple organs; thus, there is a great need for specifi c, highly effi cient MDMX antagonists. Retinoblastoma is ideal for local delivery of a targeted chemotherapeutic agent such as nutlin - 3, because the eye is readily accessible for drug delivery. Subconjunctival administration of chemotherapies for retino-blastoma can also avoid the side effects of systemic administra-tion and achieve higher intraocular concentrations.

Abramson et al. conducted a clinical study in which they injected as much as 2 ml carboplatin (10 mg/ml) subconjuncti-vally to treat intraocular retinoblastoma [91] . Some patients were also given cryotherapy to increase drug delivery. The mean number of injections was 2.8 per eye. Following treatment, regression of the solid retinal tumors and vitreous seeds was evaluated. Of the fi ve eyes with solid retinal tumors that were treated, two responded, and three remained stable. Of the fi ve

blocks p53 activity by binding to its transactivation domain and ubiquitinates p53 for degradation [84] . MDMX does not have ubiquitin ligase activity but effectively inhibits p53 activity by binding to its transactivation domain [84] . In retinoblastomas with MDMX gene amplifi cations, the levels of MDMX mRNA and protein were also increased [82] . This correlated with a decrease in p53 and p21 proteins, as previously shown in breast tumors with MDMX amplifi cations [84] . • These data suggest that amplifi cation of MDMX and, to a lesser extent, MDM2 suppresses the p53 response to increased p14 ARF levels as a direct result of activation of the oncogenic stress pathway following RB1 inactivation in retinoblastoma. Genetic analyses of human tumors have shown that disruption of one component of the p53 pathway relieves the selective pressure to inactivate other components of the same pathway [85] . For example, p53 mutations and MDM2 amplifi cations tend to be mutually exclusive [86] . Indeed, subsequent studies confi rmed that p53 and many of the direct downstream effectors of the p53 pathway are intact in retinoblastoma cell lines [82] . By modulat-ing the p53 and MDMX levels in retinoblastoma cells maintained in culture, predictable perturbation of p53 - mediated cell death and cell cycle exit occurred [82] .

On the basis of these data, it has been hypothesized that RB1 - defi cient retinoblasts sustain an MDMX/MDM2 genetic amplifi -cation that allows them to suppress p53 - mediated cell death and clonally expand to form retinoblastoma. To recapitulate this process in vivo , these changes have been engineered in a mouse model and in explants of human fetal retinae grown in cultures with the following fi ndings. • In the mouse model, the retinal cells that lacked Rb and p107 but expressed MDMX had greater proliferation and survival than did cells lacking Rb and p107 [96] . Importantly, invasive and aggressive retinoblastoma similar to that observed in Chx10 - Cre;Rb Lox/ – ;p107 – / – ;p53 Lox/ – mice now developed in the Rb Lox/Lox ;p107 – / – pups with ectopic expression of MDMX [82] . • In human fetal retinae, it was demonstrated that MDMX pro-motes human retinoblastoma by electroporating primary human fetal retinae (FW 14 - 15) with RB1 siRNA, MDMX cDNA, and a green fl uorescent protein (GFP) reporter gene. After 10 days in culture, these cells failed to differentiate, and the immature cells organized into rosettes similar to those seen in retinoblastoma [82] . Cells expressing Rb1 siRNA alone induced p14 ARF and initiated p53 - mediated apoptosis. Increased MDMX expression blocked cell death and increased proliferation by clonal expansion. • The specifi city of the MDMX effect on p53 was demonstrated by using an MDMX allele that has a single amino acid substitu-tion (G57A) that blocks its ability to bind p53 [82, 84] . These data challenge the long - held belief that retinoblastoma is the exception to the principle that the Rb and p53 pathways must be inactivated for cancer progression, and demonstrate that inac-tivation of the p53 pathway is likely to be the second genetic perturbation that occurs in human retinoblastomas after the loss of RB1 .


315

in the 1970s to the present day success rates of over 95% [50, 51] . However, the treatment of metastatic disease and the preserva-tion of vision and avoidance of enucleation for patients with more advanced or bilateral disease are continued challenges for the expert teams that treat children with retinoblastoma. This chapter has highlighted the novel approaches to therapy that are currently being developed, both in terms of local delivery of con-ventional chemotherapy agents and new therapies that target p53 interactions that appear to be important in the pathogenesis of retinoblastoma. Determining the feasibility of novel drugs will require systematic evaluation of ideal delivery techniques to max-imize drug delivery allowing for effi cacy, with every attempt made to minimize adverse effects and complications. Ease of the appli-cation will be essential if these techniques are to eventually be translated to widespread use outside of specialized academic centers. This is a promising fi eld for the treatment of eye diseases in general and as more therapeutics becomes available, drug delivery will likely become the major hurdle to pass as the fi eld progresses.

