RetinoblastomaHelen Dimaras1, Timothy W. Corson2, David Cobrinik3, Abby White4, Junyang Zhao5, Francis L.Munier6, David

Abramson7, Carol Shields8, Guillermo Chantada9, Festus Njuguna10 and Brenda L. Gallie11[Au: Please ensure all names are as you would like them fo

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| Beijing Tongren Eye Centre, Beijing, China6| Jules-Gonin Eye Hospital, Lausanne, Switzerland7| Memorial Sloan Kettering Cancer Center, New York, USA8| Wills Eye Hospital, Philadelphia, USA9| Hospital JP Garrahan, Buenos Aires, Argentina10| Moi Teaching & Referral Hospital, Eldoret, Kenya

Retinoblastoma

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

Abramson7, Carol Shields8, Guillermo Chantada9, Festus Njuguna10 and Brenda L. Gallie11[Au: Please ensure all names are as you would like them for indexing (e.g. in PubMed).]

1| Department of Ophthalmology & Vision Sciences, The Hospital for Sick Children& University of Toronto, Toronto, Canada 2| Eugene and Marilyn Glick Eye Institute, Departments of Ophthalmology, Biochemistry and Molecular Biology, and Pharmacology and Toxicology, and Simon Cancer Center,Indiana University, Indianapolis, USA 3| 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 USA 4| Daisy's Eye Cancer Fund, Oxford, UK 5| Beijing Tongren Eye Centre, Beijing, China 6| Jules-Gonin Eye Hospital, Lausanne, Switzerland 7| Memorial Sloan Kettering Cancer Center, New York, USA 8| Wills Eye Hospital, Philadelphia, USA 9| Hospital JP Garrahan, Buenos Aires, Argentina 10| Moi Teaching & Referral Hospital, Eldoret, Kenya 11| Department of Ophthalmology & Vision Sciences, The Hospital for Sick Children& University of Toronto, 555 University Ave, Toronto, Ontario M5G1X8, Canada [Au: Please check and update affiliations. Every affiliation should contain the Department (which needs to be updated for all authors), Institution, City, State and Country. Only the corresponding author has to supply the full postal address.]

11| Department of Ophthalmology & Vision Sciences, The Hospital for Sick Children& University of Toronto, 555 University Ave, Toronto, Ontario M5G1X8, Canada[Au: Please check and update affiliations. Every affiliation should contain the Department (which needs to be updated for all authors), Institution, City, State and Country. Only the corresponding author has to supply the full postal address.]

Abstract 173/200

[Au: Abstracts have no line/paragraph breaks]

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 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, MYCN oncogene, Intra-

arterial chemotherapy, Intra-vitreal chemotherapy, Cone photoreceptor, Cancer therapy, Global health

Competing interests

[Au: Please insert the competing interest statement for each author here, or in the online submission form

with your revised manuscript.]

[Au: Pleasemakesureofficialnomenclatureisusedforprotein(Uniprot)andgene(Hugoanditalics)namesandthattheyarestyledaccordingtospecies(capitalsforhumans,onlyfirstlettercapitalformice). Also make sure that abbreviations are consistently used throughout the manuscript (N-Myc, MYCN, etc.)]

[Au: I’ve inserted HX notations at all the headings to indicate the level of heading, three of which are

allowed.]

[H1] Introduction(BLG)319/300

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

retinoblastomatumours 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 intraretinal white tumour develops after

genomic changes lead to uncontrolled proliferation — the tumour is visible through the pupil (the most common

first sign) (Figures 3, 9) or blocks vision and impedes central visual fixation (the second most common sign).4

When noticed early, prompt treatment usually cures. Later 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 withdiffering severity); too few patients to interest the

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

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

response in the intraocular tumour have been quickly embraced, despite absence of rigorous clinical trials.

The Internet has opened many avenues for retinoblastoma: parents make the diagnosis themselves; colleagues

discuss and share patients around the globe; centres of retinoblastoma excellence can be mapped (Figure 2); and

a common database for all children no matter where they live is within reach. These developments could

empower a learning health system to achieve an evidence base for retinoblastoma care. In this Primer, we

review retinoblastoma at a time when new science, new ideas, new therapies and global collaboration are

unprecedented.

[H1] Epidemiology (HD,GC, JZ, FN) 762/500

[H2] Globalpatients, resources and outcomes[Au: edited to fit within a 42 character limit (which includes white spaces)]

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 decline 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 Rb 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 programme sbenefit 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,I mposing 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). 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.REFurl 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) 1574/1500

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).{Knudson, 1971 #591} 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).{Comings, 1973 #21098} Chromosomal deletions in some

patients pointed to a chromosome 13q14 locus. Loss of heterozygosity of 13q14 polymorphic markers in 70% of

retinoblastoma tumours{Cavenee, 1983 #62} implied that the second hit involved the same locus. The

breakthrough came when one retinoblastoma was missing the sequence of a 13q DNA clone,{Dryja, 1986

#10404} that turned out to be a conserved, exonic sequence of RB1.{Dryja, 1986 #10404;Friend, 1986

