Retinoblastoma XXX/8000Helen Dimaras, Junyang Zhao, Timothy W. Corson, David Cobrinik, Francis L. Munier, David Abramson, Carol Shields, Guillermo Chantada, Festus Njuguna, Abby White, Brenda Gallie

Abstract 225/200

Retinoblastoma is the rare cancer of infant retina that revealed the genetic basis of human cancer. Tumors form when both RB1 alleles mutate in a susceptible retinal cell, likely a cone photoreceptor precursor cell. The tumor suppressor functions of pRB are related to cell division and genomic stability, but the molecular basis for tissue specificity remains unknown.

Although retinoblastoma is the same disease in all 8000 children diagnosed each year, >95% survive in high-income countries, but <30% may survive globally. Collaborations of stakeholders are improving outcomes, by increasing awareness for earlier diagnosis, sharing expertise, and developing guidelines.

Two new therapies have emerged to salvage eyes with retinoblastoma: intra-arterial and intra-vitreal chemotherapy. Retinoblastoma can respond dramatically to these therapies. We look forward to international collaboration replacing the multiple different classifications of eye involvement with clear definitions, to facilitate rigorous assessment of eligibility, efficacy and safety.

Heritable retinoblastoma survivors are at risk for second cancers. We look forward to the molecular basis of RB1 retinal specificity that may explain specificity of second cancers and lead to cancer prevention.

The Internet empowers a world map (1rbw.org) and retinoblastoma-specific medical record. We look forward to new kinds of evidence: a learning health system can mine previous outcomes to provide evidence for the next child’s management. We look forward to optimizing care for every child with retinoblastoma, wherever they live.

Keywords

Retinoblastoma, Pediatric oncology, Pediatric ophthalmology, Tumour suppressor gene, MYCN oncogene, Intra-arterial chemotherapy, Intra-arterial chemotherapy, Enucleation, Cancer therapy, Global health

Introduction 333/300

Gallie

Retinoblastoma is a rare cancer initiated by mutation of the retinoblastoma gene (RB1) in a specific developing retinal cell, resulting in cell division rather than differentiation. Biological processes first revealed in retinoblastoma led to recognition that all cancer is initiated and progresses, by altered genes. Cost-effective translation of this knowledge has improved outcomes for affected families.

The number of children affected depends on the birth rate and infant death rate, available for each country. The disease starts in an RB1-/- susceptible retinal cell that fails differentiation (retinoma) (Figure 1). Genomic changes release proliferation to intraretinal white tumor that is visible through the pupil of the eye (most common first sign) or block vision causing the eye to lose central visual fixation (second most common sign). When these signs are first noticed, prompt treatment likely cures. If not, the cancer invades beyond the eye, (into the optic nerve, brain; usually incurable) or metastasizes through blood (particularly bone marrow; may be curable with modern medicine). Clinical trials in retinoblastoma are difficult for multiple reasons: too few patients in high income countries; complex disease presentation (two eyes of different severity); too rare to interest the pharmaceutical industry; multidisciplinary collaboration is necessary; and eyes and vision have high value in society and blindness is poorly understood. New technologies showing a dramatic primary response in the intraocular tumour have been quickly embraced for eye salvage. Overall world survival may be as low as 30%; but with resources retinoblastoma survival is >95%.

The Internet has opened many avenues for retinoblastoma: parents make the diagnosis themselves; colleagues discuss and share patients around the globe; centers of retinoblastoma excellence are mapped (Figure 2); and a common database for all children no matter where they live is within sight, that could empower a learning health system to achieve an evidence base for retinoblastoma care. We review retinoblastoma now, at a time when new science, new ideas, new therapies and global collaboration are unprecedented. The concept of One Retinoblastoma World (1RBW) is a reality.

Epidemiology 789/500

Dimaras, Festus, Zhao, Chantada

We first explore through the lens of epidemiology, “the branch of medicine that deals with the incidence, distribution and control of disease”.

Distribution of patients & resources

The expected retinoblastoma patients annually per country can be calculated by multiplying the retinoblastoma incidence (1 in 16,000-18,000 live births) by forecast births (forecast births = population x birth rate x [1 - infant mortality rate]). (Table 1, Extended Data Table 1).1-3

Global retinoblastoma patients reside in high-income (11%), middle-income (69%) and low-income (20%) countries. Most centres are in middle- and high-income countries, but most children are in middle- to low-income countries, creating a gap in healthcare access (Figure 2).

The 1RBW Map of Treatment Centers (www.1rbw.org) (Figure 2, Table 2) aims to connect affected families to expert care; promote evidence-based retinoblastoma treatment; and facilitate enhanced collaboration. Understanding where and how retinoblastoma children are worldwide can empower multicenter referral and co-management, keeping children close to home while optimizing their access to advanced therapies.

Solutions for global retinoblastoma

In epidemiology and global health, income is a surrogate (reliable figures exist, e.g. World Bank) for non-economic measures of quality of life (e.g. life expectancy, child mortality, education). Retinoblastoma in low-income countries is associated with low survival (30%4, 5) compared with high-income countries (>95%6), but solid data is lacking.

Poor outcome correlates with late presentation, difficulty accessing retinoblastoma-specific health care, and socio-economic issues leading to poor compliance (family decline of enucleation and abandonment of therapy).7-9 Primary enucleation is the most expeditious treatment that minimizes on going interventions, enabling high-risk pathological features to guide further treatments. Without timely diagnosis and appropriate treatment, difficult-to-cure metastatic disease may ensue. The good news is that with early diagnosis, many eyes can be safely treated to support a lifetime of good vision, pointing to key elements to focus on globally: awareness, collaboration, and affordable expert care.

Guidelines

In 2009, Canadian National Rb Strategy (NRbS) published the first-ever retinoblastoma clinical practice guidelines,6 adapted by the Kenyan National Retinoblastoma Strategy (KNRbS) and published in 2014 in partnership with the Kenyan Department of Health10. In 2013, the Pediatric Oncology in Developing Countries Committee of the SIOP (International Society of Pediatric Oncology) published a consensus guideline for management of retinoblastoma in countries with limited resources11 with clear ideas that can shape resource development.

Twinning programs

Peer-to-peer collaborations and twinning programs build 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.12 Retinoblastoma-specific twinning programs include partnerships between St. Jude’s Children’s Research Hospital (USA) and the Middle-East,13 Central America14 and Mexico,15 and between the Institut Curie and a centre in Bamako, Mali.16 The Central American Association of Pediatric Hematology Oncology (AHOPCA) created a cooperative group and implemented multicentre protocols for retinoblastoma treatment,14, 17-19 a major achievement, not yet paralleled in developed countries. The AHOPCA funding is now 90% local. Twinning programs 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 programs.20, 21

National retinoblastoma strategies

The Canadian NRbS and the KNRbS include all concerned about retinoblastoma (ophthalmologists, oncologists, pediatricians, pathologists, nurses, Child Life22 specialists, parents, survivors and others) and facilitate collaboration among the institutions that treat retinoblastoma. A major was publication of the KNRbS Guidelines10 by the Kenyan Ministry of Health. The Mexican National Strategy15 and the Brazilian SOBOPE (Brazilian Society of Pediatric Oncology)23 guidelines are also applicable at a national level with governmental support for treatment.

