· Web viewRetinoblastoma. Helen Dimaras1, Tim W. Corson2, David Cobrinik3, Abby White4, Junyang Zhao5, Francis L. Munier6, David H. Abramson7, Carol L. Shields8, Guillermo L ...

Retinoblastoma

Helen Dimaras1, Tim W. Corson2, David Cobrinik3, Abby White4, Junyang Zhao5, Francis L. Munier6, David H.

Abramson7, Carol L. Shields8, Guillermo L. Chantada9, Festus Njuguna10 and Brenda L. Gallie11

1| Department of Ophthalmology & Vision Sciences, The Hospital for Sick Children& University of Toronto, Toronto, Canada2| Eugene and Marilyn Glick Eye Institute, Departments of Ophthalmology, Biochemistry and Molecular Biology, and Pharmacology and Toxicology, and Simon Cancer Center,Indiana University, Indianapolis, USA3| The Vision Center and The Saban Research Institute Children's Hospital Los Angeles, Los Angeles, CA USA; and Department of Ophthalmology, Department of Biochemistry and Molecular Biology, The USC Eye Institute, and Norris Comprehensive Cancer Center, Keck School of Medicine of the University of Southern California, Los Angeles, CA USA4| Daisy's Eye Cancer Fund, Oxford, UK5| Department of Ophthalmology, Beijing Children’s Hospital, Capital Medial University, Beijing, China6| Department of Ophthalmology, Jules-Gonin Eye Hospital, Lausanne, Switzerland7| Department of Ophthalmlogy, Memorial Sloan Kettering Cancer Center, New York, USA8| Ocular Oncology Service, Wills Eye Hospital, Philadelphia, USA9| Hospital JP Garrahan, Buenos Aires, Argentina10| Department of Pediatric Oncology, Moi Teaching & Referral Hospital, Eldoret, Kenya11| Department of Ophthalmology & Vision Sciences, The Hospital for Sick Children& University of Toronto, 555 University Ave, Toronto, Ontario M5G1X8, Canada

Abstract 174/200

The genetic basis of human malignancy was determined through the study of retinoblastoma, a rare cancer of

infant retina. Tumours form when both RB1 alleles mutate in a susceptible retinal cell, likely a cone

photoreceptor precursor. The tumour suppressor functions of the retinoblastoma protein, pRB, relate to cell

division and genomic stability, but the key biochemical and molecular basis of tissue specificity remain

unknown. Retinoblastoma is diagnosed in 8,000 children each year worldwide, yet patient survival is >95% in

high-income countries, but <30% globally, depending on the socio-economical context. Stakeholder

collaboration is improving outcomes by increasing awareness for earlier diagnosis, sharing expertise, and

developing guidelines. Intra-arterial and intra-vitreal chemotherapy have emerged as a promising way to salvage

eyes. Ongoing international collaboration will replace the multiple different classifications of eye involvement

with standardized definitions that will facilitate assessment of eligibility, efficacy and safety of treatments.

Heritable retinoblastoma survivors are at risk for second cancers; defining the molecular basis of RB1 retinal

specificity may explain tissue specificity of second cancers, opening the path to cancer prevention.

Keywords

retinoblastoma, pediatric oncology, pediatric ophthalmology, tumour suppressor gene, RB1 gene, MYCN

oncogene, Systemic chemotherapy, Intra-arterial chemotherapy, Intra-vitreal chemotherapy, Cone

photoreceptor, Cancer therapy, Global health

Competing interests

There is NO competing interest.

[H1] Introduction(BLG) 291/300

Retinoblastoma is a rare cancer initiated by biallelic mutation of the retinoblastoma gene (RB1) in a single

susceptible developing retinal cell. Inheritance of one mutant RB1 allele strongly predisposes to retinoblastoma

tumours that form when the second RB1 allele is mutated.1 Although RB1 loss initiates cancer in specific

susceptible tissues, it is lost with progression in almost all human cancers.2 The principals discovered by study

of retinoblastoma led to the recognition that altered genes broadly underlie cancer initiation and progression.

Retinoblastoma starts when the second RB1 allele is damaged in susceptible retinal cell (RB1-/-) that undergoes

limited proliferation to form a non-malignant retinoma (Figure 1).3 An intra-retinal tumour develops after

genomic changes lead to uncontrolled proliferation — most commonly, the white tumour is visible through the

pupil (leukocoria) (Figures 3, 9) or blocks vision.4 When noticed early, prompt treatment usually cures. Delayed

diagnosis can lead to incurable invasion of the optic nerve and brain, or metastasis, sometimes cured by

extensive intervention.

Rigorous clinical trials in retinoblastoma are difficult for multiple reasons: too few patients in high-income

countries; often complex presentation (two eyes with differing severity); too few patients to interest the

pharmaceutical industry; multidisciplinary collaboration required; and high societal value on eyes and vision

imposes considerations beyond curing the cancer. New technologies showing dramatic primary treatment

response have been quickly embraced, without rigorous clinical trials.

The Internet has opened many avenues: parents diagnose retinoblastoma themselves; colleagues share patients

around the globe; centres of excellence are mapped (Figure 2); and a common database for all children no

matter where they live is within reach. These developments are set to empower a learning health system that

build and evidence base for care. This retinoblastoma Primer introduces unprecedented new science, ideas,

therapies and global collaboration.

[H1] Epidemiology (HD,GC, JZ, FN) 769/500

[H2] Global patients, resources and outcomes

The expected number of patients with retinoblastoma annually per country can be calculated by multiplying the

global retinoblastoma incidence (1 in 16,000-18,000 live births) by forecast births (Table 2).5-7 This predicts

approximately 8,000 new cases each year.

Of all affected children, 11% reside in high-income, 69% in middle-income and 20% in low-income countries.

Although the prevalence is higher in middle- to low-income countries, most retinoblastoma treatment centres are

in middle- and high-income countries, creating a gap in healthcare access (Figure 2). Consistent with income

being a surrogate for non-economic measures of standard of living, retinoblastoma in low-income countries is

associated with low patient survival (~30%8, 9) compared with high-income countries (>95%10), but

comprehensive nation-wide data is lacking.

Poor outcome correlates with late presentation, difficulty accessing retinoblastoma-specific health care, and

socio-economic issues leading to poor compliance, including family refusal to remove the affected eye and

abandonment of therapy.11-13 Without timely diagnosis and appropriate treatment, difficult-to-cure metastatic

disease may occur. Fortunately, with early diagnosis, many eyes can be safely treated to support a lifetime of

good vision, pointing to key elements for global focus: awareness, collaboration, and affordable expert care.

[H2] Solutions for global retinoblastoma

A number of initiatives address the inequality in retinoblastoma treatment between developing and developed

worlds. In 2009, the Canadian National Retinoblastoma Strategy (NRbS) published the first-ever retinoblastoma

clinical practice guidelines,10 adapted by the Kenyan National Retinoblastoma Strategy (KNRbS) and published

in 2014 in partnership with the Kenyan Department of Health.14 The KNRbS achieved standardization of

pathology processing and reporting to support treatment decisions and discussion of prognosis with families.

Adoption of upfront (first-line) enucleation with implants and immediate prosthetic eyes (sourced from India),

parent to parent interactions to allay uninformed fears, and standardization of information provided to parents

has reduced the rate of non-compliance with treatment.14, 15 In 2013, the Paediatric Oncology in Developing

Countries Committee of the SIOP (International Society of Paediatric Oncology) published a consensus

guideline for the management of retinoblastoma in countries with limited resources16 with clear ideas that can

shape resource development. The Mexican National Strategy17 and the Brazilian SOBOPE (Brazilian Society of

Pediatric Oncology)18 guidelines are also applicable at a national level with governmental support for treatment.

Peer-to-peer collaborations and twinning programmes have built a framework for knowledge and expertise

exchange, filling gaps in specialized training, and source donations of equipment and resources with the ultimate

aim of sustainable local capacity.19 Retinoblastoma-specific twinning programmes include partnerships between

St. Jude’s Children’s Research Hospital (USA) and the Middle-East,20 Central America21 and Mexico,17and

between the Institut Curie and a centre in Bamako, Mali.22 The Central American Association of Pediatric

Hematology Oncology (AHOPCA) created a cooperative group and implemented multicentre protocols for

retinoblastoma treatment,21, 23, 24 a major achievement, not yet paralleled in developed countries. The AHOPCA

funding is now 90% local. Twinning programmes benefit from strong participation of both non-governmental

and governmental organizations. However, where government may prove volatile and unpredictable, sustainable

local capacity is a challenge. Less formal cooperation between developed and less developed countries can also

result in highly efficient programmes.25, 26

A successful national strategy has been developed in China. About 1,100 newly diagnosed cases are forecast

annually, scattered over 32 provinces, imposing high travel costs. Before 2005, enucleation was the only

available treatment for most children. For better treatment options and follow-up, centres classified by expertise

and resources were established in 28 hospitals covering 25 provinces (over 90% of the population)27. The

improved efficiency and collaboration from 2006 to 2014 has led to standardized classification and treatment of

2,097 newly diagnosed patients with retinoblastoma on common protocols with gains in survival (unpublished

data, JZ). In Argentina, a single centre with high expertise coordinates affiliated retinoblastoma clinics. This

collaboration between governmental hospitals, local and international NGO’s with prospective protocols has

significantly improved survival in two decades.28 In addition, translational research and population-based

incidence and survival studies are reported.29, 30

In a rapidly changing world, Internet based strategies are opening up global collaboration. The One

Retinoblastoma World map (Figure 2) leads families to the nearest centre with known expertise. The

retinoblastoma-specific point-of-care database (eCancerCareRB, eCCRB) summarizes the medical record for

retinoblastoma for each child (Figures 3, 4 and 9). eCCRB on the Internet is freely accessible to every

retinoblastoma centre with local ethics and privacy approvals. The Disease-specific, electronic Patient

Illustrated Clinical Timeline (DePICT) is language-independent, well understood by parents, and supports fully

informed care choices.31 With guardian consent, fully identified data is accessible to those in the circle of care of

each child. Ultimately, the de-identified data will be fuel for a learning health system.

