Format of the review article:

- A word limit of 5,000 words;

- Less than 80 references;

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- An unstructured abstract of ≤ 250 words;

- The maximum number of authors: 6

Genetics and Molecular Diagnostics in

Retinoblastoma - An Update

Authors:

Sameh E. Soliman, MD

Chengyue Zhang, MD.

Hilary Racher, PhD

Heather MacDonald

Brenda L. Gallie.

Affiliations:

Department of Ophthalmology and Vision Sciences, University of Toronto, Ontario, Canada

Department of Ophthalmology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt.

Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University.

Impact Genetics, Bowmanville, Ontario.

Corresponding author:

We confirm that this manuscript has not been and will not be submitted elsewhere for publication, and

all coauthors have read the final manuscript within their respective areas of expertise and participated

sufficiently in the review to take responsibility for it and accept its conclusions. No authors have any

financial/conflicting interests to disclose.

This paper received no specific grant from any funding agency in the public, commercial or not-for-

profit sectors.

2

Unstructured abstract

Abstract: mmmmmmm

Key Words: retinoblastoma, RB1 gene,

3

INTRODUCTION [JEFFRY]

Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.

{Dimaras, 2015 #14249} Tumors are initiated by biallelic mutation of the retinoblastoma tumor

suppressor gene (RB1) in a precursor retinal cell. The first RB1 mutation is present in constitutional cells

in nearly 50% of patients, who are thereby predisposed to developing retinoblastoma after the second RB1

allele is damaged in a somatic cell. The incidence of retinoblastoma is constant at one case in 16,000 live

births, translating to about 8,000 new cases per year worldwide.{Seregard, 2004 #10380;Dimaras, 2015

#14249}

Asia and Africa have the highest mortality, with >70% of affected children dying of retinoblastoma,

compared with <5% in developed countries.{Chantada, 2011 #7549;Canturk, 2010 #7342} Delayed

diagnosis and treatment due to lack of retinoblastoma knowledge by ophthalmologists and parents,

socioeconomic{Soliman, 2015 #11668} and cultural factors are major causes of high mortality. Broad

understanding of retinoblastoma genetics and genetic counseling can contribute to reducing mortality

from retinoblastoma. In this review, we highlight the RB1 mutation types, advanced molecular diagnosis

and genetic counseling.

Clinical presentation [Sameh]

Natural History

Start with retinoma and molecular features…..

Retinoblastoma starts as a rounded white retinal mass that gradually increases in size. Centrifugal

tumor growth results in small tumors being round; more extensive growth produces lobular growth ,

likely related to genomic changes in single (clonal) cells, that provide a proliferative advantage.

{Murphree, 2005 #12287;Balmer, 2006 #8615} Tumor seeds float free of the main tumor into the

4

subretinal space or the vitreous cavity as a result of poor cohesive forces between tumor cells, appearing

as dust, spheres or tumor clouds.{Munier, 2014 #11566} Advanced vitreous tumor seeds can migrate to

the anterior chamber producing a pseudo-hypopyon. Enlarging tumor can push the iris lens diaphragm

forward causing angle closure glaucoma. Rapid necrosis of tumor can cause an aseptic orbital

inflammatory reaction resembling orbital cellulitis, sometimes showing central retinal artery occlusion.

{Balmer, 2007 #8612;Balmer, 2006 #8615;Murphree, 2005 #12287} Untreated, retinoblastoma spreads

into the optic nerve and brain, or hematogenous spread occurs through choroid, particularly to grow in

bone marrow. Direct tumor growth through the sclera can present as orbital extension and proptosis.

Retinoma is a premalignant precursor with characteristic clinical features: translucent white mass,

reactive retinal pigment epithelial growth and calcific foci.{Gallie, 1982 #5686} Pathology of retinoma

reveals fleurettes structures that are not proliferative. Genetic analysis of retinoma and adjacent normal

retina and retinoblastoma shows loss of both RB1 alleles, and early genomic copy number changes that

are amplified further in the adjacent retinoblastoma. It can transform to retinoblastoma even after many

years of stability.{Theodossiadis, 2005 #5649}

Clinical Features

Leukocorea (white pupil) is main clinical presentation usually detected by parents either directly or in

photographs (photo-leukocorea). Strabismus due early macular involvement is the second most common.