The KNRbS achieved standardization of pathology processing and reporting which supports treatment decisions and discussion of prognosis with families. Adoption of upfront enucleation with implants and immediate prosthetics 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.10, 24

A unique model has developed in China. About 1100 newly diagnosed cases are forecasted annually, scattered over 32 provinces imposing high costs related to travel. Before 2005, enucleation was the only treatment available for most children. For the better treatment and follow-up, multiple centres classified by expertise and resources were established in 28 hospitals covering 25 provinces (over 90% of the population). With efficiency and collaboration, 2097 new diagnosed retinoblastoma patients were classified and treated on common protocols from 2006 to 2014 with improvements of survival (unpublished data).

In different socio-economic situation, a single centre with high expertise coordinated affiliated retinoblastoma clinics in Argentina. This collaboration of governmental hospital, and local and international NGO’s, significantly improved survival in two decades with prospective protocols.25 In addition, translational research programs and population-based incidence and survival studies are reported.26, 27

Disease mechanisms/pathophysiology 1640/1500

Corson, Cobrinik, Gallie

Retinoblastoma has provided numerous insights into tumorigenesis mechanisms. In 1972, Knudson plotted the age at diagnosis of bilateral vs. unilateral patients and discovered that bilateral patients were diagnosed earlier. Thus, retinoblastoma formation was consistent with one rate-limiting event in bilaterally affected individuals (i.e., in those with heritable disease), and with two events in unilaterally affected children with no family history.28 Based on these results, Comings proposed that hereditary cancers result from a heritable germline mutation (first hit) and an acquired somatic mutation (second hit) in a single transformation suppressor gene in the cell of origin.29

Subsequently, this two-hit model guided investigators in their search for the retinoblastoma gene, RB1. Chromosomal deletions in some patients led investigators to chromosome 13q14. Loss of heterozygosity (LOH) of 13q14 polymorphic markers in 70% of retinoblastoma tumours30, 31 implied that the second, somatic mutation involved the same genetic region,

consistent with two hits in the same gene. Eventually, Southern blotting showed complete loss from a tumour of a 13q probe31 that was expressed in mRNA and turned out to be a conserved, exonic sequence of RB1.31-34 While RB1 was the first tumour suppressor gene to be cloned, similar searches for genomic LOH led to discovery of many suppressors of heritable cancers.35

RB1 is a large (190 kbp) gene with 27 exons. Many different mutations knock out function, including point mutations, promoter methylation, and small and large deletions.36 RB1 encodes a 4.7 kb mRNA, translated into the 928 aa protein, pRB. The A/B “pocket” region37 is best characterized, mediating protein-protein interactions and harboring missense mutations. The A/B pocket is conserved in two other pRB family members or “pocket proteins”, p107 (RBL1) and p130 (RBL2). These proteins are rarely mutated in cancer, although a large region of chromosome 16q containing p130 is lost in some retinoblastoma and other cancers.

pRB is best known as a cell cycle regulator, with function mediated by binding to E2F transcription factors, repressing cell proliferation-related genes. Hyperphosphorylation of pRB by cyclin-dependent kinases in response to mitogenic signals relieves repression and promotes the G1 to S phase transition. Notably, p107 and p130 regulate distinct E2Fs and cell cycle genes, suggesting that the few genes that are uniquely regulated by pRB have special importance.38 However, additional pRB functions in the cell cycle, maintenance of genomic stability, and apoptosis could also contribute to tumour suppression.37

Although biallelic loss of RB1 is necessary to initiate most retinoblastomas, this is not sufficient; RB1-/- retinal cells likely either undergo apoptosis, or proliferate in a limited fashion to form the benign retinal lesion, retinoma.39 Further genetic or epigenetic lesions are associated with malignant transformation.40 Comparative genomic hybridization studies identified common regions of DNA gain or loss in retinoblastoma, which led to identification of candidate oncogenes: the mitotic kinesin KIF14 and p53 regulator MDM4 (1q32), transcription factors E2F3 and DEK (6p22), and the oncomiR clusters miR-106b~25 (7q22.1) and miR-17~92 (13q31). Loss at 16q22 led to identification of cadherin-11 (CDH11) as a tumour suppressor gene, while whole-genome sequencing identified inactivating mutations in the transcriptional corepressor BCOR in some samples.41 Epigenetic alterations may also drive retinoblastoma formation: the oncogenic kinase SYK has an activated histone signature in retinoblastoma.41 Numerous other genes show altered expression in retinoblastoma compared to normal retina,40 including those altered at the DNA level (above), several microRNAs, and the multidrug resistance gene (MDR1). Gene expression profiles may segregate RB1-/- retinoblastomas into two subtypes, perhaps consistent with histology and cytogenetic aberrations. However, it remains uncertain whether the tumours instead display a spectrum rather than a dichotomy of phenotypes.42, 43.

While nearly all retinoblastomas have mutation of both copies of RB1, 1.4% of unilateral patients have tumours with no detectable RB1 mutation, and instead primary high-level amplification of the oncogene MYCN (MYCNA) drives retinoblastoma initiation.44 While RB1+/+MYCNA retinoblastomas share certain secondary genomic changes with RB1-/- tumours, they show very early onset and a distinct histology, reflecting a unique subtype.

Cone-precursor circuitry sensitizes to RB1 loss

Since pRB is expressed in most if not all cell types, the retina’s unique sensitivity to pRB loss has been perplexing. Identifying the retinoblastoma cell-of-origin could solve this conundrum

by identifying cell type-specific circuitry collaborating with RB1 inactivation. In prior studies, detection of diverse retinal cell type markers in retinoblastoma suggested a multipotent cell-of-origin, yet could also represent normal RB1-positive retinal cells within the tumour mass.45 Moreover, single retinoblastoma cells co-express RNAs characteristic of diverse retinal cell types,42 suggesting “hybrid” gene expression of a multipotent origin. However, the expression of diverse retinal cell markers could also simply reflect oncogenic transformation, rather than cell-of-origin properties. Mouse retinoblastoma models have not clarified the cell-of-origin, since they require loss of Rb1 plus p107, p130, or p2746 and express different retinal markers, and may not extrapolate to humans.47

Apart from analyses of cell type-specific markers,48, 49 the first suggestion that retinoblastoma originate in cone precursor cells came from a study showing that emerging tumors have a topographic distribution that mimics the horizontal visual streak characteristic of red/green cones.50 Further 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 prominently express oncoproteins (MDM2 and N-Myc) that could collaborate with RB1 loss to enable retinoblastoma growth.45 Moreover, direct experimental depletion of RB1 induced cone precursor proliferation, and orthotopic xenografts elicited tumours with histology, protein expression, and lack of cytogenetic changes typical of differentiated retinoblastomas.51 Proliferation depended upon cone precursor N-Myc and MDM2 high expression, cone-specific transcription factors RXR and TR2, and a down-regulation of p2751 associated with cone precursor maturation.52 These features suggest that RB1 is needed to counter an oncogenic program that is intrinsic to cone precursor maturation.