#4934;Lee, 1987 #333;Fung, 1987 #21743}

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.{Lohmann, 1999 #4359} 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.{Dick, 2013 #10937;Lee, 2006 #11229} Several “low penetrance” RB1 mutations encode proteins with

minimal ability to bind E2F, and predispose carriers to fewer retinoblastomas than RB1 null alleles.{Lohmann,

1994 #10195} 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 Increasing pRB expression is associated with decreasing p27 during cone precursor

maturation.42 However, pRB N-terminus functions may also be important.45, 46

Biallelic RB1 inactivation is necessary to initiate most retinoblastomas, but is not sufficient; RB1-/- retinal cells

likely undergo apoptosis or limited proliferation to form the benign retinal lesion, retinoma (Figure 4).3 Further

genetic or epigenetic changes are likely needed for malignant transformation.47 Comparative genomic

hybridization studies identified common regions of DNA gain that led to several candidate retinoblastoma

oncogenes: the 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). Consistent loss of 16q22 led

to cadherin-11 (CDH11) as a tumour suppressor gene; whole-genome sequencing identified inactivating

mutations in the transcriptional corepressor BCOR.48 Epigenetic alterations might also drive retinoblastoma

formation by inducing H3K4me3 and H3K9/14ac marks and expression of the SYK oncogene.48 Besides SYK,

many other genes and microRNAs show altered expression in retinoblastoma compared to normal retina.47 Gene

expression profiles may segregate RB1-/-retinoblastomas either into two subtypes or into a spectrum of

phenotypes correlating with histologic and cytogenetic aberrations.49, 50

Although nearly all retinoblastomas have mutation of both RB1 alleles, 1.4% of unilateral tumours have no

detectable RB1 mutation, but instead have high-level amplification of the oncogene MYCN (MYCNA).51

RB1+/+MYCNA are diagnosed at a much younger age than unilateral RB1-/- tumours and have a distinct histology,

reflecting a unique subtype. The trend of increasing MYCN copy number with age at diagnosis and the very

young age of presentation of very large tumours, places the cell of origin of RB1+/+MYCNA tumours earlier in

development than that of RB1-/-tumours.51 Another 1.5% of unilateral non-familial retinoblastomas have

apparently normal both RB1and MYCN genes; an unidentified factor or mechanism may inactivate pRB.

[H2] Cone circuitry sensitizes to RB1 loss

or - 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.

Intracellular signaling specific to the retinoblastoma cell-of-origin lost RB1 inactivation could explain the retinal

tropism. Detection of diverse retinal cell type markers and RNAs in retinoblastomas pointed to a pleuripotent

cell, but could also represent normal RB1+/+ cells, or aberrant expression of oncogenic transformation.{Xu, 2009

#14691;McEvoy, 2011 #16182} Rb1 plus p107, p130, or p27,53 express different retinal markers, and may not

extrapolate to humans.54

Apart from the detection of cone markers that might have beeninduced during transformation],55, 56the first

suggestion that retinoblastomas originate in cone precursors came from evidence that emerging tumourshave a

topographic distribution that mimics the horizontal visual streak characteristic of red and green cones .44Further

support came from evidence that RB1-/- retinoblastomas show consistent expression of cone photoreceptor but

not other retinal cell type-specific proteins and that maturing cone precursors have unusually high expression

ofoncoproteins (MDM2 and N-myc)that could collaborate with RB1 loss.52, 55, 56 Moreover, experimental

depletion of RB1induced cone precursor cell proliferation in vitro and development of experimental tumours

with histological, protein expression, and lack of cytogenetic changes typical of differentiated retinoblastomas 57 Proliferation depended upon high levels of N-Myc and MDM2, cone-specific transcription factors RXR and

TR2, and a down-regulation of p27 that normally occursduring cone precursor maturation.5742 These features

suggest that RB is needed to counter an oncogenic programme associated with cone precursor maturation.

Notably,smalltumours detected via optical coherence tomography (OCT) appeared to be centred in the inner

nuclear layer of the retina, not the outer nuclear layer where mature cones reside.58 However, they also extended

into the outer nuclear layer, and the smallest tumour detected to date had a significant outer nuclear layer

presence (Figure 5c).The apparent growth of early tumours within the inner nuclear layer58 could reflect a

retinoblastoma cell requirement to interact with blood vessels and retinal astrocytes, which are present only in

the inner retina and ubiquitous in tumours, and promote retinoblastoma cell growth in vitro.59Despite progress, it

remains uncertain whether cone precursors originate all RB1-/- retinoblastomas, or only highly differentiated

tumours that have few cytogenetic changes.50

[H2] Translating knowledge of pathogenesis

Understanding retinoblastoma signalling pathways could lead to treatment and prevention opportunities. For

example, pathways specific to embryoniccone precursors could be targeted postnatally .Oncoproteins such as N-

myccould be targeted60 both in RB1+/+MYCNAtumours51 and in the RB1-/- tumours that are also N-myc-dependent. 52 Furthermore, studies based on molecular discoveriesshowed thatexposing retinoblastoma-prone murine

fetuses to small molecule inhibitors of E2f or Cdk inhibited subsequent tumorigenesis without disrupting normal

retinal development53.