{Balmer, 2007 #8612} In developing countries, buphthalmos and proptosis due to advanced and

extraocular disease respectively represents a higher percentage.{Canturk, 2010 #7342} Less common

presentations include; heterochromia irides, neovascular glaucoma, vitreous hemorrhage, hypopyon or

aseptic orbital cellulitis.{Balmer, 2007 #8612} Retinoblastoma (unilateral or bilateral) might be

associated with a brain tumor in the pineal, suprasellar or parasellar regions (Trilateral retinoblastoma)

{Popovic, 2007 #11607;Antoneli, 2007 #14202} that starts early; with the median age of onset 17 months

after retinoblastoma is diagnosed and before the age of 5 years.{Popovic, 2007 #11607;Antoneli, 2007

#14202;de Jong, 2015 #14413} Retinoblastoma might present in a syndromic form (13q deletion

5

syndrome) associated with some facial features as high and broad forehead, thick and everted ear lobes,

short nose, prominent philtrum and thick everted lower lip, bulbous tip of the noseassociated with various

degrees of hypotonea and mental retardation.{Baud, 1999 #18925;Bojinova, 2001 #18926;Skrypnyk,

2004 #18927} The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary

vitreous and ocular toxicariasis.{Balmer, 2007 #8612}

Retinoblastoma Cancer Staging

Treatment and prognosis depend on the stage of disease at initial presentation. Factors predictive of

outcomes include size, location of tumor origin, extent of subretinal fluid, presence of tumor seeds and

the presence of high risk features on pathology.{Mallipatna, 2017 #18013} Multiple staging systems

have predicted likelihood to salvage an eye without using radiation therapy; the International Intraocular

Retinoblastoma Classification (IIRC){Murphree, 2005 #12287} has been recently the most reliable, but

published evidence is confusing because significantly different versions have emerged.{Dimaras, 2015

#14249} The 2017 TNMH classification is based on international consensus and evidence from an

international survey of 1728 eyes, with algorithms evaluating initial features and outcomes by 5 different

eye staging systems.{Mallipatna, 2017 #18013} (Table X) Retinoblastoma is the first cancer in which

staging recognizes the impact of genetic status on outcomes: presence of a positive family history,

bilateral or trilateral disease or high sensitivity positive RB1mutation testing, is H1; without these features

or testing of blood, HX; and H0 for those relatives who are shown to not carry the proband’s specific RB1

mutation.{Mallipatna, 2017 #18013} We propose H0* for patients with 2 RB1 mutant alleles in blood that

are not detectable in blood, reducing risk of a heritable RB1 mutation to <1%.

Treatments

Multiple treatments are now available and the choice depends on the laterality of disease and the

grouping of the tumor. Chemotherapy (systemic or intraarterial chemotherapy) to reduce the size of the

tumor followed by consolidation focal therapies (Laser therapy or cryotherapy) is the main stay of

treatment. Enucleation for eyes with advanced tumors or in unilateral disease where the other eye is

6

normal is more appropriate and definitive. Other therapies include; intravitreal chemotherapy for vitreous

disease, plaque radiotherapy or periocular chemotherapy. External beam radiation therapy has extremely

limited indications nowadays due to its extensive cancer risks and complications.{Dimaras, 2015

#14249}

Metastasis and Second Cancers

Germline retinoblastoma carry the risk of development of second primary cancers most commonly

osteosarcoma and fibrosarcoma. Sometimes it might be confused with metastatic retinoblastoma. Fine

needle aspiration cytopathology has minimal role in differentiation as both metastasis and second cancers

appear as blue round cell tumors. molecular analysis might help to differentiate.{Racher, 2016 #17799}

Inheritance pattern [Hilary]

Knudson two-hit hypothesis

In most cases, retinoblastoma develops when both copies of the RB1 gene are inactivated. This

concept was first formulated in 1971, when Knudson used retinoblastoma as the prototypic cancer to

derive the two-hit hypothesis.{Knudson, 1971 #8685} In heritable retinoblastoma, the first mutational

event is inherited via the germinal cells, while the second event occurs in the somatic cells. In

nonheritable retinoblastoma, both mutation events occur in the somatic cells. Heritable retinoblastoma

encompasses 45% of all reported cases.{MacCarthy, 2009 #8669;Moreno, 2014 #18928;Wong, 2014