Despite progress, it remains uncertain whether cone precursors originate all RB1-/- retinoblastomas, or only highly differentiated tumors that also have few cytogenetic changes.43 Moreover, the cone precursor origin of retinoblastoma is challenged by detection by optical coherence tomography (OCT) of very early tumours in infants carrying an inherited RB1 mutation. The smallest tumours are centered in the inner nuclear layer of the retina, not the outer nuclear layer where mature cones reside (Fig. x).53 Perhaps RB1-deficient cone precursors migrate inappropriately when they are born or begin to divide. The location of these early tumours 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.54 However, such inward migration still requires experimental support.

The pRB functions that normally suppress retinoblastoma with such strong specificity for one embryonic cell type remain unknown. In addition to E2F suppression, pRB can up-regulate p27 via stabilization of SKP2, and is implicated in cell differentiation, apoptosis, and genomic integrity.37 pRB seems unlikely to be required to sustain p27, since increasing pRB expression is associated with decreasing p27 during cone precursor maturation.52 It is tempting to speculate that pRB is primarily needed to suppress E2F. However, several “low penetrance” RB1 mutations encode proteins that have minimal ability to bind E2F, yet predispose carriers to only one or no retinoblastomas55 in contrast to the mean of ~ 6 tumours in carriers of RB1 null alleles.50 Such E2F-binding defective alleles may suppress retinoblastomas that would form with RB1 null alleles through an E2F-independent mechanism. Clearly, there is opportunity to further define tumour suppressor functions in the clinical retinoblastoma scenario.

RB1+/+MYCNA retinoblastomas are diagnosed much younger age than RB1-/- tumours, even those that arise in predisposed persons carrying RB1 mutations. The trend of increasing

MYCN copy number with age at diagnosis, and the very young age of presentation of very large tumours, suggests that the cell of origin of RB1+/+MYCNA tumours is developmentally earlier than that of RB1-/-tumours.44

Translating advances in retinoblastoma pathogenesis

Knowledge of retinoblastoma signalling pathways could lead to treatment and prevention opportunities. For example, pathways specific to maturing cone precursors could be targeted after retinal maturation is complete. Oncoproteins such as MYCN could be targeted56 both in RB1+/+MYCNA tumours44 and in the RB1-/- tumours that are also MYCN-dependent.45 Furthermore, murine studies suggest that retinoblastoma development can be prevented by blocking some effects of pRB loss.46 It would be very exciting if targeting pathways that collaborate with pRB loss in retinoblastoma could also reduce the second cancer risk for persons carrying RB1 germline mutations.

These new opportunities require cell line and mouse models that accurately reflect retinoblastoma cell responses. Most cell culture studies of retinoblastoma have used just two cell lines, Y79 and WERI-Rb1, developed in the 1970s.57, 58 Many other lines exist59 and with further characterization could provide insights into the therapeutic consequences of different genomic changes. Primary retinoblastoma cells form xenografts in immune deficient mice60 and can bypass the need to establish cell lines, as recently used to evaluate SYK inhibitors as preclinical therapies.41

Genetically engineered mouse models are also powerful for treatment testing.61 While Rb1+/- mice do not develop retinoblastoma, retinal deletion of Rb1 (using Pax6a, Nestin, or Chx10 promoters) in mutant p107, p130 or p27 backgrounds achieves retinal tumour formation. These models have been used to examine genetic interactions in vivo, such as the role of miR-17~92 overexpression in Pax6a-Cre;Rb1lox/lox; p107-/- mice.62 However, since the mouse tumours require different collaborating mutations and seem not to originate from the same retinal cell type, their ability to predict human retinoblastoma treatment responses is not certain. Viral oncoproteins can promote murine retinoblastoma, such as by delivery of adenovirus E1A to the retinas of p53-/- mice, and by expression of Simian Virus 40 T-antigen in developing Müller cells.63 Subconjunctival topotecan 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.18 Thus, much may be learned and translated to the bedside with available systems while awaiting models that more precisely simulate retinoblastoma pathogenesis.

Diagnosis, screening and prevention 1824/1500

This section describes diagnosis (primary detection by a healthcare professional), screening (prospective procedures to diagnose as early as possible) and prevention (pre-empt the development) of retinoblastoma and second cancers.

Clinical Diagnosis

The most common first sign worldwide of retinoblastoma is leukocoria (white pupil), due to light reflecting directly off white tumor into the observer’s eye. The second most common sign is strabismus, or misaligned eyes, due to tumour blocking central vision. Signs of advanced disease include change in iris color, enlarged eye due to glaucoma, and orbital cellulitis. Proptosis (protrusion of the eye from the socket) is very late presentation, but

common where awareness and resources are poor. Often parents notice leukocoria, but cannot convince their health advisors that there is a problem. Importantly, as awareness efforts are being implemented globally, advanced retinoblastoma is declining.

Unlike most cancers where pathology provides definitive diagnosis, retinoblastoma diagnosis is clinical; biopsy incurs high risk of metastasis. Observation of leukocoria necessitates rapid referral to an expert. A dilated eye exam with indirect ophthalmoscope or retinal camera, and ultrasound usually narrows the differential diagnosis. Calcification is diagnostic of retinoblastoma, well demonstrated using 10 MHz b-scan. However, infiltration of the optic nerve, choroid or sclera is not well resolved by b-scan ultrasound. MRI screens for optic nerve invasion and trilateral tumor (pinealoblastoma, primitive neuroectodermal tumor). CT Scan is now avoided if possible due to oncogenic toxicity of radiation in these children. The OPTOS is a non-mydriatic, wide field camera of 200° may be prove useful for a rapid view of the retina, with minimal intrusion on the awake child. 

Detailed retinal exam under anaesthetic (EUA) almost always confirms the diagnosis and facilitates classification of severity of intraocular disease. The hand-held fundus camera (RetCam®) is used to view and record the whole retina, with views of posterior pole, superior, inferior, nasal and temporal fields taken with the wide field lens (130° or 120°). Scleral depression provides visualization of the ora serrate, critical for accurate classification.

Additional imaging under EUA includes high frequency ultrasound (50 MHz), ultrasound biomicroscopy (UBM) to image the anterior extension of tumor behind the iris,64 and optical coherent tomography (OCT) can discover invisible tumors in infants with familial disease (Figures 1 and 4).65 Such imaging supports classification of each eye and cancer stage, documents treatment responses, supports consultation with colleagues, and helps parents to understand treatment options. Intravenous fluorescein angiogram (IVFA) with the RetCam® can evaluate vascular abnormalities, suspicious residual or recurrent tumour, new vessel activity in scars, and areas of non-perfusion. However, IVFA does not show very tiny intra-retinal tumors because they are not yet vascularized; they are better discovered with OCT.