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

cell responses. Most in vitro studies have used Y79 and WERI-Rb1 cell lines, which were developed in the

1970s.61, 62Many other lines exist63 and but require further characterization. Primary retinoblastoma cellscan be

implanted asxenografts in immunodeficient mice6448However, caution is needed, becausexenografts implanted

subretinallyor intravitreallyhave a different anatomy relative to the eye’s natural barriers, potentially affecting

drug distribution and limiting translation to patients. Genetically engineered mouse models can also be used to

assess novel treatments.65 While Rb1+/- mice do not develop retinoblastoma, retinal deletion of Rb1 (using

Pax6a, Nestin, or Chx10 promoters) in p107, p130 or p27mutant backgrounds achieves retinal tumour

formation.54 These 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.66 However, since the mouse

tumours require different collaborating mutations and may not originate from the same retinal cell type, their

ability to predict human treatment responses is not certain. Viral oncoproteins can promote murine

retinoblastoma development, such as adenovirus E1A expressed in retinas of p53-/- mice, and Simian Virus 40

T-antigen expressed in developing Müller cellsdirected by the transgene insertion site.67, 68

Subconjunctivaltopotecan was tested in this latter model and is now in the clinic, and Cdh11 was confirmed as a

tumour suppressor by crossing a knockout into this model.69 Thus, much may be learned and translated to the

bedside with available systems while awaiting models that more precisely simulate retinoblast1oma

pathogenesis.

[H1] Diagnosis, screening and prevention(HD, GC, BLG) 1720/1500

Diagnosis is usually clear from presenting signs70 and clinical examination. Biopsies are not performed on

presumed intraocular retinoblastoma due to risk of seeding tumour outside of the eye.71-73]Differential diagnosis

for Coats disease, persistent fetal vasculature, and vitreous haemorrhage is needed.74

[H2] Clinical Diagnosis

Diagnosis is usually clear from presenting signs70and clinical examinationThe most common first sign of

retinoblastoma is leukocoria (white pupil), which is caused by light reflecting directly off the tumour(Figure 6)

Often parents notice leukocoria, but cannot convince their health advisors that there is a problem. The second

most common sign is strabismus, or misaligned eyes, due to tumour blocking central vision. Signs of advanced

disease include change in iris colour, enlarged eye due to glaucoma, and orbital cellulitis, a dangerous eye

infection.75[?]Proptosis (protrusion of the eye from the socket)[?]presents very late, but commonlyin countries

where awareness and resources are poor. Importantly, as awareness efforts are being implemented globally,

advanced retinoblastoma is declining.

Unlike most cancers for whichpathology provides definitive diagnosis, diagnosis of retinoblastoma is wholly

clinical; biopsy incurs risk of metastasis.71-73Observation of leukocoria necessitates rapid referral to an expert. A

dilated eye exam (dilation of the pupil) with indirect ophthalmoscope (retinal camera) is performed to provide a

clear view of the retina, macula and optic nerve. ?The OPTOS® (Optos, UK) ],a non-mydriatic, wide field

camera of 200°, might be useful for a rapid view of the retina, with minimal intrusion on the awake

child. Calcification, a typical characteristic of retinoblastoma , is easily detected by ultrasonography(b-scan) .

MRI is used to detect infiltration of the optic nerve and trilateral tumours (intracranial tumours associated with

RB1 mutations such as pinealoblastoma and primitive neuroectodermaltumou][.. CT scansshould beavoided if

possible owingto oncogenic toxicity of radiation in these children.

Differential diagnosis for Coats disease, persistent fetal vasculature, and vitreous haemorrhage is

needed.74Detailed retinal examination under general anaesthesia almost always confirms the diagnosisand

facilitates classification of severity of intraocular disease.The hand-held fundus camera (RetCam®, Clarity,

USA ) is used to view and record the whole retina (Figures 7, 8), with views spanning all the retina fields with

the wide field lens (130° or 120°). Scleral depression(physical manipulation of the sclera) to visualize

theoraserrata(the junction between the retina and the ciliary body)is critical for accurate classification of the

retinoblastoma , andoptic nerve arterial pulsations are watched for as indication of excessive pressure on the

eye.When available,high frequency (50 MHz)ultrasound biomicroscopymonitors,76and OCT can discover

invisible tumours in infants with familial disease (Figures 5).77Intravenous fluorescein angiogram with the

RetCam®can evaluate vascular abnormalities, suspicious residual or recurrent tumour, new vessel activity in

scars, and areas of non-perfusion. Very tiny intra-retinal tumours not yet vascularized are better discovered with

OCT. Imaging 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 classification78for intraocular retinoblastoma predicted outcomes of external beam

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

common primary treatment to salvage eyes, new schemes were developed (Figure 7, Extended Data 2). An

international collaboration, led byMurphree, developedthe International Classification of Intraocular

Retinoblastoma (IIRC) which better predicted responses to IVC than did the Reese-Ellsworth classification

.79Eyes are classified by the IIRC with groups ranging from A to E, with E being the most severe. However,

subsequent modifications of the IIRC group definitions without an overt change to the Classification Scheme

name (Figure 7).The subsequent Shields version of the IIRC 80, 81 results in discrepant classification of 25% of

the most severely involved eyes when compared to the original IIRC definitions (Figure 7).82, 83Subsequently, the

Children’s Oncology Group (COG) used a variation84 with minor measurement differences with the IIRC

(Figure 7).