[H1] Mechanisms/pathophysiology (TWC, DC, BLG) 1069/1500

The search for the genetic basis of retinoblastoma provided the two-hit model of tumor suppressor gene

inactivation. In 1971, Knudson discovered that the age of diagnosis of retinoblastoma is consistent with one

rate-limiting event in bilaterally affected patients (who have heritable disease), and two events in unilaterally

affected patients with no family history (usually non-heritable).32 Others proposed that heritable retinoblastomas

result from a germline mutation (‘first hit’) and an acquired somatic mutation (‘second hit’), and non-heritable

retinoblastomas arise when two somatic mutations in the same transformation suppressor gene in a susceptible

cell (Figure 4).33 Chromosomal deletions in some patients pointed to a chromosome 13q14 locus. Loss of

heterozygosity of 13q14 polymorphic markers in 70% of retinoblastoma tumours34 implied that the second hit

involved the same locus. The breakthrough came when one retinoblastoma was missing the sequence of a 13q

DNA clone,35 that turned out to be a conserved, exonic sequence of RB1.35-38

RB1 is a large (190 kb) gene with 27 exons, encoding a 4.7 kb mRNA that translates into a 928 amino acid

protein, pRB. Many modifications impair pRB function, including point mutations, promoter methylation, and

small and large deletions.39 The A/B “pocket” region40 harbours most missense mutations.

The pRB functions that normally suppress retinoblastoma within the retina remain unknown. pRB is best known

as a cell cycle regulator that binds to E2F transcription factors to repress cell proliferation-related genes.

Hyperphosphorylation of pRB by cyclin-dependent kinases in response to mitogenic signals normally relieves

repression and promotes the G1 to S phase transition. pRB loss relieves this suppression in the absence of

mitogenic signals to enable tumourigenesis. It is tempting to speculate that pRB is primarily needed to suppress

E2F.40, 41 Several “low penetrance” RB1 mutations encode proteins with minimal ability to bind E2F, that that

result in fewer tumours than RB1 null alleles.42, 43 Such defective E2F-binding alleles may function to block

retinoblastoma development through an E2F-independent mechanism. pRB also up-regulates p27, implicated in

cell differentiation, apoptosis, and genomic integrity.40 Increased pRB expression is associated with decreasing

p27 during cone precursor maturation.41 pRB N-terminus functions may also be important.44, 45

Biallelic RB1 inactivation is necessary to initiate most retinoblastomas, but is not sufficient; the benign retinal

lesion, retinoma, has lost both RB1-/- alleles (Figure 4).3 Further genetic or epigenetic changes are likely needed

for malignant transformation.46 Comparative genomic hybridization studies identified several candidate

retinoblastoma oncogenes (mitotic kinesin KIF14 and p53 regulator MDM4 (1q32), transcription factors E2F3

and DEK (6p22), and the onco-miR clusters miR-106b~25 (7q22.1) and miR-17~92 (13q31)) and the cadherin-

11 (CDH11) tumour suppressor gene (16q22 loss). Whole-genome sequencing identified inactivating mutations

in the transcriptional corepressor BCOR.47 Epigenetic alterations might drive retinoblastoma formation by

inducing H3K4me3 and H3K9/14ac marks and expression of the SYK oncogene.47 Other genes and microRNAs

also show altered expression in retinoblastoma compared to normal retina.46 Gene expression profiles may

segregate RB1-/-retinoblastomas either into two subtypes or into a spectrum of phenotypes correlating with

histologic and cytogenetic aberrations.48, 49

Nearly all retinoblastomas have mutation of both RB1 alleles, but 1.4% of unilateral tumours show evidence of

RB1 mutation, but have high-level amplification of the oncogene MYCN (MYCNA).50 These RB1+/+MYCNA

tumours are diagnosed in much younger children than unilateral RB1-/- tumours with distinct histology,

reflecting a unique subtype. Another 1.5% of unilateral non-familial unexplained retinoblastomas have

apparently normal both RB1and MYCN genes.50

[H2] The retinoblastoma cell of origin

Since pRB is expressed in most if not all cells, the retina’s unique sensitivity to pRB loss is perplexing. Diverse

retinal cell type markers and RNAs suggest a pleuripotent cell of origin, aberrant expression of oncogenic

transformation or normal intra-tumour RB1+/+ cells.48, 51 Mouse models have not clarified the cell-of-origin, since

they require loss of Rb1 plus p107, p130, or p27,52 express different retinal markers, and may not extrapolate to

humans.53

The topographic distribution of emerging retinoblastomas mimics the horizontal visual streak characteristic of

red and green cones, evidence for a cone precursor to be the cell of orgin.43 RB1-/- retinoblastomas show

consistently express cone photoreceptor but not other retinal cell type-specific proteins; and maturing cone

precursors have unusually high expression of oncoproteins (MDM2 and N-myc) that could collaborate with RB1

loss.51 Moreover, experimental depletion of RB1 induced cone precursor cell proliferation in vitro and in vivo

experimental tumours typical of differentiated retinoblastomas.54 Proliferation depended upon high levels of N-

Myc and MDM2, cone-specific transcription factors RXR and TR2, and down-regulation of p27 associated

with cone precursor maturation.41, 54 pRB may counter an oncogenic programme associated with cone precursor

maturation.

Notably, small tumours detected via optical coherence tomography (OCT) appear to be centred in the inner

nuclear layer of the retina, not the outer nuclear layer where mature cones reside.55 However, they also extend

into the outer nuclear layer (Figure 5c). Perhaps blood vessels and retinal astrocytes in inner retina promote

retinoblastoma cell growth in the inner nuclear layer55 and in vitro.56 However, gene expression studies suggest

several different classes of RB1-/- retinoblastomas, that that may arise in different cell types.49

[H2] Translating knowledge of pathogenesis

Understanding retinoblastoma molecular pathways could lead to treatment and prevention opportunities.

Oncoproteins such as N-myc could be targeted57 in both RB1+/+MYCNA tumours50 and RB1-/- tumours that have

progressed to N-myc-dependence.51 Furthermore, studies based on molecular discoveries showed that exposing

retinoblastoma-prone murine fetuses to small molecule inhibitors of E2f or Cdk inhibited subsequent

tumorigenesis without disrupting normal retinal development.52

These new translational opportunities require cell lines and animal models that accurately reflect retinoblastoma

cell responses. Most in vitro studies have used two old cell lines, but many others are available.58 Primary

xenografts in immunodeficient mice59 are useful47 but will not reflect the cell and immune environment of

natural retinoblastoma, potentially limiting translation to patients. Genetically engineered mouse models can

also be used to assess novel treatments.60 While Rb1+/- mice do not develop retinoblastoma, retinal deletion of

Rb1 (using Pax6a, Nestin, or Chx10 promoters) in p107, p130 or p27 mutant backgrounds achieves retinal

tumour formation.53 Such models have been used to examine genetic interactions in vivo, such as the role of

microRNA miR-17~92 overexpression in Pax6a-Cre;Rb1lox/lox; p107-/- mice.61 However, experimental murine

tumours have different collaborating mutations and cell type origin, reducing their ability to predict human

treatment responses. Viral oncoproteins, such as Simian Virus 40 T-antigen, promote murine retinoblastoma

specifically in developing Müller cells.62, 63 There is lots of room for models that more precisely simulate

retinoblastoma1 pathogenesis, to develop novel therapies that will target RB1-/- cancers.

[H1] Diagnosis, screening and prevention (HD, GC, BLG) 1506/1500

[H2] Clinical Diagnosis

Diagnosis of retinoblastoma is usually clear from presenting signs and clinical examination.4 The most common

sign is leukocoria (white pupil), when light reflecting directly off the tumour is noticed by parents, often in

photographs (Figures 6, 9). When parents report a strange reflection in the child’s eye, retinoblastoma should be

at the top of the differential diagnosis. The second most common sign is strabismus (misaligned eyes) when

central vision is lost. Advanced disease may present with iris colour change, enlarged cornea and eye from

pressure, or non-infective orbital inflammation. Very late, the eye may bulge from the orbit, a common

presentation where awareness and resources are inadequate.

Diagnosis of retinoblastoma does not rely on pathology, since biopsy incurs risk of metastasis.64 With the pupil

pharmacologically dilated and the indirect ophthalmoscope the diagnosis is usually clear to an eye specialist.