Digital cameras are useful to document external features including the face, both eyes, the socket, implant extrusions, granulomas and features related to the ocular prosthesis. The photo slit lamp will also provide high magnification the socket and adnexa. External photography by parents is often the first clue of the diagnosis of retinoblastoma.

Clinical Eye Classification and Cancer Staging

The first classification for intraocular retinoblastoma was the Reese-Ellsworth (RE) classification.66 It predicted outcomes of external beam radiation largely based on size, retinal location of tumour and vitreous seeding. When intravenous/systemic chemotherapy (IVC)/focal therapy became the common primary treatment to salvage eyes, Murphree led international collaboration toward the International Classification of Intraocular Retinoblastoma (IIRC) which better predicted responses to IVC than the RE scheme.67 The subsequent Shields version (also called IIRC)68, 69 results in discrepant classification of 25% of the most severely involved eyes (Figure x: Inconsistent Classification of Intraocular Retinoblastoma).70, 71 Subsequently, the Children’s Oncology Group (COG) used a variation (also called IIRC)72 with minor measurement differences (Figure x: Inconsistent Classification of Intraocular Retinoblastoma). For this Nature Reviews Disease Primer, we choose the Murphree IIRC.67

Similarly, for extraocular retinoblastoma, a number of staging systems co-exist and none has gained uniform acceptanceREF The St Jude Staging System, reported decades ago is still used by some groups, especially in Latin America. In a coordinated action with the Murphree international IIRC collaboration, the International Retinoblastoma Staging System focused on the overall staging of cancer for retinoblastoma.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 not widely used. There is a strong need for a single, collaborative, consensus classification of eyes and cancer staging.

Pathology

Retinoblastomas are a “small, round, blue cell tumour”. Well-differentiated regions form rosette structures: Flexner-Wintersteiner rosettes, virtually pathognomonic of retinoblastoma; and Homer Wright rosettes, common in diverse neural cancers (Figure 3).75 Retinomas feature more differentiated photoreceptor-like clusters of cells termed fleurettes.39 Poor prognosis histopathological features increasing the risk of metastasis include invasion of tumour into the optic nerve, choroid, or sclera..

For clinical IIRC67 Group E eyes, enucleation and histopathology is important to assess risk for metastases and need for adjuvant therapy.76, 77 Pathology specimens are staged using the pathology TNM (pTNM) (Supplementary pdf) and the International Retinoblastoma Staging System.74, 78 The preparation and examination of the enucleated eye have been optimized to completely evaluate risk features.79 However, too frequently retrospective examination finds a previously un-noticed risk, after metastatic disease is diagnosed. Highly retinoblastoma-specific stains such as CRX80 and NeuGc-GM381 may play facilitate observation of a few tumours cells lurking in a high-risk location. High risk features start with invasion of the uvea and extension into sclera; this may co-exist with invasion of optic nerve past lamina cribrosa. Extensive tumor causes neovascular glaucoma and then enlargement of the eye (buphthalmus) and/or optic nerve. Tumor cells are pushed to central nerve and subarchnoid space seeding the CSF. The tumor also can extends directly though the sclera or by vortex veins into orbit and through the choroid to bone marrow.

Genetic Diagnosis

The majority (94%) of patients with retinoblastoma are the first individuals to be diagnosed in a family (141/2141 probands (6%) indicated one or more family members previously diagnosed with retinoblastoma when tested for RB1 mutations, data from Impact Genetics, January 2015). Approximately 50% of people with retinoblastoma carry one RB1 mutation in their constitutional cells (all bilateral and 15% of unilateral patients).82 The remaining unilateral retinoblastomas arise by somatic mutational events (biallelic RB1 loss or somatic amplification of the MYCN oncogene) with no additional cancer risks.

Knowledge of the proband’s RB1 mutation enables precise screening of relatives and subsequent generations. The mutation can be detected prenatally and labour induced near term to detect and treat retinoblastoma tumours early. Without genetic testing, it is recommended that children who may (e.g. unilateral, positive family history) continue to undergo multiple exams under anaesthetic. After genetic testing, 85% of unilateral retinoblastoma patients will test negative with <1% residual risk for undetectable low level mosaicism, reducing the intensity of surveillance and accurate determination of cancer risks

of family members.6 Pre-implantation genetic diagnosis offers the family the option for unaffected children when the mutation of a parent is identified. 83, 84

The unique RB1 mutations of the eye tumor (if tumor DNA is available) can also be 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 different from germline mutation, or the child is not germline carrier),85 the M3-Mn signature86 and CRX.80

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.87 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.88

Vision Screening / 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 clear childhood screening test doesn’t mean this child is clear since a tumour m appear later, or may have been in the periphery and not visible.

Leukocoria is most often first noted by parents, 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. Awareness campaigns bring children to attention, but are 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.89 Another pioneering project is developing software to enable cameras to detect photoleukocoria.90

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 (Figure: Lancet photos or example photo). The global imaging industry could play a role in early diagnosis of retinoblastoma.

Second Cancer Surveillance

Individuals with germline RB1 mutation and/or treated with radiotherapy have an elevated risk of developing specific second cancers, including leiomyosarcoma, osteosarcoma, melanoma, lung and bladder cancer.91 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.92 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.93

Prevention

Lifestyle counseling 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.

The future of retinoblastoma care is to move from prediction to prevention. Retinoblastoma survivors are desperate for a solution that would eliminate cancer risk for themselves and their family members. Transgenic murine retinoblastoma can be prevented.46 As molecular research further elucidates the markers of the cell-of-origin, and the molecular paths in susceptible cells are elucidated, targeted therapies to block development will emerge. Familial newborn infants (known RB1 mutation carriers) present the opportunity for a clinical trial of this new therapy, potentially targeted to eyes alone. Clinical trial of this ocular therapy could be randomized to one eye, the other a control, and effect determined by counting emerging tumours in each eye. Such a trial would only take a few years, since retinoblastoma develops quickly; this therapy we dream of could then be considered to reduce risk of second cancers as well.

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(only 1, we have 3 potentials, but perhaps we could refer to another section for some of these, if overlap)

Table X: Staging Differences (from Brenda’s TNM database table) Table Y: Impact Genetics numbers on familial cases Figure: Leukocoria image?