Similarly, for extraocular retinoblastoma, a number of staging systems co-exist and none has gained uniform

acceptance. The St Jude Staging System, reported decades ago, is still used, especially in Latin America. In a

coordinated action with the originalMurphreeIIRC 79collaboration, the International Retinoblastoma Staging

System focused on the overall staging of cancer including [retinoblastoma (Extended Data 2).85 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 retinoblastoma86 is not widely

used.There is a strong need for a single, collaborative, consensus classification of eyes and cancer staging.

[H2] Pathology

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

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

Wright rosettes, common in diverse neural cancers (Figure 4)[.87Retinomas feature more differentiated

photoreceptor-like clusters of cells termed fleurettes (Figure 4).3

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

optic nerve, post lamina cribosa, cut end of nerve; invasion of uvea ≥3 mm dimension; or both optic nerve and

uveal invasion)and establish pathological staging. []88-92 High-risk features are observed in 17% of Group D and

24% of Group E eyes90using Shields classification, and 15% Group D and 50% Group E88using the Murphree

classification.79 The documentation of high-risk pathological features is important to recognize need foradjuvant

systemic chemotherapy to reduce risk of metastatic relapse.93

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

features.92, 94Pathology specimens are staged using the pathology TNM (pTNM) and the International

Retinoblastoma Staging System (Extended Data 2).86, 92Still, 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)95 and N-

glycosylated ganglioside (NeuGc-GM3)96 mayfacilitate observation of a few tumour cells lurking in a high-risk

location.

[H2] Genetic Diagnosis

The majority (94%) of patients withretinoblastoma are the first individuals to be diagnosed in a family, only141

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

retinoblastomawhen tested for RB1 mutations,(data from Impact Genetics, January 2015). Approximately 50%

of people with retinoblastoma carry one RB1 mutation in their constitutional cells (100% ofbilateral and 15% of

unilateral patients).97The remaining unilateral retinoblastomas arise by somatic mutational events (biallelicRB1

loss or somatic amplification of the MYCN oncogene) with no additional cancer risks.

Knowledge of the patient’sRB1mutation enables precise screening of relatives and subsequent generations. 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. Without genetic testing, it is recommended that children who are at risk( already one eye affected ,and/or

positive family history) continue to undergo multiple exams under anaesthesia.Whengenetic tests canconfirm

that no mutant RB1 gene was inherited , 85% of patients with unilateral retinoblastoma will test negative with

<1% residual risk for undetectable low level mosaicism, which informs the decision to reducetheintensity of

surveillance and accurate determination of cancer risks of family members.10Pre-implantation genetic diagnosis

offers the family the option to selectunaffected embryoswhen the mutation ina parent is identified.98, 99

Characterization ofunique RB1 mutations of the tumourDNA (if available) can providea highly sensitive marker

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

are the RB1tumour mutation (when mutation is not present or different from the germline alleles),100the

signature of post-RB1 genomic gains and losses 101and CRX expression.95

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

counsel families. Parentsneed 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.102 The authors inferred that the probandsdid 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 inheritancewould inform counselling approaches that meet the needs

of families.103

[H2] Screening

[H3] Screening througheye examination[Au: edits OK? Dashes are not allowed]

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, onenegativechildhood screening

test doesnot mean this child will remain tumour-free since a tumourmight 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[Au: reference?]. Photoleukocoria refers

to the appearance of leukocoria on flash photographs (Figure 6). Awareness campaigns bring children to

attention, but are lose effectiveness with time. An innovative study called PhotoRed in India trained healthcare

professionals to used flash photography to identify childhood eye diseases, including retinoblastoma.104Another

pioneering project is developing software to enable cameras to detect photoleukocoria.105A 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 radiotherapyhave an elevated risk of

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

cancer.106 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).107 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.108

[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 ideaswill prevent second cancers

is unknown.

[H1] Management(FLM, CS, DA, GC, FN, JZ, BLG) 4012/3000

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 opportunitiesfor

the family, and accesstoexpert care.70, 109

[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.109 In order of approximate frequency of

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

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

whentumours aresmall at diagnosis. External beam radiotherapy (EBRT)is no longer recommended for primary

intraocular retinoblastoma, since radiation incurs a very high risk of second cancers on persons carrying an

RB1mutation, especially in the first year of life.110, 111The preferred primary treatment depends on the severity of

disease of each affectedeye(Figure 8).Bilateral retinoblastoma in much of the developing world is best treated

with bilateral enucleation, where there is lack of expertise, equipment and resources, and especially when there

are difficulties with close monitoring.4, 16Manybilaterally enucleated retinoblastoma survivors lead active,

productive and satisfying lives because they were treatedby timely surgery as infants.