Calcification, characteristic of retinoblastoma, is detected by ultrasonography (b-scan). MRI is used to assess

invasion the optic nerve and trilateral retinoblastoma (pinealoblastoma and primitive neuroectodermal

intracranial tumours associated with RB1 mutations) (Figure 6). CT scans are now avoided because radiation

induces second primary cancers in people carrying RB1 mutations.65

Detailed retinal examination under general anaesthesia is required to distinguish the differential diagnoses

(Coats disease, persistent fetal vasculature, and vitreous haemorrhage) and classify the severity of intraocular

disease. The wide-angle, hand-held fundus camera is used to view and record all the retina, while as expert

depresses the sclera to bring the most anterior retina into view, while watching for optic nerve arterial pulsations

indicative of excessive pressure. Very useful are high frequency (50 MHz) ultrasound biomicroscopy,66 and

OCT67 to discover invisible tumours in infants with familial disease (Figure 1). Good imaging of the whole

retina supports eye classification and cancer staging, documents treatment responses, supports consultation with

colleagues, and helps parents understand treatment options.

[H2] Classification and Staging

The Reese-Ellsworth classification68 for intraocular retinoblastoma predicted outcomes of external beam

radiation. When intravenous chemotherapy (IVC), systemic chemotherapy and focal therapy became the

common primary treatment to salvage eyes, new schemes were developed (Figure 7). An international

collaboration led by Murphree developed the International Classification of Intraocular Retinoblastoma (IIRC)69

which better predicted responses to IVC than the Reese-Ellsworth classification.69 Eyes are classified by the

IIRC Groups A to E, with E being the most severe. However, subsequent modifications70 results in two similarly

named but significantly different classifications: 25% of the most severely involved (dangerous) eyes are

classified differently (Figure 7).71 Subsequently, the Children’s Oncology Group (COG) insert a further minor

variation.72 This confusion undermines research in retinoblastoma.

A number of staging systems also exist for extraocular retinoblastoma. In a coordinated action with the original

IIRC69, the International Retinoblastoma Staging System focused on overall staging.73 The American Joint

Committee on Cancer (AJCC)/International Union Against Cancer (UICC) staging system (TNM, defined by

primary tumour (T), lymph node extension (N), and distant metastasis (M) for retinoblastoma74 is under

revision, has wide cancer acceptance in general, and offers the potential to achieve a single, collaborative,

consensus classification of eyes and cancer staging, critical to clinical research.

[H2] Pathology

Retinoblastomas consist of small, round cells that stain blue on haematoxylin staining. Well-differentiated

regions form rosette structures: Flexner-Wintersteiner rosettes, highly indicative of retinoblastoma; Homer

Wright rosettes common in diverse neural cancers (not shown); and differentiated clusters of photoreceptor-like

cells termed fleurettes, characteristic of retinoma (Figure 4).3 The RB1+/+MYCNA tumors are histologically very

distinct, without rosettes (Figure 4).

Histological study of the enucleated eye is the only way to evaluate high-risk features (tumour invasion into the

optic nerve beyond the confines of the eye or to the cut end of nerve; more than 3 mm of invasion of the

vascular layer (choroid) under the retina; invasion of sclera) to establish pathological staging.75-79 High-risk

features are observed in 17% of Group D and 24% of Group E eyes using Shields classification,77 and 15%

Group D and 50% Group E75 using the Murphree classification.69 Documentation of high-risk pathological

features is critical to recognize need for adjuvant systemic chemotherapy and reduce risk of metastatic relapse.80

The preparation and examination of the enucleated eye have been optimized to completely evaluate risk

features.79, 81 Pathology specimens are staged using the pathology TNM (pTNM) and the International

Retinoblastoma Staging System.74, 79 Still, too frequently retrospective examination finds a previously unnoticed

risk after metastatic disease is diagnosed. Highly retinoblastoma-specific stains such as staining for the cone-rod

homeobox transcription factor (CRX; not normally seen outside the eye)82 and N-glycosylated ganglioside

(NeuGc-GM3)83 may facilitate observation of a few tumour cells lurking in a high-risk location.

[H2] Genetic Diagnosis

The majority (94%) of patients with retinoblastoma are the first individuals to be diagnosed in a family, only

141 out of 2,141 patients (6%) indicated that one or more family members were previously diagnosed with

retinoblastoma when tested for RB1 mutations.84 Approximately 50% of people with retinoblastoma carry one

RB1 mutation in their constitutional cells (100% of bilateral and 15% of unilateral patients).85 The remaining

unilateral patients develop retinoblastoma by somatic biallelic RB1 loss or somatic amplification of the MYCN

oncogene have no additional cancer risks.

Knowledge of the patient’s RB1 mutation enables precise screening of relatives and subsequent generations.

Without genetic testing, all children at risk (already one eye affected, and/or positive family history) undergo

multiple exams under anaesthesia in the first 3 years to detect small, easily treated tumors. When the RB1

mutation of the family is discovered by genetic testing, children at risks can be tested for that mutation: 85% of

patients with unilateral retinoblastoma will test negative with <1% residual risk for undetectable low level

mosaicism, which informs the decision to reduce the intensity of surveillance and accurate determination of

cancer risks of family members.10 The mutation can be detected prenatally from DNA in amniotic fluid when

amniocentesis can be safely performed during pregnancy, and, if necessary, labour can be induced near term to

detect and treat retinoblastoma tumours early. Pre-implantation genetic diagnosis for the known mutation of a

parent offers the option for a family to have an unaffected child.86, 87

Characterization of unique RB1 mutations of the tumour DNA (if available) can provide a highly sensitive

marker to screen CSF, bone marrow and harvested stem cells for minimal residual disease. Markers that have

been used are the RB1 tumour mutation (when mutation is not present or different from the germline

alleles),88the signature of post-RB1 genomic gains and losses89 and CRX expression.82

Best outcomes in familial retinoblastoma depend on the effectiveness of the healthcare team to identify and

counsel families. Parents need appropriate genetic counselling and understanding of the risks and actions for

each at-risk pregnancy. Alarmingly, a study of retinoblastoma in several developing countries observed that

familial cases were diagnosed later than non-familial probands.90 The authors inferred that the probands did not

understood the risks to their children, but they also might be frightened that there would be no treatment

available. Alternatively, socioeconomic or geographic barriers may have reduced desired healthcare access.

Clearly, study of social determinants of health, such as health seeking behaviour, perceptions of medical care,

and sociocultural issues related to cancer inheritance would inform counselling approaches that meet the needs

of families.91

[H2] Screening

[H3] Screening through eye examination

To detect retinoblastoma as early as possible, vision screening and eye examination have been developed.

General recommendations for childhood vision screening with effective training to detect signs of

retinoblastoma do occasionally identify a child with retinoblastoma. However, one negative childhood screening

test does not mean this child will remain tumour-free since a tumour might appear later, or may have been in the

periphery and not visible (false negative).

Leukocoria is most often first noted by parents (Figure 6), rarely first by physicians. Too often health workers

fail to take the parent’s complaint seriously, due to lack of awareness. Photoleukocoria refers to the appearance

of leukocoria on flash photographs (Figure 6). Awareness campaigns bring children to attention, but lose

effectiveness with time. An innovative study called PhotoRed in India trained healthcare professionals to use

flash photography to identify childhood eye diseases, including retinoblastoma.92 Another pioneering project is

developing software to enable cameras to detect photoleukocoria.93 A camera’s Red Eye Reduction technology

constricts pupils with a pre-photograph flash, limiting photoleukocoria detection. Red-eye and pet-eye

correction tools also enable unsuspecting parents to remove photoleukocoria. The global imaging industry could

play a role in early diagnosis of retinoblastoma.

[H3] Second Cancer Surveillance

Individuals with germline RB1 mutation and/or have been treated with radiotherapy have an elevated risk of

developing specific second cancers, including leiomyosarcoma, osteosarcoma, melanoma, lung and bladder

cancer.65 Surveillance screening for second cancers is a pressing need in the opinion of retinoblastoma survivors.

The first study to evaluate annual whole body MRI surveillance for individuals with predisposing RB1 mutation

showed it was feasible to detect second cancers but with modest sensitivity (5 out of 25 patients screened).94

This is an important area for further research so that early intervention can reduce mortality from second

cancers, as has been demonstrated in Li-Fraumeni syndrome.95

[H2] Prevention

Lifestyle counselling educates survivors on ways to mitigate their second cancer risk. In addition to being

vigilant about reporting unexplained lesions, they are encouraged to avoid an unnecessary radiation and

carcinogens (e.g. cigarettes and excessive alcohol). The extent to which these ideas will prevent second cancers

is unknown.