Management 4624/3000

Munier, Abramson, Shields, Chantada, Njuguna, Gallie, Zhao

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 accessibility to expert care where the child lives.94, 95 Diagnosis is usually clear from presenting signs95 (See DimarasDx section) and clinical examination. Biopsies are not performed on presumed intraocular retinoblastoma due to risk of seeding tumour outside of the eye. The leading simulators of retinoblastoma include Coats disease, persistent fetal vasculature, and vitreous hemorrhage.96

INTRAOCULAR RETINOBLASTOMA PRIMARY TREATMENT

Choice of primary treatment reflects first, consideration of patient survival, and second, eye salvage and ultimate visual potential, both weighed against short term and long term complications of treatment.94 In order of approximate frequency of use globally, primary treatment options for presumed intraocular disease include enucleation, intravenous chemotherapy (IVC) with focal therapy (laser, cryotherapy), intra-arterial chemotherapy (IAC) with focal therapy, and focal therapy alone for patients/eyes with small tumours at diagnosis. External beam radiotherapy (EBR) is now reserved for refractory cancer in the last

eye with a chance for useful vision, since radiation incurs a very high risk of second cancers on persons carrying an RB1mutation, especially in the first year of life.97, 98

Treatments considered for intraocular retinoblastoma are dependent on the classification of severity of disease (section diagnosis…). We use the Murphree IIRC67 for this Review (Figure x: Inconsistent Classification of Intraocular Retinoblastoma;Table x: Treatments for Retinoblastoma Based on Classification and Stage). The availability of treatments globally varies directly with expertise and resources. The safest treatment for cure, available everywhere, is enucleation. Eye salvage can be achieved using conservative methods. Choices of primary treatment for each child are best arrived at by fully informed discussion of all factors with the parents and guardians.

Primary treatment options depend on the IIRC of each eye (Table x: Primary Treatment for Intraocular Retinoblastoma). Group A eyes can be treated with laser/cryo focal therapy only. If the other eye is Group B, C or D, laser to the Group A eye may be delayed to allow the systemic chemotherapy to reach the tumour before the blood supply is cut off by laser. Group B eyes require several chemotherapy cycles followed by focal laser/cryo to optimize vision by minimizing focal therapy near the macula and optic nerve. Group C eyes require several chemotherapy cycles because minimal extension beyond the retina is evident as vitreous or subretinal seeding; intravitreal melphalan (IViC) for vitreous seeds may be included in the primary plan, after systemic chemotherapy. Isolated Group B, C eyes with one tumour may occasionally be appropriate for primary radioactive plaque therapy.

Group D eyes can be managed by enucleation, IVC or IAC. Enucleation is the fastest and cheapest treatment for such eyes99, 100 but the eye is lost. Eye salvage with either IVC or IAC, or both, followed by repeated EUAs for focal therapy offers hope to save the eye safely. The success to save a Group D eye with ICV is 47% with high dose vincristine, etoposide and carboplatin (VEC) (Table x: Chemotherapy drugs and doses.) and optimal focal therapy.101 The success rate for IAC for Group D eyes (Shields IIRC)68, 102 was 94%.102 The major reason for failure of IVC and IAC was recurrence of subretinal or vitreous seeding within 3 years.103,

104 Since vitreous seeds at diagnosis predicts recurrence, IViC may be included in the initial treatment plan, to be delivered after control of the source of seeds by IVC or IAC105.

IIRC67 Group E eyes have clinical features that suggest risk for imminent or actual extension of retinoblastoma beyond the confines of the eye, with 10% risk of metastatic death.106-108 Attempts to salvage Group E eyes have a negative impact on survival,108 since pre-enucleation treatment may mask the high risk for metastases.108 When high risk features are documented, adjuvant chemotherapy may abort incipient metastases.106, 109-112 Metastatic disease is reported after IAC in a Group E eye.113 Evaluation of the present literature is difficult, since the Shields IIRC69 Group E includes many Murphree IIRC67 Group D eyes.

Bilateral retinoblastoma in much of the less developed 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.11, 114 Many bilaterally enucleated retinoblastoma survivors lead active, productive and satisfying lives because they were cured by timely surgery as infants.

Intraocular Retinoblastoma: 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 rate104 and long drawn out attempts to salvage an eye incur high costs of many kinds for the child and family.115, 116

Second line treatments have included focal therapies including peri-ocular chemotherapy,117 repeated systemic chemotherapy,117, 118 repeated IAC,104 iodine or ruthenium brachytherapy,119,

120 EBRT101, 121, 122 and tantalum ring localization123 or whole-eye124 proton beam radiotherapy. Criteria for secondary enucleation after to salvage an eye are not well defined but are dominated by refractory subretinal and vitreous seeding,118, 125 complications such as vitreous hemorrhage and secondary neovascular glaucoma suggesting risk of extraocular extension,124 and socio-economic and psychological fatigue to save an eye with poor vision.116 Unlike pathologic risk factors following primary enucleation, scleral invasion in secondarily enucleated eyes was most associated with extra-ocular relapse.126

Prior to the advent of IAC, over 60% of eyes with advanced retinoblastoma treated by chemoreduction and focal therapy failed (required salvage external beam radiotherapy and/or enucleation).127 First-line IAC reduced the recurrence rate in group D eyes.128 Combination of IAC and intravitreal chemotherapy (IViC) can play an important role to save eyes that have failed IVC.102, 128, 129 However, extensive treatments to save an eye may increase risk for metastases.{Suzuki, 2011 #16292;Francis, 2015 #21614;Mathew, 2015 #21631}

Despite advances in the conservative management of advanced retinoblastoma, the major cause of failure remains the persistence or recurrence of vitreous seeding. Pharmacokinetic studies have shown poor vitreous levels of drugs administered by either systemic chemotherapy or IAC.131 The highest drug bioavailability in the vitreous, is achieved by IViC using a safety-enhanced injection technique in carefully selected eligible eyes.132 Following control of the source of seeds, IViC achieved two-year Kaplan-Meier estimates of 98.5% and 90.4% event-free survival for target seeds and ocular survival respectively.105, 132, 133 The number of IViC treatments to attain control of vitreous seeds is dependent on the type and extent of seeding.105 The efficacy of IViC has eliminated the need for EBR, and decreased patient exposure to salvage systemic or intra-arterial chemotherapy. The toxicity of IViC is limited to localized peripheral salt-and-pepper retinopathy, the extent of which is technique-dependent.134

Combinations of new routes for therapy can target salvage therapy to the site and extent 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, it is clear that therapies that have already failed to control the intraocular tumour are unlikely to succeed as salvage therapy for the same eye.

Ocular Therapies

Enucleation

Enucleation is a first-line therapy for the majority of eyes with retinoblastoma globally. The procedure is readily available wherever there are ophthalmologists. Since the majority of children with intraocular retinoblastoma have Group D or E eyes at diagnosis, and more than 50% have unilateral disease with another normal eye, cure can be achieved with enucleation.

All IIRC67 Group E eyes require enucleation since by definition, they carry risk of extraocular extension, determined only by pathology. 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;135 Comparative studies have shown enhanced prosthetic eye motility with the myoconjunctival approach, which is affordable world-wide.136, 137 Complex 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,138 observed in Kenya to help the next family accept enucleation for their child.