[H3] Second-Line (Salvage) Therapy

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

failed the first plan. All retinoblastoma treatments involve multiple modalities, and a range of modalities is

appropriate for second line therapy. However, each subsequent plan has a lower success rate112 and long drawn

out attempts to salvage an eye incur high costs of many kinds for the child and family.113, 114

Second line treatments have included focal therapies including peri-ocular chemotherapy,115 repeated systemic

chemotherapy,115, 116 repeated IAC,112 iodine or ruthenium radioactive plaque (brachytherapy),117, 118 EBRT119-121

and tantalum ring localization122 or whole-eye123Criteria for secondary enucleation are not well defined but are

dominated by refractory subretinal and vitreous seeding,116, 124 complications that suggest risk of extraocular

extension123such as vitreous haemorrhage and secondary neovascular glaucomaand socio-economic and

psychological fatigue to save an eye with poor vision.11479 eyes, high risk pathology is uncommon. However,

secondarily enucleated eyes showing scleral invasion many developextra-ocular relapse;125and Group E79eyes

treated with neo-adjuvant systemic chemotherapy before enucleation have significant risk for death from

retinoblastoma.126

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

and/or enucleation).121127128and a combination of repeat IAC and intravitreal chemotherapy (IViC) may playan

important partin saving eyes that have failed IVC.128-130 However, extensive treatments to save an eyeincrease

risk for metastases.112, 131, 132

Despite advances in the eye-salvage therapiesforadvanced retinoblastoma, the major cause of failure has

remainedvitreous seeding. Pharmacokinetic studies show poor vitreous levels of drugs administered by either

systemic chemotherapy or IAC.133 The highest drug bioavailability in the vitreous is achieved by IViC using a

safety-enhanced injection technique in carefully selected eligible eyes.134 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.134-136134The 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 salt-and-

pepper retinopathy.137

Combinations of new administration routes 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. Conversely, eyes with relapse touching the optic nerve head and/or vision-

critical regions such as the maculo-papillary bundle, and eyes with diffuse retinal/subretinal recurrence,

represent good indications for IAC, which might achieve better visual outcome than focal treatments. However,

therapies that have already failed to control the intraocular tumour are unlikely to succeed as salvage therapy for

the same eye.

[H2] Ocular Therapies

[H3] Enucleation

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

cheapest treatment for theseeyes138, 139. Since the majority of children with intraocular retinoblastoma have

Group D or E diseaseat diagnosis, and more than 50% have unilateral disease with other eye unaffected, cure

can be achieved with enucleation (Figures 3 and 8). All IIRC79 Group E eyes require enucleationbecause,by

definition, they carry risk of extraocular extension; however, this can only be confirmed by thepathological

assessment of the enucleated eye. Best cosmetic outcome is achieved by replacement of the volume of the eye

with an implant buried in the orbit, and provision of a prosthetic eye, worn in the conjunctival sac. Many

different reconstruction techniques are used worldwide.140 Comparative studies have shown enhanced prosthetic

eye motility with the myoconjunctival approach, which is affordable world-wide.141, 142Complex integrated

implants are commonly used but have a higher rate 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,143

observed in Kenya to help the closefamily accept enucleation for their child.

[H3] Intravenous chemotherapy and focal therapy

Since 1996 first-line therapy to control IIRC Groups B, C and D diseasehas 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).124, 144, 145Sometimes high dose acute cyclosporineis

added to modulate multidrug resistance.146, 147Eyes in groups B and C do well with CEV and focal therapy. With

follow-up of 54 months, 47% of IIRC79Group D eyes121 and 47% of Reese-Ellsworth Group V124 avoided

enucleation or EBRT(Figure 9).81

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.148 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),149 second non-ocular cancer risk with alkylating agents150, 151 and secondary acute myeloid leukaemia

following intense chemotherapy including topoisomerase inhibitors, doxorubricin and alkylating agents.152, 153

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

under[Au:general?]anaesthesiais very important (Figure 9).154-156Tumours in the macular region, which threaten

vision,are particularly at risk of recurrence without focal therapy.157

Following control of retinoblastoma with IVC, 50% of patients have visual acuity (acuteness and clearness of

vision) at 5-years of 20/20-20/40; 67% have 20/200 or better.158, 159Foveal involvement with tumour or subretinal

fluid at presentation contribute to poor vision. There is no documented local toxicity of IVC to the eye.

[H3] Intra-arterial chemotherapy

Intra-arterial chemotherapy [Au: IViC deleted since it is not discussed in this section; OK?]has had a great

impact on retinoblastoma management as it enables saving of eyes that were previously deemed unsalvageable.