[H1] Management(FLM, CS, DA, GC, FN, JZ, BLG) 2559/3000

Management of retinoblastoma depends on extent of disease at diagnosis (classification of intraocular disease,

stage of systemic disease), status of the opposite eye, overall health of the child, socioeconomic opportunities

for the family, and access to expert care.4, 16, 96

96, 97[H2] Intraocular retinoblastoma

[H3] Primary Treatment

Choice of primary treatment is based on likelihood of cure (patient survival), eye salvage and ultimate vision–

weighed against short term and long term complications of treatment.96 In order of approximate frequency of

global use, primary treatments for intraocular disease include enucleation, intravenous chemotherapy (IVC) with

focal therapy (laser, cryotherapy), intra-arterial chemotherapy (IAC) with focal therapy, and focal therapy when

tumours are small at diagnosis. External beam radiotherapy (EBRT) is no longer recommended for primary

intraocular retinoblastoma, since radiation, especially in the first year of life, imposes on persons carrying an

RB1 mutation a very high risk of second cancers.98, 99 The preferred primary treatment depends on the severity of

disease of each affected eye (Figure 8). Recent advances in eye salvage technologies are very significant, but

clinical studies with clear eligibility and outcomes are not reported. For the simplest situation of unilateral

salvageable eyes (Groups69 B, C, D) comparison of primary treatment with enucleation, IVC, and IAC is

provided in Table 4. Bilateral advanced retinoblastoma where there is lack of expertise, equipment and

resources, and difficulties with close monitoring is best treated with bilateral enucleation.4, 16 Many bilaterally

enucleated retinoblastoma survivors lead active, productive and satisfying lives because they were treated by

timely surgery as infants.

[H3] Second-Line (Salvage) Therapy

Second line salvage means initiating a new plan of therapies, in a second attempt to save an eye that has failed

the first plan. A range of modalities is appropriate for second line therapy. However, each subsequent plan has a

lower success rate100 and long drawn out attempts to salvage an eye incur high costs of many kinds for the child

and family, including metastasis and death.101-103

Second line treatments have included focal therapies,104 104repeated systemic chemotherapy,104, 105 repeated

IAC,100 brachytherapy,106, 107, EBRT108-110 and 107whole-eye 111radiation with proton beam.111 Criteria for

secondary enucleation are not well defined but are dominated by refractory subretinal and vitreous seeding,105, 112

suggestion of risk of uncontrolled cancer111 such as vitreous haemorrhage and secondary neovascular glaucoma,

and socio-economic and psychological fatigue to save an eye with poor vision.102 Following primary enucleation

of Group69 D eyes, high risk pathology is uncommon. However, secondarily enucleated eyes showing scleral

invasion may develop extra-ocular relapse;113 and Group69 E eyes treated with systemic chemotherapy delaying

enucleation, have significant risk for death from retinoblastoma.114

Of Group69 D eyes treated with systemic chemotherapy and focal therapy 53% failed (required salvage EBRT

and/or enucleation).110, 115 First-line IAC has reduced the recurrence rate in Group69 D eyes116 and a combination

of repeat IAC and intravitreal chemotherapy (IViC) may play an important part in saving eyes that have failed

IVC.116-118 However, extensive treatments to save an eye increase risk for metastases and death.100, 103, 119

However, the major cause of failure from all primary treatments has remained vitreous seeding.

Pharmacokinetic studies show poor vitreous levels of drugs administered by either systemic chemotherapy or

IAC.120 The highest drug bioavailability in the vitreous is achieved by IViC using a safety-enhanced injection

technique in carefully selected eligible eyes.121 Following control of the source of seeds, IViC achieved two-year

Kaplan-Meier estimates of 98.5% control of target seeds and 90.4% event-free ocular survival.121-123 The efficacy

of IViC has eliminated the need for EBRT and decreased patient exposure to salvage systemic or IAC. The

toxicity of IViC is limited to technique-dependent localized peripheral retinal toxicity.124

Combinations of technologies can target salvage therapies to the site of relapse. For relapse confined to the

retina and/or vitreous, salvage therapy can consist of focal therapy and/or IViC, as long as whole-eye therapy is

not required. Relapses touching the optic nerve head and/or vision-critical regions such as the maculo-papillary

bundle, and diffuse shallow retinal/subretinal recurrence, represent good indications for IAC, which might

achieve better visual outcome than focal treatments.

[H2] Ocular Therapies

[H3] Enucleation

Enucleation is a first-line therapy for the majority of eyes with retinoblastoma globally; it is the fastest and least

costly treatment125, 126. Since the majority of children without a family history with intraocular retinoblastoma

have Group69 D or E disease at diagnosis, and more than 50% have unilateral disease with other eye unaffected,

cure can be achieved with enucleation (Figures 3 and 6). All Group69 E eyes require enucleation because by

definition they carry risk of extraocular extension which can only be confirmed by the identification of high risk

pathological features in the enucleated eye. Best cosmetic outcome is achieved by replacement of the volume of

the eye with an implant deep in the orbit, and provision of a prosthetic eye, worn in the conjunctival sac behind

the eyelids. Many different reconstruction techniques are used worldwide.127 Comparative studies have shown

best artificial eye motility with a simple implant and muscles sutured to conjunctiva to form the pockets to hold

the artificial eye (myoconjunctival approach), which is affordable world-wide.128, 129 Porous implants that

become vascularized with muscles sutured to the implant are commonly used in developed countries but have

higher rates of infection and extrusion and are more costly. Provision of a temporary prosthetic eye at the time

of enucleation has a positive psychological impact on families,130 observed in Kenya to help the family accept

enucleation for their child.

[H3] Intravenous chemotherapy and focal therapy

Since 1996 first-line therapy to control Groups69 B, C and D disease has been IVC with different combinations,

doses, schedules, and durations of carboplatin, etoposide and vincristine (CEV) followed by focal therapy to

consolidate the chemotherapy responses(Table 3).112, 131, 132Sometimes high dose acute cyclosporine is added to

modulate multidrug resistance.133, 134Eyes in Groups69 B and C do well with CEV and focal therapy. With follow-

up of 54 months, 47% of Group69 D eyes110 and 47% of Reese-Ellsworth Group V112 avoided enucleation and

EBRT(Figure 9).70

Fundamental principles for systemic cancer therapy apply to retinoblastoma: optimized outcomes are achieved

by high dose intensity and combination of several agents with complementary mechanisms of action. This is

illustrated by the reduced effectiveness of single agent, low dose carboplatin for retinoblastoma.135 Acute

toxicities of IVC for retinoblastoma are as for other paediatric cancers, including short-term transient

pancytopenia (reduction in red blood cells, white blood cells and platelets), hair loss, vincristine-induced

neurotoxicity, and infections. Long term toxicities include carboplatin-induced ototoxicity (toxicity to the

ear),136 second non-ocular cancer risk with alkylating agents137, 138 and secondary acute myeloid leukaemia

following intense chemotherapy including topoisomerase inhibitors, doxorubricin and alkylating agents.139, 140

IVC alone rarely eradicates the retinoblastoma entirely, and focal therapy with repeated examinations under

[Au:general?]anaesthesia is very important (Figure 9).141-143Tumours in the macular region, which threaten

vision, are particularly at risk of recurrence without focal therapy.144

Following control of retinoblastoma with IVC, location of tumour is the most important predictor of final visual

acuity (near-normal visual acuity ≥0.5 in 22% of eyes with macular tumour, 67% of eyes with extrafoveal

tumour).145, 146 In children with two eyes, patching of the better eye to force use-dependent development of the

eye with macular involvement (occlusion therapy) achieved near-normal vision in 53% and useful vision in

73%.147 There is no documented local toxicity of IVC to the eye.

[H3] Intra-arterial chemotherapy

IAC [Au: OK?] is a recently optimized technique in which the patient is heparinized and a micro-catheter is

inserted into the femoral artery and passed up to the orifice of the ophthalmic artery (but not into the artery)

where drugs or combination of drugs (typically melphalan, carboplatin, and [Au:OK?]topotecan) are infused in

a pulsatile fashion over many minutes.148Alternatively, a catheter with an inflatable balloon near the tip can be

used to ensure selective treatment to the eye. During the procedure, the internal carotid artery is temporarily

occluded by inflating the tip and melphalan is injected over a few seconds below the balloon with the intention

of having the drug directedinto the ophthalmic artery(so-called selective ophthalmic treatment)119 Cannulation

via OAC has been successful in almost 99% of cases.OAC is usually performed via the internal carotid artery

but sometimes also through the external carotid artery and [Au:OK?]through the middle meningeal artery (15%

of cases); in 10% of the procedures a modification of the balloon technique is used149Also, tandem therapy in

which both eyes are treatedin the same, one hour session has successfully been performed.150OAC is currently

performed in more than 30 countries worldwide of which nearly half in developing nations.151

Of the more than 2,500 infusions and >800 patients in the literature using OAC with a mean follow-up of less

than 5 years, only two patients have died of metastatic retinoblastoma[Au: over all groups, A-E?].In the USA,

patient survival was 100% at 5 years[Au: over all groups, A-E?].117 However, death from metastatic disease

can occur more than five years after any treatment. The longest follow-up study described for selective

ophthalmic treatment in Japan (including death from metastatic retinoblastoma and second cancers) showed an

overall survival of 95% at 15 years.119However, the 8 deaths from metastases of 343 patients cannot be clearly

assigned to IAC alone since only a minority of the patients received IAC exclusively.On the other hand, similar

figures of deaths from metastasis have been reported with other conservative therapeutic modalities.143