Histological study of the enucleated eye the only way to evaluate high-risk features and establish pathological staging139 (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).77, 107, 111, 139 High-risk features are observed in 17% of IIRC67 Group D eyes and 24% of Group E eyes.77, 107 The role of adjuvant systemic chemotherapy to reduce risk of metastatic relapse in patients with high-risk pathological features is reviewed.111, 112

Intravenous chemotherapy (IVC) and focal therapy Since 1996 first-line therapy to control IIRC Groups B, C and D eyes has been IVC with different combinations, doses, schedules, and durations of carboplatin, etoposide and vincristine (CEV) (Table x. Chemotherapy for retinoblastoma) followed by focal therapy to consolidate the chemotherapy responses.125, 141, 142 One group used high dose acute cyclosporine (CSA) to modulate multidrug resistance.143, 144 The Groups B and C eyes do well with CEV and focal therapy. With follow-up of 54 months, 47% of IIRC67 Group D eyes101 and 47% of RE Group V125 avoided enucleation or external beam radiation.69 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.145 Acute toxicities of IVC for retinoblastoma are as for other pediatric cancers, including short-term transient pancytopenia, hair loss, vincristine-induced neurotoxicity, and infections. Long term toxicities include carboplatin-induced ototoxicity,146 second non-ocular cancer risk with alkylating agents147, 148 and secondary acute myeloid leukemia following intense chemotherapy including topoisomerase inhibitors, doxorubricin and alkylating agents.149, 150

IVC alone rarely eradicates the last retinoblastoma cell in the eye, and focal therapy consolidation is very important with repeated EUAs.103, 140, 151 Tumours in the macular region are at risk of recurrence without focal therapy152 and better treatments are required for these visually threatening tumours.

Following control of retinoblastoma with IVC, 50% of patients have visual acuity at 5-years of 20/20-20/40; 67% have 20/200 or better.153, 154 Foveal involvement with tumour or subretinal fluid at presentation contribute to poor vision. There is no documented local toxicity of IVC to the eye.

Intra-arterial chemotherapy (IAC)

Intra-arterial chemotherapy and intra-vitreal chemotherapy for intraocular retinoblastoma have had the greatest transformative effect on retinoblastoma management since the introduction of radiation more than 100 years ago as they have allowed saving eyes that were previously unsalvageable.

Method of delivery: There have been 3 major breakthroughs in intra-arterial treatment for retinoblastoma. It was first done in the USA sixty years ago where 54 patients were treated in via intra-carotid injections of TEM (called “intra-arterial chemotherapy”).155 This was followed 30 years later in Japan where more than 300 patients were treated via a catheter with an inflatable balloon near the tip which was fed into the internal carotid artery. During the procedure the tip was inflated, temporarily occluding the internal carotid artery and Melphalan injected “over a few seconds” below the balloon with the intention of having the drug go into the ophthalmic artery-(this was called “selective ophthalmic treatment”).130 In 2006 a technique was introduced in the USA in which a micro catheter (450 microns in diameter) was inserted into the femoral artery after heparinization and passed up to the orifice of the ophthalmic artery (but not into the artery) where drug or combination of drugs (Melphalan, Carboplatin, Topotecan) were infused in a pulsatile fashion over many minutes (called “Superselective ophthalmic artery infusion or ophthalmic artery chemosurgery (OAC)).156 In both the Japanese and USA techniques, cannulation has been successful in almost 99% of cases. Currently, OAC is performed in more than 30 countries and nearly half of these are in developing nations.157

OAC is usually done via the internal carotid artery but at times treatment can only be delivered through the external carotid artery through the middle meningeal artery (15% of cases) or a modification of the balloon technique (10% of cases).158

Although the initial group of patients who received OAC were unilateral, bilateral treatment is often done in some centers at the same session-within one hour (utilizing combinations of drugs in the two eyes) with similar success-this has been called “tandem therapy”.159

Patient survival: Of the more than 2,500 infusions and >800 patients in the literature using OAC only two patients have died of metastatic retinoblastoma. In the USA, patient survival was 100% at 5 years.102 However, Retinoblastoma death from metastases can occur more than five years after any treatment. The longest follow-up study is described for the Japanese series using IA delivery (not OAC) (including death from metastatic retinoblastoma and second cancers) with an overall survival of 95% at 15 years.130 In this paper, the 8 deaths from metastases of 343 patients cannot be clearly assigned to IAC alone since a minority of the patients received exclusively IAC. On the other hand, similar figures of deaths from metastasis have been reported with other conservative therapeutic modalities.140

Ocular Survival: Many patients treated with intrarterial chemotherapy had advanced 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 eyes (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 3 different IIRC67, 68, 72 (Table x), which prevents a clear-cut comparison of the results between centers, especially regarding Group D and E eyes. A universal classification should be proposed and adopted in the near future in order to avoid confusion regarding the effective salvage rates, and to clarify the indications of IAC.

Ocular survival with the Japanese technique was: Group C (65%) and Group D (45%). 130 Using OAC in the USA, ocular survival was 96% in both Groups B and C, and 94% in Group D.102, 160 Eyes with neovascular glaucoma, pthisis bulbi, and anterior chamber involvement were universally enucleated primarily.

Ocular survival is highest in naive eyes with extensive retinal detachments (Figure 1). With OAC eyes with >50% retinal detachments demonstrated ocular survival (Kaplan-Meier) of 87.9% at two years and 76% had complete retinal reattachment as a result of IAC alone.161

The most common reason for secondary enucleation of eyes with retinoblastoma in developed countries has always been the presence of extensive vitreous seeding.162 Only 20% of such eyes can be salvaged with external beam irradiation.163 With OAC 74% of eyes with seeding have been salvaged (Figure 2).102, 104, 129

Ocular complications: Complications following OAC are currently few and include both short-term and long-term effects. In an analysis of 198 catheterizations of the ophthalmic artery in 70 consecutive eyes with retinoblastoma, minor transient complications included transient eyelid edema (5%), blepharoptosis (5%), and forehead hyperemia (2%).102 More lasting complications included vitreous hemorrhage (2%), branch retinal artery obstruction (1%), ophthalmic artery spasm with reperfusion (2%), ophthalmic artery obstruction (2%), partial choroidal ischemia (2%), and optic neuropathy (< 1%).102 These complications are minimized at experienced centers.

Doses and drugs: Melphalan by OAC has been given at doses of 2.5-7.5 mg based on eye size, extent of disease and whether the disease was naive or recurrent tumor.164 Because the ophthalmic artery has laminar flow, the drug is delivered in pulses manually given over 10-30 minutes in equally divided doses a minute apart. Carboplatin has also been used as a single agent in doses of 25-50 mg.165-167 The two-year KM ocular survival was 89.9%. A two-drug regimen has been used in selected cases. For very advanced eyes a three-drug regimen has been used (Melphalan, Carboplatin and Topotecan).168 The drugs are delivered in the same session within an hour. These are more advanced eyes (92% Reese-Ellsworth V) and for naive eyes 86% of them were salvaged (Figure 3).168

Number of cycles: In the initial report on OAC patients were treated as few as 2 times (and as many as 9) with complete response. Subsequently it was realized that some patients (even recurrent cases) achieved complete responses with as little as one treatment session.160, 164, 169-

171

Consolidation: Historically consolidation was necessary in the majority of radiated 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.169, 170

Bridge Therapy: Although this procedure has been performed in children as young as three weeks of age, most centers withhold canulation 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) intravenous chemotherapy 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 radiation.172

ERG/Vision: In the Japanese experience, 58% of children with foveal tumors retained a visual acuity of >0.01 and for those without foveal tumors 51% retained visual acuity of >0.5 with 36% >1.0 (2). ERG monitoring of OAC patients156, 166, 173-175 demonstrated remarkable stability of the ERG.