IAC is usually performed by ophthalmic artery chemosurgery (OAC, also called superselective ophthalmic

artery infusion)[Au: OK?]whereby, after the patient has been administered heparin,a micro-catheter isinserted

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) areinfused in a pulsatile

fashion over many minutes.160Alternatively, a catheter with an inflatable balloon near the tip can be used to

ensure selective treatment to the eye.During the procedure, theinternal 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).131Cannulation 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 used.161Also, tandem therapy in which both

eyes are treatedin the same, one hour session has successfully been performed (so-called tandem

therapy).162OAC is currently performed in more than 30 countries worldwide of whichnearly half in developing

nations.163

Of the more than 2,500 infusions and >800 patients in the literature using OAC, 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?].129 However, death from metastatic diseasecan occur more than five years after any

treatment. The longest follow-up study described forselective ophthalmic treatmentin Japan (including death

from metastatic retinoblastoma and second cancers) showedan overall survival of 95% at 15 years.131However,

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.156

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 classifications79, 80, 84 (Figure 7, Extended Data

2), which prevents a clear-cut comparison of the results between centres, especially regarding Group D and E

cases. We need a universally endorsed single classification in order to avoid confusion regarding eligibility and

salvage rates for new treatments. Ocular survival with the selective ophthalmic treatment65% for Group C

and45% for Group D in one Japanese study [Au:OK?].131 Using OAC in the USA, ocular survival was 96% in

both Groups B and C, and 94% in Group D.129, 164 Eyes with neovascular glaucoma, pthisisbulbi (a shrunken,

non-functional eye) , and anterior chamber involvement were universally enucleatedup 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 be87.9% at two years and 76% had complete

retinal reattachment as a result of IAC alone.165 The most common reason for second-line [Au:OK?]enucleation

of eyes with retinoblastoma in developed countries has always been the presence of extensive vitreous

seeding.166 Only 20% of such eyes can be salvaged with EBRT.167With OAC, 74% of eyes with seeding have

been salvaged(Figure 10b).112, 129, 130With the selective ophthalmic treatment, 58% of children with foveal

tumours retained a visual acuity of >0.01[Au: unit necessary? Earlier in the article, the 20/20 etc scale was

used for acuity. Please be consistent with which scale you use to avoid confusion] and for those without

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

OK?]monitoring of OAC patients160, 168-171 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.172[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%).129Lasting complications included vitreous haemorrhage (2%), branch retinal artery

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

choroidalischaemia (2%), and optic neuropathy (< 1%).129These complications are minimized at experienced

centres.

Historically, consolidation therapy (continued treatment once remission has been achieved)[Au: definition for

nonexperts OK?]was necessary in the majority of irradiated eyes and in nearly 100% of the eyes initially

treated with systemic chemotherapy 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.173, 174

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.175

[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 IIRC Group A cancersand consolidation therapy for residual or recurrent small-volume, active

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

atrophic or calcified.

Transpupillary thermotherapyinvolves 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 freezethetumour through the sclera with a nitrous oxide probe; the tumour

is directly visualized and duration of freeze judged to completely eliminatethe 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 delivertrans-scleral radiotherapy withan 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 recurrenttumour in a location that will not compromise vision.

Paraocularchemotherapy has been usefulunder conjunctiva or in tenon’s capsule for small volume recurrences

and vitreous seeds.176-178

[H3] Intravitreal Chemotherapy

Vitreous seeds are the major cause of failure (enucleationor 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.134, 135, 137, 179, 180[Au: Sentence in green

moved up]The procedure involves lowering theintraocular pressure with an anterior chamber paracentesis or by

digital massage after induction of anesthesia.Intravitrealmelphalanis 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.135Ultrasound biomicroscopymay be used to evaluate the otherwise

hidden ciliary region behind the iris,76 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. Children with

orbital retinoblastoma, which may be massive and disfiguring, benefit from up-front adjuvant chemotherapy.

The preferred chemotherapeutic agents are carboplatin, etoposide and vincristine, as for intra-ocular

retinoblastoma; other agents that are useful include cisplatin, cyclophosphamide and anthracyclines.16Those who

present with overt extraocular disease have a low chance of survival, especially in low-income settings.

Chemotherapy followed byenucleation, orbital radiation, adjuvant chemotherapy, intrathecal chemotherapy and

high dose chemotherapy with stem cell rescue have potential for cure.

[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 diseasehas good outcomes.89, 91 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.126, 132 Bone marrow metastasis without central nervous system

involvementhas potential for cure with extensive therapy including stem cell transplant. However, extension of

retinoblastoma into the brain has a very low likelihood of cure.

[H2] Palliation [Au: should this be a subheading under ‘Management’ in general, or under ‘Extraocular

retinoblastoma’? I think the former, but wasn’t certain]

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 even when no curative intent is pursued. These children often present with severe

emaciation needing prompt medical treatment. Easily available, moderate intensity chemotherapy should be

offered to these children since life prolongation will be likely achieved and their quality of life will significantly

improve. Options include the combination of cyclophosphamide (which may also be administered orally) and

vincristine, or carboplatin and etoposide, which will seldom cause severe toxicity. Radiotherapy may also be

helpful, especially for a CNS relapse or for the treatment of massive orbital extension. EBRT and chemotherapy

are useful for pain control in palliation but are often unavailable in low-income countries.[Au: references?]