Many patients treated with IAC have advanced-stage disease and would have been candidates for enucleation

but for cultural reasons, families (and often physicians) refused this option. Despite this, ocular survival exceeds

all other approaches for advanced-stage retinoblastoma (which represent the majority of eyes at diagnosis

worldwide). It should be stressed that a comprehensive analysis of the published literature on eye survival

following IAC is confounded by the concomitant use of different classifications69, 70;Group, 2011 #21798}

(Figure 7, Extended Data 2), which prevents a clear-cut comparison of the results between centres, especially

regarding Group69 D and E eyes. We need a universally endorsed single classification in order to avoid

confusion regarding eligibility and salvage rates for new treatments. Ocular survival with IAC was 65% for

Group69 C and 45% for Group69 D in one study;119 a more recent study showed 96% in both Groups69 B and C,

and 94% in Group69 D.117, 152 Eyes with neovascular glaucoma, pthisis bulbi (a shrunken, non-functional eye),

and anterior chamber involvement were universally enucleated up front. Ocular survival is highest in treatment-

naive eyes with extensive retinal detachment (Figure 10a). With eyes with >50% retinal detachments, ocular

survival (Kaplan-Meier) was demonstrated to be 87.9% at two years and 76% had complete retinal reattachment

after IAC alone.153 The most common reason for second-line enucleation of eyes with retinoblastoma in

developed countries has been vitreous seeding.154 Only 20% of such eyes can be salvaged with EBRT.155 With

OAC, 74% of eyes with seeding have been salvaged(Figure 10b).100, 117, 118 With the selective ophthalmic

treatment, 58% of children with foveal tumours retained a visual acuity of >0.01 and for those without foveal

tumours 51% retained visual acuity of >0.5 with 36% >1.0 (2). Electroretinographic[Au: OK?]monitoring of

OAC patients148, 156-159 demonstrated remarkable stability of.OAC decreases the appearance of subsequent, new

(usually peripheral) intra-ocular tumours that commonly develop after systemic chemotherapy or radiation in

genetic[Au: patients with heritable retinoblastoma? All cancers are genetic.] cases resulting in fewer overall

treatments for the children.160[Au: Paragraph in Green moved up.]

Complications following OAC are currently few and include both short-term and long-term effects. In an

analysis of 198 catheterizations of the ophthalmic[Au: OAC technique?] artery in 70 consecutive eyes with

retinoblastoma, minor transient complications included transient eyelid oedema (5%), blepharoptosis (5%), and

forehead hyperaemia (2%).117 Lasting complications included vitreous haemorrhage (2%), branch retinal artery

obstruction (1%), ophthalmic artery spasm with reperfusion (2%), ophthalmic artery obstruction (2%), partial

choroidal ischaemia (2%), and optic neuropathy (< 1%).117 These complications are minimized at experienced

centres.

Historically, consolidative focal therapy (focal treatment once response to cytoreductive chemotherapy has been

achieved)[Au: definition for nonexperts OK?] was necessary in the majority of the eyes initially treated with

systemic chemotherapy (even including EBRT) so it was initially used in almost all of the original patients

treated with OAC. Subsequently, experience has shown that consolidation was not needed in 23-33% of

cases.161, 162

Although this procedure has been performed in children as young as three weeks of age, most centres withhold

cannulation until the patient is at least 3 months of age and 6-7 kg in weight because of concerns about repeated

puncture of the femoral artery. As a result, these very young children are given single agent (carboplatin) IVC in

modest doses (18.7 mg/kg) as an outpatient until they attain the 6-7 kg/3 month goal when they are suitable for

OAC. This approach is called “bridge therapy” and 94.7% of such eyes (Kaplan-Meier) have been salvaged

without the need of radiotherapy.163

[H3] Focal therapy [Au: Please add references to this section.]

Focal therapy is the local application of anti-cancer therapy – thermo-, cryo-, radio-, or chemotherapy – to the

eye, under direct visualization through the pharmacologically dilated pupil. This approach is useful for primary

treatment of Group69 A cancers and consolidation therapy for residual or recurrent small-volume, active tumours

after IVC or IAC. In general, focal therapy is repeated monthly until the tumour is completely atrophic or

calcified.

Transpupillary thermotherapy involves laser treatment through the dilated pupil. Photocoagulation treatment

with 532 nm, 810 nm or continuous wave 1064 nm laser is directly applied by multiple short (0.7 s) burns to

small volume active or suspicious tumour, starting at a sub-coagulation power intensity and increasing to attain

white, opaque coagulation. Both laser treatments are repeated monthly until the tumour is flat, atrophic or

calcified. Cryotherapy is performed to freeze the tumour through the sclera with a nitrous oxide probe; the

tumour is directly visualized and duration of freeze judged to completely eliminate the tumour. Since tumour

cells die when thawing, one minute is allowed for each thaw between freezing cycles. Cryotherapy effectively

destroys small primary tumour(s) or recurrences in the periphery of the retina. Plaque radiotherapy involves the

position of a radioactive probe on the eye to deliver trans-scleral radiotherapy with an apex dose of 35 Gy over

4-7 days.[Au: edits OK?]Plaque focal radiation has not been associated with second primary tumours and is

effective for treatment of a single primary or recurrent tumour in a location that will not compromise vision.

Paraocular chemotherapy has been useful under conjunctiva or in tenon’s capsule for small volume recurrences

and vitreous seeds.164-166

[H3] Intravitreal Chemotherapy

Vitreous seeds are the major cause of failure (enucleation or external beam radiation) of primary treatments.

IViC is adjunctive to many other treatments, initiated after source of the seeds is controlled, with promising

results. IViC using a safety-enhanced injection technique in carefully selected eligible eyes has shown excellent

responses with the most difficult to control form of retinoblastoma.121, 122, 124, 167, 168[Au: Sentence in green

moved up]The procedure involves lowering the intraocular pressure with an anterior chamber paracentesis or

by digital massage after induction of anesthesia. Intravitreal melphalan is injected through the conjunctival,

sclera, and pars plana with a small-gauge needle. On needle withdrawal, the injection site is sealed and sterilized

with cryotherapy and the eye is shaken gently to distribute the drug though the vitreous. Three classes of

vitreous seeds have been identified with significantly different median times to regression, mean number of

injections and cumulative and mean melphalan dose.122Ultrasound biomicroscopy may be used to evaluate the

otherwise hidden ciliary region behind the iris,66 to confirm that the injection site is tumour-free prior to IViC

treatment.

[H2] Extraocular retinoblastoma

[H3] Extraocular at presentation

Retinoblastoma may present with evident extraocular disease, especially in low-income countries., Orbital

extension of an intraocular retinoblastoma may be detected clinically in children with proptosis or a fungating

mass or occasionally by imaging studies. , Treatment includes neo-adjuvant chemotherapy, including

carboplatin, etoposide and vincristine, as for intra-ocular retinoblastoma; other agents that are useful include

cisplatin, cyclophosphamide and anthracyclines.16This is followed by enucleation or limited exenteration, orbital

radiation, adjuvant chemotherapy. Intrathecal chemotherapy may also be used and high dose chemotherapy with

stem cell rescue may be added in metastatic disease.. Children with orbital disease and those with metastatic

spread without CNS invasion may be cured with intensive therapy.

[H3] Adjuvant therapy for high-risk pathology

Extraocular retinoblastoma can develop despite initial diagnosis of intraocular disease. Recognition of high-risk

pathological features of primarily enucleated eyes followed by adjuvant chemotherapy with tight surveillance

for metastatic disease has good outcomes.76, 78 Enthusiasm to salvage eyes increases metastatic risk by masking

primary extraocular disease, and potentially continuing attempted salvage too long so that extraocular disease

develops that was not there at primary diagnosis.103, 114

[H2] Palliation

Palliation includes pain management, symptom relief, nutritional support, and psychosocial support for the child

and families.16Untreated retinoblastoma is highly sensitive to most chemotherapy agents. Children presenting

with orbital retinoblastoma are usually in severe pain and discomfort that may be alleviated with judicious use

of anticancer therapy, including conventional chemotherapy, even when no curative intent is pursued. These

children often present with severe emaciation needing prompt medical treatment. Radiotherapy may also be

helpful, especially for a CNS relapse or for the treatment of massive orbital extension, but it isoften unavailable

in low-income countries.169[Au: references?]

[H1] Quality of life(AW, HD, BLG) 844/500

[Au: I think this section would benefit from a short introduction that outlines what the major

QOL issues in retinoblastoma survivors are. For example, radiotherapy on a young brain can

lead to developmental issues, cognitive decline, etc. This will set up the rest of the section nicely

and need only be a sentence or two.]

“Quality of life” describes the level of physical, emotional and psychological wellbeing experienced by an

individual. Cancer, its treatment and effects on the developing body, brain and mind can significantly decrease

Quality of Life, with implications for treatment decisions, supportive and long-term follow up care.