Prevention of new tumors: OAC decreases the appearance of subsequent, new (usually peripheral) intra-ocular tumors which commonly develop after systemic chemotherapy or radiation in genetic cases resulting in fewer overall treatments for the children.176

Focal Therapy

Focal therapy is local application of anti-cancer therapy to the eye, under direct visualization through the pharmacologically dilated pupil. This approach is useful for primary treatment of IIRC Group A eyes and “consolidation” therapy for residual or recurrent small volume active tumour after systemic or intra-arterial chemotherapy. Focal Therapy generally is repeated monthly until the tumour is completely atrophic or calcified.

Transpupillary thermotherapy is 810 nm diode laser delivered through the dilated pupil at sub-photocoagulation level for a period of 3-5 minutes per spot. 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 freezing of tumour through the sclera with a nitrous oxide probe; the tumour is directly visualized and duration of freeze judged to completely encompass the tumour. However, since tumour cells die when thawing, one minute is allowed for each thaw, between freezes. Cryotherapy is effective to destroy small primary tumour(s) or recurrences in the periphery of the retina.

Plaque radiotherapy is trans-scleral radiotherapy to deliver an apex dose of 35 Gy to an isolated single intraocular tumour or recurrence, over 4-7 days. Plaque focal radiation has not been associated with second primary tumours. Plaque radiotherapy is effective for treatment of a single primary or recurrent tumour in a location that will not compromise vision.

Paraocular Chemotherapy

Although chemotherapy has been used under conjunctiva or in tenon’s capsule, it is effective only for very small volume recurrences and vitreous seeds.177-179 It may now be replaced by IViC, which shown to be highly effective and safe.105, 132, 134

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.105, 132, 134, 180, 181

UBM may be used to evaluate the otherwise hidden ciliary region behind the iris,64 to confirm that the injection site is tumor-free prior to IViC treatment.

After induction of anesthesia, the intraocular pressure was lowered with an anterior chamber paracentesis or by digital massage. Intravitreal melphalan (20-40 µg in 0.05 to 0.15 ml) is

injected through the conjunctival, sclera, and pars plana with a 32- or 33-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.105

Extraocular retinoblastoma

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.11

Those who present with overt extraocular disease have a low chance of survival, especially in low-income settings. Chemotherapy followed by enucleation, orbital radiation, adjuvant chemotherapy, intrathecal chemotherapy and high dose chemotherapy with stem cell rescue have potential for cure.

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, 111 Enthusiasm to salvage eyes increases metastatic risk by masking primary extraocular disease.108, 113 Bone marrow metastasis without central nervous system disease has potential for cure with extensive therapy including stem cell transplant. However, extension of retinoblastoma into the brain has a very low likelihood of cure.

Palliation

Palliation includes pain management, symptom relief, nutritional support, and psychosocial support for the child and families.{Chantada, 2013 #18862} Untreated 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. External beam radiotherapy and chemotherapy are useful for pain control in palliation but are often unavailable in low income countries.

Role of being at home; pain control; oral morphine; short cycle chemo? Benefit or not? Etc.

Treatments in the pipeline

Preclinical studies are crucial to identify new molecular targets to block the drivers of tumourigenesis in retinoblastoma and for pharmacokinetic evaluation of new agents alone or in combination185 before their incorporation to the clinics and for assessing their toxicity

profile. However, very few translational studies have actually resulted in significant changes in clinical practice for retinoblastoma. In most instances, preclinical studies have helped in optimizing treatments already available in the clinic, mostly by providing information on the ocular pharmacokinetics of drugs by comparing different routes186. In addition, preclinical studies have been carried out for the development of innovative delivery systems to the vitreous for the treatment of vitreous seeding such as devices for sustained released preparations or metronomic administration for periocular or intravitreal routes. Fibrin-sealant carboplatin and topotecan179, episcleral implants187 or nanoparticles188 and exoplants have been evaluated, but only the first one is currently used in clinical practice albeit for restricted indications. Other agents such as those targeting the tumour vasculature189 or hypoxia190 have been evaluated with some degree of detail in preclinical models but they have not yet progressed to clinical use. As new biomarkers for molecular dissemination of retinoblastoma outside the eye are identified, treatments may be targeted based on that information.191

One of the earliest targeted therapies developed for retinoblastoma that was based on preclinical information generated by transgenic mice was reported for Nutlins.192 Nutlin-3 showed promising activity in combination with topotecan for retinoblastoma control in preclinical models. Nutlin-3targets the MDM2/MDMXpathway as a negative regulator of p53 resulting in apoptotic cell death mediated by p53.This drug combination is currently undergoing evaluation in a prospective study in combination with local chemotherapy. Based upon information obtained from sequencing the whole genome and the epigenome of retinoblastoma tumours, thespleen tyrosine kinase (SYK), an upregulated proto-oncogene required for retinoblastoma cell survival has been identified as a potential new target for the treatment of retinoblastoma.41 The SYK antagonist R406,wasconsidered a promising candidate from preclinical studies, but later it was found that its ocular pharmacokinetics was not favorable for clinical useby periocular administration.193 Drugs targeting MYCN, which has been recently identified as a candidate driver for retinoblastoma tumourigenesis in cases with no RB1 gene mutation,44 may also be considered for targeted therapy but this is still under development.

Transgenic mice models of retinoblastoma do not entirely recapitulate the tumourigenic steps of human retinoblastoma, so drugs targeting specific molecular pathways may behave differently in these models. On the other hand, xenografts from patient-derived specimens show a different eye anatomy since injected human retinoblastoma cells in the vitreous or the subretinal space modify the natural anatomic barriers of the eye potentially affecting drug distribution and limiting translation to the patient.

In summary, novel targeted agents undergoing intensive investigation for retinoblastoma, may significantly change future treatment.