#18931} The clinical presentation of heritable retinoblastoma consists of 80% bilateral and 15-18%

unilateral.{Dimaras, 2015 #14249} In non-heritable retinoblastoma the majority (98%) of cases have

somatic biallelic RB1 loss in the tumor, while the remaining 2% have no mutation in either copy of RB1

but instead have somatic amplification of the MYCN oncogene.{Rushlow, 2013 #11249}

7

Heritable Retinoblastoma and Penetrance

In heritable retinoblastoma, each offspring of a patient has a 50% risk of inheriting the RB1

pathogenic change. Typically, nonsense and frame-shift germline mutations, which lead to absence of

RB1 expression or truncated dysfunctional RB1 protein, show nearly complete (90%) penetrance. Often

the second mutational event in the retinal cell is loss of the second RB1 allele (LOH, loss of

heterozygosity). In these families the presentation is typically unilateral multifocal or bilateral

retinoblastoma. In a smaller subset of hereditary retinoblastoma, reduced expressivity and reduced

penetrance is observed . In these families, when retinoblastoma develops, it is often late onset and less

severe, presenting as unilateral, unifocal (reduced expressivity) and in some carrier family member

retinoblastoma never develops (reduced penetrance). The types of reported RB1 mutations that result in

reduced expressivity or penetrance are diverse. Many consist of mutations that reduced RB1 protein

expression. Examples include, (1) mutations in exons 1 and 2,{Sanchez-Sanchez, 2007 #18933} (2)

mutations in exons 26 and 27,{Mitter, 2009 #7347} (3) intronic mutations{Schubert, 1997

#18936;Lefevre, 2002 #18938} and (4) missense mutations.{Scheffer, 2000 #18939;Cowell, 1998

#18940} In addition, large deletions encompassing RB1 gene and MED1 gene cause reduced

expressivity/penetrance.{Dehainault, 2014 #18941;Bunin, 1989 #18950} Dehainault et al showed that

RB1-/- cells cannot survive in the absence of MED4. This can explain why patients with 13q14 deletion

syndrome more often have unilateral tumors, in comparison to patients with gross deletions with one

breakpoint in the RB1 gene whom typically present with bilateral disease.{Mitter, 2011

#19016;Matsunaga, 1980 #19020;Albrecht, 2005 #19022} The severity of risk can be evaluated through

the disease-eye-ratio (DER) calculated by taking the number of eyes affected with tumors divided by the

total number of eyes of carriers within the family.{Lohmann, 1994 #19003}

In some instances of hereditable reduced expressivity/penetrance retinoblastoma, the parental origin

impacts whether or not an individual develops retinoblastoma and subsequently whether their carrier

offspring are at risk to develop retinoblastoma, a phenomenon termed the parent-of-origin effect.{Klutz,

8

2002 #19004;Schuler, 2005 #19011;Eloy, 2016 #19015} Eloy et al{Eloy, 2016 #19015} proposed a

potential molecular mechanism to explain the parent-of-origin effect. Using the c.1981C>T

(p.Arg661Trp) reduced penetrance/expressivity missense mutation, the researchers discovered that

differential methylation of the intron 2 CpG85 skews RB1 expression in favor of the maternal allele. In

other words, when the p.Arg661Trp allele is maternally inherited there is sufficient tumor suppressor

activity to prevent RB development and 90.3% of carriers remain unaffected. However, when the allele is

paternally transmitted, very little RB1 is expressed, leading to haploinsufficiency and RB development in

67.5% of cases. A similar inheritance pattern was also reported for intron 6 c.607+1G>T substitution.