[H1] Quality of life(AW, HD, BLG) 881/500

[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 181, 182in short and long-term verbal memory, verbal learning

and verbal reasoning abilitiescompared with those diagnosed at over 1 year of age.181, 182 In bilaterally affected

children, whole brain radiation exposure at any age was significantly associated with poorer verbal

memory[Au:reference?].181, 182In children followed up from diagnosis to age 5 years, trajectories of

developmental functioningdeclined over time.182 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.183No 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.184, 185181182There is clinical and animal evidence that

repeated anaesthesia of young children may impair neurocognitive development.138, 186, 187Repeat 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 anaesthesiaandage.

[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 survivorsinto peer-support communities. Research processes that deepen understanding of quality of

lifefollowing retinoblastoma are needed to learn from these valuable evidence sources.

[H3] Coping during treatmentand 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.188-194Repeated

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.113, 195-201at 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,

106, 202-211

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, manydescribe 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.181 However, reduced vision causes some children to become

frustrated by their inability to keep up with peers, damaging self-esteem and confidence.212-214

[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 lifeafter radiotherapy.[Au: reference?]

[H2] Second cancer risk

Individuals at risk of second primary cancers require life-long oncology follow up.150, 215, 216Lack of agreed

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

compounded by primary doctors who are not awareof late effects and lifelong implications of RB1

mutation.Full information about the patient’scancer history, genetic status and life-long risks will empower

survivors to be advocates for their own and their children’s health.217-220

180211-213

[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) 1332/1000

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

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, responding to the immediate obvious targets for improvements, and setting the stage for

innovative discoveries in retinoblastoma.

[H2] Patient-centered, global research focus[Au:OK?]

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. [Au:

Deleted description of AHOPCA to avoid repetition]Today we are faced with sweeping change in treatment

of retinoblastoma usingIAC 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. For

example, AHOPCA created and implemented multicentre protocols for retinoblastoma treatment,21, 23, 24 a major

achievement not yet paralleled in developed countries.

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 trialswill 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 achieving 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 and replace the current clinical staging

schemes that confuse analysis or eligibility or outcomes.79, 80, 86

We are poised to collect uniform data on all children with retinoblastoma. 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”,221 to assist in

selection of evidence-based care for the next affected child.

[H2] Cause of every death from retinoblastoma

We propose to determine the most likely cause and key risk factors of every death from retinoblastoma.Similar

in concept to the groundbreaking Million Death Study,222 this information will guide us to target preventable

deaths quickly, by simply developing awareness of the disease, clinical research, and strengthening of health

systems.223The 1RBW.org map documents staff, equipment [Au:OK?], and spaces, and provides an alternative

system of global collaboration, perhaps unique to small neglected diseases that fall through the cracks even in

the most developed health care system.Implementation of this knowledge can help tostreamlinereferral and the

speed of pathology reporting.

[H2Targetedtherapies

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,wasNutlin-3.224 Nutlin-3 targets the negative regulation of p53 by MDM2/MDM4, resulting

in p53-mediated apoptotic cell death. Nutlin-3 showed promising activity in combination with topotecan for

retinoblastoma control in preclinical models. This drug combination is currently undergoing evaluation in a

prospective study in combination with local chemotherapy.As mentioned, gene expression and epigenetic

analyses of retinoblastomas identified the spleen tyrosine kinase (SYK) as required for retinoblastoma cell

survival.48Although the SYK antagonist R406was considered a promising candidate based onpreclinical studies,

its ocular pharmacokinetic profile following peri-ocular administration rendered it unfavourable for clinical

use.225 Preclinical studies have sought to identify new molecular targets and new agents to block the drivers of

tumourigenesis in retinoblastoma, and to evaluate pharmacokinetics of new agents alone or in combination226

before clinical deployment. However, few translational studies have actually resulted in significant changes in

retinoblastoma clinical practice.227 Innovative delivery systems to the vitreous to targetvitreous seeding have

been evaluated and include devices for sustained release preparations or metronomic administration

viaperiocular or intravitreal routes,episcleral implants228 or nanoparticles229. They are not currently used in

clinical practice albeit for restricted indications. Other agents such as those targeting the tumour vasculature230

or hypoxia231 have been evaluated in preclinical models but have not yet progressed to clinical use. Alsonew

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

targeted threatments[Au: OK?].232

Improved understanding of retinoblastoma pathogenesis (Figure 1) could yield additional agents that with

appropriate clinical evaluation may significantly change future treatment.

[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) Biallelic loss of RB1 may block differentiation of a cone precursor

cell, as itfails to localize normally. b) Genomic instability might leadto an intraretinalbenign 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 tumourcan progress to form a retinoblastoma if additional mutations are acquired [Au:OK? Or do all the

retinomas progress to retinoblastomas], and seeds under the retina and into the vitreous; d) invades past the

lamina cribrosa of the optic nerve, uvea, or sclera to constitute high risk pathologic features. e) Finally, the

tumor mightextend extraocularly into orbit, brain (direct or subarachnoid to CSF) or metastasizes 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.31 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;79 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[Au: inactivating?YES]inactivatingmutation (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 transition to

retinoblastoma. Non-heritable, RB1 mutant retinoblastomas follow a similar progression, except both M1 and

M2 occur in a susceptible retinal cell. MYCN-amplified (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. Conversely,

MYCNAretinoblastoma show a distinct histology with rounded nuclei and prominent nucleoli.[Au: Can you

please provide a short explanation for the histological images?]