[H2] Measured life-long impact

Analysis of life-long treatment impact shows that 170, 171overall, survivors diagnosed under 1 year of age

performed significantly better in short and long-term verbal memory, verbal learning and verbal reasoning

abilities compared with those diagnosed at over 1 year of age.170, 171 In bilaterally affected children, whole brain

radiation exposure at any age was significantly associated with poorer verbal memory[Au:reference?].170, 171In

children followed up from diagnosis to age 5 years, trajectories of developmental functioning declined over

time.171 However, verbal IQ of age-matched persons with the early infancy form of retinoblastoma was

significantly above[Au: Explanation for the higher IQ?]normal sighted and non-retinoblastoma blind

persons.172No explanation has been identified for this finding, though hypotheses of a genetic cause have been

proposed. There is altered development of visual, auditory and multisensory brain morphology in adults who

lost one eye to retinoblastoma in early life, suggesting that the remaining eye acquired increased contralateral

visual cortical connections.173, 174There is clinical and animal evidence that repeated anaesthesia of young

children may impair neurocognitive development.125, 175, 176Repeat anaesthesia is necessary in eye salvage and in

very young children to discover and treat small tumours. Going forward, one outcome to record in

retinoblastoma clinical studies is number of examinations under anaesthesia and age.

[H2] Direct insight from survivors[Au: This section contains 22 references. Please try to delete some,

since the reference list needs to be cut to 200 references. ]

Most important are insights from the retinoblastoma survivors themselves. Social media brings retinoblastoma

parents and survivors into peer-support communities. Research processes that deepen understanding of quality

of life following retinoblastoma are needed to learn from these valuable evidence sources.

[H3] Coping during treatment and psychosocial outcomes

A mother describes her son’s response to radiotherapy aged 16 months: “In the beginning he was extremely

combative. At the end of the treatment course, he was a broken child, withdrawn and passively accepting what

was happening to him. The long term damage caused took years of therapy to start to heal"[Au: source

reference?].Children’s perception of pain and medical interventions changes over time.177-183Repeated

procedures cause anticipation anxiety and intolerance of even minimally invasive experiences and mild pain.

The child’s initial strong emotions may be suppressed as the child gives up, and re-emerge as depression, post-

traumatic stress or developmental trauma disorder.101, 184-190Child Life interventions at any age and with any

treatment help children thrive during treatment, reduce treatment costs, ease family stress and improve long-

term mental health(Figure 11).5, 65, 191-200

Retinoblastoma treatment is often the child’s only life experience, forming the centerpiece of their earliest

memories. While adult survivors may not remember being anaesthetized, many describe acute fear of their

mouth and nose being covered. One adult describes how the scent and taste of strawberries makes her nauseous

– her mask was always coated with strawberry scent.

Extended isolation during therapy may impact social functioning. While most adult survivors perform well

socially, many report low confidence and intense anxiety, especially in large groups and crowded environments.

Most survivors are high cognitive performers.170 However, reduced vision causes some children to become

frustrated by their inability to keep up with peers, damaging self-esteem and confidence.201-203

[H2] Radiotherapy late effects

While radiotherapy is now rarely used for retinoblastoma, thousands of adult survivors live with its long-term

effects. Many feel neglected and demoralized by lack of follow up and prospective management. Facial

deformity causes low self-confidence and social anxiety. Reconstructive surgery is a painful process that may

impact remaining vision, but its cosmetic effects can dramatically improve quality of lie. Dry eye is very

painful, and corneal vascularization reduces already limited vision. Use of ocular lubricants may prevent

complications, and should be started early before pain and vision loss occur. Chronic primary headaches,

hormone dysfunction and seizures also impact quality of life after radiotherapy.[Au: reference?]

[H2] Second cancer risk

Individuals at risk of second primary cancers require life-long oncology follow up.137, 204, 205 Lack of agreed

protocols causes confusion, frustration and fear as adults struggle to access informed follow up care,

compounded by primary doctors who are not aware of late effects and lifelong implications of RB1 mutation.

Full information about the patient’s cancer history, genetic status and life-long risks will empower survivors to

be advocates for their own and their children’s health.206-209

[H2] Family planning

Many adult survivors have little knowledge of retinoblastoma genetics, genetic counselling or testing, their

status, or of options for their baby. Cost and availability of genetic testing and pre-implantation genetic

diagnosis is often prohibitive. Lack of an agreed screening protocol for at-risk babies causes anxiety among

survivor-parents. Profound anger and guilt about somehow being responsible for the child’s cancer is amplified

when diagnosis is delayed by inadequate screening. Agreed screening protocols for at-risk children will reduce

survivor-parent anxiety and enhance early diagnosis to achieve minimally invasive therapy.

[H1] Outlook(All) 977/1000

Retinoblastoma is curable if diagnosed early. We propose that within 10 years retinoblastoma can be a zero-

death cancer. Our anti-retinoblastoma arsenal will consist not only of curative treatments, but also measures to

prevent development of disease in predisposed individuals. To achieve this vision, critical efforts are needed to

build upon the current state-of-the-art for retinoblastoma as presented in this Primer. Now, we dream big with

key action items [BOX], responding to the immediate obvious targets for improvements, and setting the stage

for innovative discoveries in retinoblastoma.

[H2] Patient-centered, global research

Retinoblastoma will benefit from clinical science at the level that has achieved dramatic change for pediatric

cancer. Because most centres have too few affected children, or many children and inadequate resources to

study them, we have lagged behind in systematic accumulation of evidence on which to base therapy. Today we

are faced with sweeping change in treatment of retinoblastoma using IAC and salvage of unresponsive vitreous

seeds by IViC. However, we do not have full data on which to judge eligibility, long-term effectiveness or

outcomes.

We have opportunities now to learn from initiatives and innovations everywhere, and to listen to the patient’s

voice to become an inclusive, participatory global network. Patients and families, empowered by social media,

will be heard in the research approaches, making them relevant, ethical, and appropriate. Stakeholders from low-

and-middle income countries will be active innovators, assuring that protocols are feasible and acceptable

Equality for retinoblastoma will come by education (Internet access), with shared collaborative care coordinated

online. Retinoblastoma expertise will be developed locally (proportional to the local burden of retinoblastoma)

but derived globally, with best practices drawing on rigorous molecular and clinical research. Retinoblastoma

clinical trials will be collaborative, high enrolment studies. Effective innovations will include all outcomes and

sustainability (cost, uptake by health ministries, social acceptability of therapy, etc.).

The global burden of retinoblastoma shows the inherent inequity in how patients are diagnosed and cared for

around the world. Documentation of human and material resources required for optimal care, drawn from

established clinical care guidelines, represent the first step towards health equity. National strategies in areas

currently underrepresented in the retinoblastoma literature are poised to fill the knowledge gap.

Progress in therapy is critically dependent on achieving consensus on a standard classification of intraocular

disease. The ongoing AJCC/UICC international survey of features of eyes at diagnosis, treatments given and

outcomes will be used to derive ONE evidence-based classification to replace the current clinical staging

schemes that confuse analysis or eligibility or outcomes.69-71, 74

We are poised to collect uniform data on all children with retinoblastoma. The 1RBW.org map documents

“staff, stuff, systems and spaces”210 and provides an alternative system of global collaboration, perhaps unique

to small neglected diseases, but perhaps the a for-runner for the most developed health care system. The eCCRB

point-of-care database summarizes the health record from diagnosis, including features of eyes and patient,

treatments, complications, and outcomes, and is available to all retinoblastoma centres. eCCRB also supports the

caregivers by graphic display of information otherwise out of reach, ensuring high motivation for good data

quality. The next phase of this Internet project (aiming at 2020) will combine the 1RBW map with eCCRB de-

identified data. “Real-time” analysis of outcomes is planned to feedback to treating doctors, a “learning health

system”,211 to assist in selection of evidence-based care for the next affected child.

We propose to determine the most likely cause of every death from retinoblastoma. Similar in concept to the

groundbreaking Million Death Study,212 each death will be an opportunity to avoid more deaths. The identified

key risk factors will lead to systematic guidelines to avert preventable deaths. BOX These will fall into

categories such as Public Awareness (public and health system understanding of the urgency of

leukocoria, utilization of Social Media), Health Services213 (Infant screening, financial support for every

child, regional retinoblastoma expertise, etc.), Clinical Research (Co-operative Groups for clinical trials,

outcomes analysis from learning health records, etc.) and Basic Research (define in available

retinoblastoma tumour cells targets for new therapy, define molecular targetable pathways for treatment

of RB1-/- tumours and prevention of RB1-/- cancers, starting with heritable retinoblastoma).

[H2] Targeted therapies

Our knowledge of the retinoblastoma cell of origin and its unique sensitivities is steadily increasing, and may

reveal strategies to prevent tumorigenesis. Moreover, ongoing work to define molecular lesions in

retinoblastoma will continue to yield novel therapeutic targets. Advances in therapeutic development and

prevention depend on these molecular studies, but we do not yet have preclinical models that accurately reflect

retinoblastoma pathogenesis and treatment.

One of the earliest targeted therapies developed for retinoblastoma, based on preclinical information generated

by transgenic mice was Nutlin-3.214 Nutlin-3 blocks MDM2/MDM4, releasing p53-mediated apoptotic cell death

and showed promising activity in combination with topotecan for retinoblastoma control in preclinical models.

Although the SYK antagonist R406 was considered a promising candidate based on preclinical studies,47 its

ocular pharmacokinetic profile following peri-ocular administration rendered it unfavorable for clinical use.215

Preclinical studies further suggest that retinoblastoma initiation and tumor growth might be suppressed by

inhibitors of MYCN, SKP2, E2F, or CDKs (Figure 12).