{Klutz, 2002 #19004}

Mosaicism

FIGURE ON MOSAICISM

RB1 gene [Hilary]

The RB1 gene, located on 13q14, encodes the RB protein, which is an important cell cycle regulator

and the first tumor suppressor gene ever discovered.{Friend, 1986 #19025} After a cell completes

mitosis, the RB protein is dephosphorylated, permitting it to bind to the promoter region of the E2F

transcription factor gene, thereby repressing transcription and inhibiting the progression of the cell cycle

from G1 to S phase.{Nevins, 2001 #19053;Cobrinik, 2005 #19059;Sage, 2012 #19061} In order for the

cell to enter S phase, cyclin-dependent kinases phosphorylate RB, which removes the ability of RB to

bind to the E2F gene promoter.{Knudsen, 2008 #19071} RB functions to regulate proliferation in most

cell types.{Cobrinik, 2005 #19059} Often, loss of RB1 is compensated by increased expression of its

related proteins, however, in certain susceptible cells, such as the retinal cone cell precursors,

compensatory mechanisms are not sufficient and tumorigenesis is initiated.{Xu, 2014 #19253}

9

RB1 Mutations

There are many ways in which the function of the RB protein is impaired including point mutations,

small and large deletions, promotor methylation and chromothripsis.{Lohmann, 1999 #19258;McEvoy,

2014 #19260} The majority of RB1 mutations are de novo, unique to a specific patient or family,

however, there are some known recurrent mutations found across many unrelated individuals. One subset

of recurrent mutations involve 11 CpG sites, which make up ~22% of all RB1 mutations.{Rushlow, 2009

#12290} The high recurrence of nonsense mutations at these sites is due to the hypermutabilty and

subsequent deamination of 5-methylcytosine.{Richter, 2003 #12288}

The origin of a de novo RB1 mutation can arise either pre- or post-conception. Most often, pre-

conception mutagenesis occurs during spermatogenesis..{Dryja, 1997 #19332;Munier, 1998 #8627}

Furthermore, advanced paternal age has been shown to increase risk for retinoblastoma.{Toriello, 2008

#19267} This might be due to the larger number of cell divisions during spermatogenesis than oogenesis

or the increased rate for base substitution errors in aging men compared to women. In cases of pre-

conception mutagenesis, the proband carries the de novo RB1 mutation in every cell within their body and

typically presents with bilateral retinoblastoma. In contrast, post-conception RB1 mutagenesis occurs

during embryogenesis. Depending on the embryological stage of development, a few or numerous tissues

may be mosaic for the RB1 mutation. If the mutational event occurs during retinal development, the

presentation is often unilateral retinoblastoma.{Dimaras, 2015 #14249}

OTHER GENOMIC CHANGES IN ADDITION TO RB1

In a small subset (2%) of unilateral patients, no RB1 mutant is identified. Instead, striking

amplification (28-121 copies) of the MYCN oncogene is detected.{Rushlow, 2013 #11249} Patients with

RB1+/+ MYCN are clinically distinct from RB-/- patients, showing much younger age at diagnosis, distinct

histological features and larger, more invasive tumors. In addition to loss of RB1 or MYCN

amplification, specific somatic copy number alterations commonly occur in the progression of the

retinoblastoma. Commonly seen are gains in 1q32, 2p24, 6p22 and losses at 13q and 16q22-24.{Corson,

10

2007 #12275} These regions contain important oncogenes (MDM4, KIF14, MYCN, DEK and E2F3) and

tumor suppressor genes (CDH11), thought to act as drivers promoting the growth of the cancer.

{Theriault, 2014 #19306}

Other less common alterations that have been identified in retinoblastoma tumors include differential

expression of some microRNAs{Huang, 2007 #19315} and recurrent single nucleotide variants/insertion-

deletions in the genes BCOR and CREBBP.{Kooi, 2016 #19325} In comparison to the genomic landscape

of other cancers, retinoblastoma is one of the least mutated.{Kooi, 2016 #19325}

Molecular diagnosis [Hilary]

The presentation of the patient helps to guide the most optimal strategy for retinoblastoma molecular

genetic testing. If the patient is bilaterally affected, the probability of finding a germline mutation in the

RB1 gene is high (example - 97% detection rate in comprehensive laboratory). For this reason, the most

optimal strategy for testing bilateral patients involves testing genomic DNA extracted from peripheral

blood lymphocytes (PBL) first. In rare instances, some patients with isolated bilateral retinoblastoma, the

predisposing RB1 mutation has occurred sometime during embryonal development. In these cases, the

RB1 mutation may only be present in some cells and may not be detected in DNA from PBL. Therefore,

in the event that no mutation is identified in the blood of a bilaterally affected patient, DNA from tumor

should be investigated.{Canadian Retinoblastoma, 2009 #18012}

The situation is different for unilateral patients. Given that approximately 15% of unilateral patients

carry germline mutations, the most optimal strategy for highest detection rate is to first test DNA

extracted from a tumor sample. Upon identification of the tumor mutations, targeted molecular analysis

can be performed on DNA from PBL to determine if the mutation is present is the patient’s germline.