Indeed, an international consensus of the current state of retinoblastoma therapy world - wide recognizes that the excellent survival fi gures of the industrialized West refl ect the complexity of multi - disciplinary care that is achievable, a situation that is seldom achievable in the developing world. The ‘ One World, One Vision ’ theme is leading to practical partnerships between aca-demic treatment centers in Europe and North America and the developing countries, lay agencies and interest groups with the aim of bringing the success story that is the treatment of retino-blastoma to children around the world [97] .

Acknowledgment

Many thanks to Dr Helen Jenkinson, Paediatric Oncologist at The Birmingham Children ’ s Hospital, UK, for the provision of the images used for Plate 18.1 , and for her advice and comments on the text of the chapter.

eyes with vitreous disease, three responded. In the fi rst eye, all vitreal seeds disappeared; in the second eye, disease regressed more than 50%; and in the third eye, all non - calcifi ed seeds disap-peared. The remaining two eyes with vitreal disease remained stable. The side effects were minimal; only one eye in one patient suffered a severe ocular side effect. The authors had previously addressed the question of systemic exposure to carboplatin fol-lowing periocular injection in a primate model: the 2 hour mean blood level after periocular injection of carboplatin was 2.9% that of the mean peak level of carboplatin after intravenous injection. Therefore, systemic exposure to carboplatin was greatly reduced by local administration of the drug [92] .

Testing of subconjunctival racemic nutlin - 3 in the setting of a preclinical animal models, an orthotopic xenograft model for retinoblastoma, has been reported with the following fi ndings [93] . • Racemic nutlin - 3 alone signifi cantly reduced tumor burden, as measured by luciferase activity [82] . • The combination of racemic nutlin - 3 with the topoisomerase inhibitor topotecan (30 – 40 nM) induces a p53 response in retino-blastoma cells. Racemic nutlin - 3 also induces a p53 response in retinoblastoma by inhibiting MDMX and MDM2 from binding to p53 [82] . • The combination of the two drugs resulted in a 20 - fold syner-gistic killing of retinoblastoma cells in vitro . Subconjunctival administration of topotecan and racemic nutlin - 3 in our ortho-topic xenograft model resulted in an 82 - fold reduction in tumor burden with no ocular or systemic side effects [82] .

Previous studies for subconjunctival chemotherapeutic treat-ment for retinoblastoma have involved administration of 25 ml of drug in mice and up to 2 ml of drug in children [94 – 96] . The stock solution of racemic nutlin - 3 used for these studies was 170 mM and showed no ocular toxicity. Thus, it is feasible to achieve the intraocular concentration of nutlin - 3 needed to inac-tivate MDM2 and MDMX; this approach should prove effective in 75% of patients with retinoblastoma with either MDM2 or MDMX gains or amplifi cations. Moreover, by combining MDM2/MDMX antagonists with drugs that induce a p53 response through DNA damage (i.e. topotecan), further enhancement of their antitumor effects may be gained. Retinoblastoma is not only a good model for studying the suppression of p53 - mediated cell death by MDMX amplifi cation, but it is also an ideal system in which to study local delivery of chemotherapy targeted to the Arf - MDM2/MDMX - p53 pathway. In addition, retinoblastoma would be easier to treat in developing countries if we could use local delivery of targeted therapy, because this approach would avoid the cost associated with managing the side effects of sys-temic broad - spectrum chemotherapy.

Summary and f uture d irections (Box 18.4 )

In industrialized Western countries, the survival following a diag-nosis of retinoblastoma has improved from approximately 85%

Box 18.4 Future directions.

• p53 inactivation important in the pathogenesis of retinoblastoma

• Some of the novel therapeutic approaches include strategies based on inhibition of MDMX/MDM2 and p53 interactions

• Preclinical studies also exploring VEGF inhibition and adenoviral/herpes simplex thymidine kinase gene activation of systemic therapies such as acyclovir

• International collaborations to improve therapy of metastatic disease and vision/ocular salvage in localized disease

• Improving the outcomes for retinoblastoma in developing countries


316

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