Quality of life 1081/500

White, A

“Quality of life” (QoL) describes the level of physical, emotional and psychological wellbeing experienced by an individual. Cancer significantly decreases QoL, with implications for treatment decisions, supportive and long-term care.194-197

Measured life-long impact

Life-long impacts of retinoblastoma and its various treatments show surprising results.198, 199 Overall, survivors diagnosed under 1 year of age performed significantly better compared with those diagnosed at over 1 year of age. Whole brain radiation exposure was significantly associated with poorer verbal memory. 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.200, 201 In children followed from diagnosis to age 5 years, trajectories of developmental functioning declined over time.199 However, study IQ of age-matched persons with the early infancy form (presumed heritable) was significantly above normal sighted and non-retinoblastoma blind persons.202

There is clinical and animal evidence that repeated anesthesia of young children may impair neurocognitive development. Repeat anaesthesia is necessary in eye salvage and in very young children to discover and treat small tumors. Going forward, one outcome to record in retinoblastoma clinical studies is number of EUAs and age.99, 203, 204

Direct insight from survivors

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 QoL following retinoblastoma are needed to learn from these valuable evidence sources. Quotes are used with author permission.

Coping During Treatment

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". Children’s perception of pain and medical interventions changes over time.205-211 Repeated 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.115, 212-218 Child Life promotes effective coping through play, preparation, education, positive-touch and self-expression activities based on natural child development.22 Child 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.91, 219-227

Treatment Choices

When only one eye is involved, choice between eye salvage (with potential long term intensive therapies) and enucleation (loss of the eye) is complex. A comparative retrospective study of socioeconomic and psychosocial impacts of attempted ocular salvage in a middle-income country concluded that primary enucleation is a good treatment for unilateral retinoblastoma.116 Challenges following enucleation range from discomfort with appearance or handling their prosthesis, to fear, shame and non-compliance. Facilitated self-expression activities that build self-esteem and confidence help children overcome these negative feelings.

Shopping for therapy at multiple centres is a complex journey that can ravage family life and finances. Delays while seeking alternatives can result in curable children dying.228-232 Prospective protocols can build collaboration, communication and efficiency, to achieve optimized care as close to home as possible, with coordinated trips for special needs.

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 QoL. Dry eye is very painful, and corneal vascularization reduces already limited vision. Use of ocular lubricants may prevent complications, best started early before pain and vision loss occur. Chronic primary headaches, hormone dysfunction and seizures also impact QoL after radiotherapy.

Second Cancer Risk

Individuals at risk of second primary cancers require life-long oncology follow up for second cancers.147, 233, 234 Lack of agreed protocols causes confusion, frustration and fear as adults struggle to access informed follow up care, compounded by inaction of primary doctors unfamiliar with late effects and lifelong implications of an RB1 mutation. Full information about their cancer history, genetic status and life-long risks will empower survivors to be advocates for their own and their children’s health.235-238 They seek honesty, compassion and support in learning about how their cancer may impact them throughout life.

Psychosocial Outcomes

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.198 However, reduced vision causes some children to become frustrated by their inability to keep up with peers, damaging self-esteem and confidence.239-241

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.

Outlooks 821/1000

Gallie, Dimaras, Corson, Munier, Zhao, and all.

[Maximum 1,000 words. What are the key outstanding research questions, and why? Where will the field focus its efforts on in the next 5–10 years? What are the advances to look out for, for bench researchers and clinicians? Will emerging technologies and advances in other fields influence the research trajectory in this disease?]

We envision a day where we can celebrate zero deaths from retinoblastoma. 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. We have suggested that progress in treatment of retinoblastoma is critically dependent on achieving consensus on the standard classification of intraocular disease. A current international survey of outcomes will compare initial clinical staging by Murphree IIRC,67 Shields IIRC68 and the 7th edition TNM74. This evidence will be used to formulate the 8th edition TNM for retinoblastoma, to achieve one cancer staging and eye classification with consensus of retinoblastoma experts, with an official cancer organization that can control versions, endorsed by eye journals.

A vast body of scientific work is steadily increasing our knowledge of the retinoblastoma cell of origin and its unique sensitivities, which may reveal strategies to prevent tumours. Moreover, ongoing work to define molecular lesions in retinoblastoma will continue to yield novel targets for therapy. Advances in therapeutic development and prevention depend on these molecular studies. Rigorous preclinical models, such as those established at St. Jude’s Children’s Research Hospital, and committed multicentre collaboration for eventual clinical trials will be essential in this regard.

Without thinking about the lifelong outlook for patients, however, all of the above endeavours become moot. One survivor observes “We're the center of the world when we go through treatment, but the moment we survive, we're last on the list, and no one wants to be anywhere near us, as if surviving cancer is the black plague, especially if you survive with vision loss – I can't imagine doing this for the rest of my life”. Patient-oriented research that aims to 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.

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 retinoblastoma care, drawn from established clinical care guidelines, represent the first step in the path towards achieving health equity. We will 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. National strategies in areas currently under-represented in the retinoblastoma literature are poised to fill the knowledge gap with new approaches and evidence.

We suggest that a coordinated interdisciplinary, international effort is required to address the global retinoblastoma burden, but this is easier said than done. The documented difficulty in achieving consensus on so many important retinoblastoma issues, including clinical and pathologic staging, and treatment approach, suggests a failure in one or more of the major components of consensus-making: inclusion, participation, cooperation, collaboration, egalitarianism. Far too often, stakeholders from low-and-middle income countries have been absent from efforts to innovate. Much of the evidence-base for care has been established in high-income countries, often not tested for feasibility or acceptability in other settings. Even when attempts were made to form consensus, such as the TNM guidelines, low-and-middle income country participants were not consulted. Add to this the potential isolating effects of twinning programs, where capacity is built on the basis of expertise of a single partner, this can further reduce the capacity to truly work together and build a joint consensus (e.g. Central America has adopted St. Jude’s classification, largely because that is who trained them).

Also absent from much of the research and development is the patient voice. The comment from the survivor above indicates that the end-beneficiaries of this work are not being heard. It naturally points to a truly inclusive, participatory global network to advance evidence, one that links clinicians and scientists to the patient and families, and efforts are pursued with the input of all.

One Retinoblastoma World (1RBW) is an attempt toward complete inclusion and participation from all those who share in the bold idea that all retinoblastoma patients and survivors can have equal opportunity to optimal care. A global collaborative, constellation-modelled approach aims to harness the power of all the individual activities, institutions and national strategies and networks, and deliver coordinated, evidence-based retinoblastoma care. The Internet links retinoblastoma experts everywhere, proportional to numbers of patients and their geographical distribution. Without prior awareness of retinoblastoma, parents now commonly self-diagnose on the Internet, arrive at the 1RBW map and discover means to directly connect them to experts, without delay and obstacles. We look to a day when equality for retinoblastoma comes by education (Internet access), with shared care coordinated online. Retinoblastoma expertise will be developed locally (proportional to local burden of retinoblastoma) but derived globally, with best practices drawing on rigorous molecular and clinical research. This will optimize outcomes for patients and families.

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Supplementary information

Extend Data Table 1. Estimated number of new retinoblastoma cases in 2012 by country

Acknowledgement

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

Where appropriate, we would like to attribute sections to individual authors, either with their initials at the end of each section or with a statement at the end of the article (referring authors by initials here, too). Please make clear author contributions in the main document.

Competing interests

The authors declare no competing interest

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