Preclinical studies have also sought to identify and evaluate pharmacokinetics of new agents alone or in

combination216 before clinical deployment. A few translational studies have resulted in changes in

retinoblastoma clinical practice.217 Innovative delivery systems to target small volume sub-retinal tumour and

vitreous seeding include devices for sustained-release, periocular and intravitreal administration, episcleral

implants218 and nanoparticles219. Approaches adjusting chemotherapy schedules and dose intensity (Metronomic

therapy)220 may particularly play an important role in palliative care for retinoblastoma. New biomarkers for

molecular dissemination of retinoblastoma outside the eye may lead to earlier diagnosis of metastasis and

targeted treatments.82, 83

[H2] QoL

Without thinking about the lifelong outlook for patients, however, all of the above endeavours become moot.

Quality of life begins in infancy, assisted by Child Life and respect for the child’s need to play. Patient-oriented

research will develop and validate life-long survivor follow-up care protocols, including centralized specialist

eye care, genetic testing, second cancer screening, ongoing psychological support and fertility care. will

educate primary physicians and survivor clinics, reduce survivor morbidity and aid research that can inform

patient management to improve quality of life during retinoblastoma treatment and throughout life.

[Au: QoL section deleted to remove overlap.]

Display Item Legends

Box 1: [Au: Please consider including a box with explanations for the specialist terms.]

Complex integrated implants

EBRT and tantalum ring localization

brachytherapy

myoconjunctival approach

oraserrata

orbital retinoblastoma

salt and pepper retinopathy

Scleral depression

Stem cell therapy?

proton beam radiotherapy

Box 2: Outstanding research questions [Au: Titles OK?]

Patient-centered, global research focus

Address the "reluctance of healthworkers to confer the true risks"(such as in Jordan where

doctors do not want to destroy families) and choice of families who know and understand the

risks not to followthrough because of the same stigma.

Define treatment success: cure (life), salvage of vision (one eye), salvage of eye (vision and

cosmetic), fewest examinations under anaesthesia (cumulative toxicitiyof anaesthesia)

Meta-analysis of all children treated primarily with IAC.

Develop a “retinoblastoma index” relating levels of awareness, access to centres with the

necessary resources and expertise, and application of evidence-based care, as predictors of

outcome.

Pathogenesis

What is the genomic progression post RB1 loss?

What molecular changes promote (early) metastasis?

Are there other RB1+/+ subtypes beyond MYCN-amplified retinoblastoma?

What dictates the tissue specificity of RB1 loss leading to cancer?

What are the unique molecular features of second cancers in RB1 mutation carriers?

Figure 1| Progression of retinoblastoma. a) Normal eye until genomic damage (yellow lightening bolt)

damages the second RB1 allele (M2) resulting in biallelic functional loss of RB1 in a developing retinal cell

(possibly a cone phototreceptor precursor cell that is dependent on pRB to stop proliferationRB1. may block

differentiation of a cone precursor cell, as itfailsthat that fails to localize normally. b) Genomic instability

might leads to an intraretinal benign retinoma. Inset: a small retinoblastoma visualized by optical coherence

tomography[Au: OK? Or do RB1-/- cells always form retinomas.] [Au: Which kind of scan is shown in the

insert? Please add information to the legend.] . c) This tumourIntra-retinal retinoblastoma arises if additional

genomic changes release uncontrolled cell proliferation; only 5% of patients show retinoma without

retinoblastoma. Inset shows a small tumour not visible except by optical coherence tomography,can progress to

form a retinoblastoma if additional mutations are acquired [Au:OK? Or do all the retinomas progress to

retinoblastomas], andThe tumour which will grow and seeds seed under thesubretinallyunder the retina retina

and into the vitreous.; d) Retinoblastoma can invades adjacent tissues: past the lamina cribrosa of into the optic

nerve, uvea, or sclera to constitute high- risk pathologic features. e) Finally,Eventually, the tumorretinoblastoma

mightextends extraocularly into orbit, brain (direct or subarachnoid to CSFcerebro-spinal fluid)) or metastasizes

to especially to bone marrow.

[Au: Can we add percentages of to the figure, showing how many % of patients will progress to the next

stage – if data are available? How many % of RB1 carriers, for example, will progress to develop

retinoblastoma?][Au: It would be great if we could have an extra panel, preferably panel A, showing the

normal eye anatomy, with labels for all the relevant anatomical terms mentioned in this legend, or the

manuscript. ]

Figure 2 | Global Retinoblastoma Prevalence. Heat Map of estimated distribution of the global retinoblastoma

patient population as assembled by the One Retinoblastoma World (1RBW) initiative. The majority of cases

reside in low and middle income countries. The 1RBW Map of Treatment Centers (www.1rbw.org) connects

affected families to expert care close to home, providing rich global data for anyone to use. Without prior

awareness of retinoblastoma, parents now commonly self-diagnose on the Internet, and the 1RBW map connects

them to experts without delays. Images from www.1rbw.org . .[Au: I think only panel A should be sufficient.

It is quite clear that most cases are in the middle/lower income countries.]

Figure 3 | Triplets with retinoblastoma tumours. These triplets developed retinoblastomas in all six eyes

illustrating the full expressivity and penetrance of the RB1-/- germline mutations that result in no protein, such as

the mutation carried by each triplet. The eCCRB timelines reveal that all eyes had individualized therapy, with

choices considering the overall impact on each child. Each child lost one eye, and each has a normal vision eye

with disease control in less than one year. The number of eye exams under anaesthesia was least for the child

who had primary enucleation. OD, right eye; OS, left eye. Eye involvement at diagnosis is indicated in the

International Intraocular classification;69 the eye labelled “Group 0” had no tumour at initial diagnosis but

develop a tumour 6 weeks later.

Figure 4 | Genetic origins of retinoblastoma. Three genetic subtypes of retinoblastoma are known. Heritable

retinoblastoma patients have a constitutive inactivating mutation (M1) in the RB1tumor suppressor gene in all

cells of their body. A second, somatic mutation (M2) in a susceptible retinal cell can lead to benign retinoma.

Further genetic and/or epigenetic events (M3…Mn) are required to transform to retinoblastoma. Non-heritable,

RB1-/- retinoblastomas progress similarly, except both M1 and M2 occur in one susceptible retinal cell.

RB1+/+MYCN-amplified (RB1+/+MYCNA) retinoblastoma is a rare, non-heritable retinoblastoma subtype driven

by amplification of MYCN with normal RB1; other changes in these tumours remain uncharacterized. Retinoma

histology shows distinct photoreceptor-like fleurettes, while RB1-/- retinoblastoma can show Flexner-

Wintersteiner (indicated) and Homer Wright rosettes. RB1+/+MYCNA retinoblastoma show distinct histology with

rounded nuclei and prominent nucleoli related to the high MYCN protein.

Figure 5 | Retinal origin of retinoblastoma tumors. a, The retina has a complex structure and contains

multiple cell types. Haematoxylin and eosin staining of post-fertilization week 19 retina shows three post-

mitotic nuclear layers in the central retina near the fovea (left) and two nuclear layers in the less mature

periphery of the same histologic section (right). Cell types in each layer are indicated. b, Cone precursors

normally have high expression of the RB protein (pRB). Immunofluorescence staining of post-fertilization week

19 retina shows especially strong pRB signal (red) in maturing cone precursors in the central retina (left,

counterstained for cone arrestin, green) and in retinal progenitor cells in the peripheral retina of the same

histologic section (right). pRB staining is less intense in DAPI-stained nuclei (blue) in other retinal cell types. c,

Optical coherence tomography of a tiny retinoblastoma tumor in a 2.5 month old infant appearing to extend

from the inner nuclear layer to the outer nuclear layer. The tumour has an uncertain epicentre making it difficult

to infer the retinal layer from which the tumor arose. This layer-of-origin may be defined in the future with more

images of higher resolution OCT of retina in very young children carrying an inherited germline RB1 mutation.

Abbreviations: ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; NBL, neuroblastic

layer; RPE, retinal pigment epithelium.

Figure 6 | Photoleukocoria to Internet diagnosis. a, Leukocoria was noticed only on this digital image. The

parents search on “white pupil” and diagnosied retinoblastoma. The left eye was enucleated two days later. The

other eye was normal at diagnosis but later developed a small tumour treated with only laser. b, One month later

the child presented with signs of high intracranial pressure, before a scheduled screening CT (today MRI would

be done to reduce radiation exposure for persons with retinoblastoma) and a large intracranial midline tumour

(trilateral disease) was diagnosed. c, The eCCRB timeline of treatment: the child received systemic chemotherapy

and high dose chemotherapy with hematopoietic rescue by stem cell transplant. Subsequently he had more than

70 injections of intrathecal (given directly into the cerebro-spinal fluid) chemotherapy through an implanted

intraventricular catheter (not all shown in timeline). At age years, d, followup MRI at age 8 years shows no

residual disease and e, the child is well.

Figure 7 | Different classification schemes for intraocular retinoblastoma confound comparison of

outcomes. The features listed determine the overall classification, ranging from small tumours not threatening

vision (“Group A”) to tumours clinically noted to have features suggesting potential spread outside the eye

(“Group E”). Most importantly, size of tumour alone does not make an eye dangerous by Murphree,69 Children’s

Oncology Group (COG),72 or TNM classification;74 but any eye with tumour >50% of eye volume is E

(advanced-stage disease) by Shields classification.70 The consequence is widespread confusion in the literature

undermining clinical research, since studies using the different classifications cannot be compared.