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

cell types. Haematoxylin and eosin staining of post-fertilization week 19 retinashowsthree 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 RB. Immunofluorescence staining of post-fertilization

week 19 retina showsespecially strong RB 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).RB 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 toextend

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

infer the retinal layer from which the tumor arose.This layer-of-origin may be definedin 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.

[Au: Where should this text go? Please take into account that legends should not exceed 200 words.]Retinoblastomas

are thought to originate in developing retinal cells that have biallelicRB1 inactivation. The prominent expression of cone-

specific proteins in retinoblastoma cells combined with high-level pRB, MYCN, and MDM2 in cone precursors and the

proliferative response of RB1-depleted cone precursors have provided compelling evidence of a cone precursor

origin34,38,39.Although cone precursors are among the earliest post-mitotic retinal cell types produced, they lie dormant for

several weeks to months prior to expressing pRB, cone phototransduction proteins, and the oncoproteins that may sensitize

to pRB loss. 34,39Hence, retinoblastomas have been proposed to arise following cell cycle re-entry of this normally post-

mitotic retinal cell type. 38

The figure displays the locations of maturing cone precursors relative to other cell types in the developing retina. Because

the retina develops in a central-to-peripheral direction, the central retina is developmentally far more advanced than the

periphery, and different retinal cell maturation states can be viewed in a single histologic section. Panels ( a) and (b) display

the different histologic appearance and pRB expression patterns in the relatively mature and post-mitotic central retina ( left)

and in the immature far periphery (right).In the central retina, postmitotic cone precursors in the outer nuclear layer highly

express pRB (red) and cone arrestin (green).However, in the immature peripheral retina, pRB is most highly expressed in the

retinal progenitor cells in the neuroblastic layer, but neither pRB nor cone arrestin is detected in immature post-mitotic cone

precursors. 39

Despite that maturing cone precursors but not other retinal cell types proliferate in response to pRB depletion 38, and that cone

precursors are located in the outer nuclear layer, the smallest and presumably earliest retinoblastomas that have been

detected in infants using optical coherence tomography have been centered in the inner nuclear layer (c).This poses a

challenge to the cone precursor origin model, yet might be consistent with a migration of RB1-deficient cone precursors from

the outer nuclear layer to the inner nuclear layer, where they could interact with astrocytes and blood vessels that enable

tumorigenesis.41

Figure 6 | Photoleukocoria led the parents to diagnose retinoblastoma using the Internet (a). The left eye

was enucleated. The other eye was normal at diagnosis and developed later a small tumour treated with only

laser. b, Subsequent MRI revealed thathe also had trilateral retinoblastoma. He was treated with systemic

chemotherapy, intrathecal(given directly into the cerebro-spinal fluid) chemotherapy and high dose

chemotherapy with hematopoietic stem cell transplant, c,The child is disease-free at age 8 years.d, His treatment

summary is illustrated in the DePICT view of eCancerCareretinoblastoma.

Figure 7 | Many different classification schemes for intraocular retinoblastoma confound comparison of

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

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

importantly, size of tumour alone does not make an eye dangerous by Murphree, Children’s Oncology Group

(COG), or TNM classification; but any eye with tumour >50% of eye volume is E (advanced-stage disease)by

Shields classification.The consequence is widespread confusion in the literature undermining clinical research,

since studies using the different classifications cannot be compared for outcomes.

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

Classification (Murphree). 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. Group A eyes can be treated with laser or

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

chemotherapy followed by focal therapy(Group B) and intravitrealchemotherapy(IViC) with melphalan(Group

C). Isolated single tumours inGroup 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 ofmetastasisrequires

adjuvant chemotherapy.

Figure 9 | Good responses to intravenous chemotherapy.a,Before (Left) 4 cycles of carboplatin, etoposide

and vincristine (CEV) + cyclosporine (CSA) chemotherapy and laser consolidation for Group B/ D[Au: Please

specify what the rows are: upper row: B level eye and lower row: D level eye?]; and after (Right) showing

good response and recovering fovea at edge of calcification.b, Before (Left) 4 cycles of CEV + CSA

chemotherapy and laser consolidation for Group D/ D; and after (Right) showing good response with minimal

focal therapy (15 years follow-up).

Figure 10 | Resolution of retinoblastoma with intra-arterial chemotherapy (IAC).a, Before (Left) and after

(Right) IAC for a retinoblastoma eye with extensive retinal detachment; b, Before (Left) and after (Right) IAC

for a retinoblastoma eye with extensive vitreous seeding; c,Before (Left) and after (Right) IAC treatment of an

eye with macular tumour.

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.

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 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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