Figure 8 | Primary treatment choices for each eye based on the International Intraocular Retinoblastoma

Classification.69 Treatment depends on the combined severity of each of the affected eyes (Eye 1 / Eye 2); the

preferred option for each eye is depicted in the blue boxes. Group69 A eyes can be treated with laser or

cryotherapy (focal therapy) alone. Group69 B and C eyes require several cycles of systemic or intra-arterial

chemotherapy followed by focal therapy (Group69 B) and, for unresponsive vitreous seeds, intravitreal

chemotherapy (IViC) with melphalan (Group69 C). Isolated single tumours in Group69 B or C eyes may

occasionally be appropriate for primary radioactive plaque therapy. For safety of the child, it is important that all

eyes with features suggesting imminent extraocular extension be removed to enable accurate pathological

examination to determine if risk of metastasis requires adjuvant chemotherapy.

Figure 9 | Good Response to Systemic Chemotherapy: a, b, White pupillary reflexes in both eyes brought this

child to attention at age 8 months. c, Right eye and d, left eye at diagnosis showed total retinal detachment and

underlying large tumours. e, eCCRB time line of shows treatment with systemic chemotherapy and focal therapy,

then only follow-up visits. After 20 years, both eyes remain stable: f, right eye (0.25 vision) and g, left eye (0.1

vision) (Vision decimal system, 1 = normal, 0.1=legal blindness).

Figure 10 | Response to Primary Intra-arterial Chemotherapy: a, Before (Left) IAC for a massive exophytic

retinoblastoma with total retinal detachment; and after (Right) showing complete response and eye salvage,

leaving a 6 mm calcified scar. b, Before (left image) IAC for an endophytic retinoblastoma eye with extensive

vitreous seeding; and after (right image) showing complete control with clinically intact fovea and visual acuity

20/25. c, Before (left image) IAC treatment for an eye with macular retinoblastoma demonstrating excellent

extramacular tumor regression; and after (right image) leaving intact fovea and hope for vision.

Figure 11 | Child Life promotes effective coping through play, preparation, education, positive-touch and

self-expression activities, based on natural child development. Child Life helps children and their families

cope with challenging healthcare issues, hospitalization and therapeutic interventions. Even in palliation, play is

the job of a child. These children play doctor and nurse, wearing gloves and stethoscopes while they examine

Puppet Kevin who had one eye removed for retinoblastoma. Play (medical play and just general play) promotes

a sense of mastery for children within their medical experience. Photo taken at the Sally Test Pediatric Centre,

Moi Teaching and Referral Hospital, Eldoret, Kenya.

Figure 12 | Proposed roles for human L/M-cone precursor circuitry in retinoblastoma tumorigenesis and

targeted therapy. Recent studies suggest that signaling circuitry specific to maturing L/M-cone precursors is

uniquely conducive to oncogenic transformation in response to Rb loss.41, 51, 54 Following cone cell specification

and cell cycle exit, nascent L/M cone precursors prominently express the cone lineage transcription factors

RXRg and TRb2, yet do not take on cone photoreceptor morphology or function. During maturation, L/M- cone

precursors express especially high levels of N-Myc, MDM2, and pRB, and appear to down regulate p27 via a

SKP2-related mechanism. pRB depletion in maturing cone precursors enabled proliferation and survival

dependent upon MYCN, SKP2, and MDM2. Mouse models suggest that E2Fs and CDKs may contribute to the

proliferative response to pRB loss52, although it is not known if they appear in human cone precursors or are

induced following pRB loss. N-Myc, MDM2, and SKP2 are also needed for proliferation and survival of

patient-derived retinoblastoma cells {Xu, 2009 #14691;Wang, 2010 #22470} suggesting that cone precursor

features needed for retinoblastoma initiation also contribute to tumor growth. In addition, SYK was highly

expressed in retinoblastoma but not normal retina{Zhang, 2012 #21116}.

The identification of circuitry mediating cone precursor proliferative response to RB loss provides opportunities

to prevent tumor initiation usingclinical grade agents based on preclinical inhibitors of MYCN expression (JQ1)

{Puissant, 2013 #22473} (Puissant,PMID 23430699), inhibitors of MDM2-mediated p53 degradation (Nutlin

3a){Laurie, 2006 #10712}, (Laurie 2006), inhibitors of SKP2-mediated p27 degradation (C25){Chan, 2013

#22472}Chan,2013 PMID: 23911321), and inhibitors of E2F and CDK activities (HLM006474 and R547,

respectively){Sangwan, 2012 #21115}., Sangwan et al, 2012). Additional targeted agents may be effective

against growing tumors, such as improved versions of the SYK inhibitor R406 (Zhang 2012, Pritchard 2014).

{Pritchard, 2014 #21452;Zhang, 2012 #21116}

Tables

Table 2: Estimated Global Distribution of Retinoblastoma

Forecast births were calculated using most recent data (2012) for population, birth rate and mortality rate

(World Bank (http://data.worldbank.org] accessed on 5 February 2015. Low (1:18,000 live births) and high

estimates (1:16,000 live births) calculated, following the example of Kivela.5[Au: it might also be useful to list

in the table footnote which countries are included in the different categories.]

Category

Population,

total[Au:

approximations

might be

better: 7.0

billion; 1.3

billion; 250

million]

Birth

rate,

crude

(per

1,000

people)

Mortality

rate, infant

(per 1,000

live births)

Forecast Live

Birthstotal[Au:

approximations

might be better

total[Au:

approximations

might be

better]

Retinoblastoma

Cases (High)

1:16,000

Retinoblastoma

Cases (Low)

1:18000

% of World

Retinoblastoma[Au:

is this based on the

high or low

approximations?]

World 7,043,105,591 19.4 34.6 131,629,862 8,227 7,313 100%

High income 1,299,489,520 11.6 5.5 14,941,320 934 830 11.4%

High income:

nonOECD 249,927,994 13.8 10.3 3,418,872 214 190

High income:

OECD 1,049,561,526 11.0 4.4 11,518,845 720 640

Low &

middle

income 5,743,616,071 21.1 38.1 116,699,203 7,294 6,483 88.7%

Middle

income 4,913,580,797 19.2 33.7 91,300,546 5,706 5,072 69.4%

Upper middle

income 2,390,210,774 14.8 16.3 34,737,317 2,171 1,930 26.4%

Lower middle

income 2,523,370,023 23.4 45.3 56,490,388 3,531 3,138 42.9%

Low income 830,035,274 32.3 54.5 25,373,890 1,586 1,410 19.3%

Table 1. Availability of treatment and imaging at retinoblastoma centres worldwide.

Retinoblastoma centres worldwide were surveyed (up to March 25, 2015) and featured on the One

Retinoblastoma World Map (1rbw.org). Information is available for 54 countries (22 High-Income

countries, 28 Middle-Income countries and 4 Low-Income countries, following World Bank

Classification).The estimate of availability of intra-arterial chemotherapy was drawn from

Grigorovskiet al's study.[Au: Only the percentages would be sufficient.]

CountriesTotal

centres

Enucleation

(#, %)

Laser

therapy

(#, %)

Cryo-

therapy (#,

%)

IVC (#, %)

Retcam

imaging (#,

%)

High Income 47 47 (100%) 43 (91%) 42 (89%) 40 (85%) 42 (89%)

Middle Income 75 75 (100%) 68 (91%) 68 (91%) 52 (69%) 47 (63%)

Low Income 10 10 (100%) 5 (50%) 2 (20%) 5 (50%) 3 (30%)

Total 132 132 (100%) 116 (88%) 112 (85%) 97 (73%) 92 (70%)

Supplementary information

Acknowledgements

TWC: NIH NCATS KL2TR001106, Research to Prevent Blindness, Inc. Can contain grant and

contribution numbers. Should be brief, and should not include thanks to anonymous referees and editors,

inessential words, or effusive comments.

Author contributions

Introduction (B.G.); Epidemiology (B.G., H.D.); Mechanisms/pathophysiology (B.G., T.C., D.C.); Diagnosis,

screening and prevention (B.G., H.D., J.Z.); Management (B.G., F.M., D.A., C.S., G.C.); Quality of life (B.G.,

A.W.); Outlook (B.G., H.D., T.C., D.C., F.M.); overview of Primer (B.G.).

[Au: I noticed some discrepancies between the information in the online submission system and the

manuscript – with regard to author contributions. I took the info of the submission system. Please check

very carefully and make changes (with tracked changes on) so that I can update the submission system.

Also, Festus Njuguna does not have any contributions in the submission system; please add information

here.]

Competing interests

In the interests of transparency, the Nature Clinical Reviews journals have a competing financial

interests policy. A detailed explanation of the policy can be found in the Nature website

(http://www.nature.com/nature/submit/policies/competing/index.html). Each author is required to

disclose any relationship, financial or otherwise, that that could be perceived as a conflict of interest.

[Au: We need to have competing interested information before we can send the manuscript for peer-

review. Please add to the system or declare that none of the authors have conflicts.]

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