When only the tumor is found to carry the mutations, this information can be very valuable for genetic

counselling, reducing the risk of recurrence in siblings and cousins. In addition, this targeted approach

11

can allow for a more sensitive assessment of the PBL DNA, which can be useful in the detection of low

level mosaic mutations, more common in unilateral cases.{Canadian Retinoblastoma, 2009 #18012}

Sample preparation impacts the quality of DNA. For best results, fresh or frozen tumor samples

should be taken, as opposed to formalin fixed paraffin embedded tumors, in which DNA is often highly

degraded, making it often too fragmented for use in some molecular diagnostic methods. With regards to

genomic DNA from PBL, it is best to collect whole blood in EDTA, as this anticoagulant has minimal

impact on downstream molecular methods.

Technologies and techniques: Given that there are many ways in which the RB1 gene can be mutated,

several molecular techniques are required to assess for the whole spectrum of oncogenic events.

DNA sequencing: Single nucleotide variants (SNVs) and small insertions/deletions can be identified

using DNA sequencing strategies including Sanger dideoxy-sequencing or massively parallel next-

generation sequencing (NGS) methods.{Singh, 2016 #19381;Li, 2016 #19404;Chen, 2014 #19419}

While both strategies function to produce DNA sequences, NGS has the add advantage of producing

millions of DNA sequences in a single run, in contrast to one sequence per reaction with Sanger.

Deciding on which technology to use depends on the clinical question being asked. When screening

family members for a known sequencing detectable RB1 mutation, targeted Sanger sequencing would be

a more cost and time effective strategy as opposed to a screen for an unknown de novo mutation, where

NGS may be the most effective screening strategy. Another added advantage to NGS is the ability to

provide deep sequencing, allowing for a much lower limit of detection (analytic sensitivity) for identify

low level mosaic mutations compared to Sanger sequencing.{Chen, 2014 #19419}

Copy number analysis: Large RB1 deletions or duplications that span whole exons or multiple exons

typically cannot be easily detected by DNA sequencing. Instead, techniques including multiplex ligation-

dependent probe amplification (MLPA), quantitative multiplex PCR (QM-PCR) or array comparative

genomic hybridization (aCGH) are often used to interrogate for large deletions (ex. 13q14 deletion

syndrome) and duplications. In addition, these techniques can also be used to identify other genomic

12

copy number alterations seen in retinoblastoma tumors, such as MYCN amplification. Recently, new

developments in bioinformatics analysis have created ways in which NGS data can be interrogated for

copy number variants

Low-level mosaic detection: Somatic mosaicism can arise in either the presenting patient or their

parent. Detecting a mosaic mutation can be difficult depending on the individual’s level of mosaicism.

NGS can be used detect low-level mosaicism (see above). In addition, allele-specific PCR (AS-PCR) is

an another strategy that can be used in situations where the RB1 mutation is known.{Rushlow, 2009

#12290} This strategy involves the generation of a unique set of primers specific to the mutation of

interest and can detect mosaicism levels as low as 1%.

Microsatellite analysis: LOH, MCC, identity,

Methylation analysis: In addition to genetic changes, epigenetic changes have been recognized as

another mechanism of retinoblastoma development. Hypermethylation of the RB1 promoter CpG island

results in transcription inhibition of the RB1 gene and has been identified 10-12% of retinoblastoma

tumors.{Richter, 2003 #12288} This epigenetic event is thought to only occur somatically and has not

been identified constitutionally in any retinoblastoma patients thus far.

RNA analysis:

Protein studies

Cytogenetic strategies: Karyotype, fluorescent in situ hybridization (FISH) or array comparative

genomic hybridization (aCGH) of peripheral blood lymphocytes can be used to identify large deletions

and rearrangements in patient’s suspected of 13q14 deletion syndrome. In parents of 13q14 deletion

patients, karyotype analysis can be used to assess for balanced translocations, which increases the risk of

recurrence in subsequent offspring.

13

Genetic Counseling (Heather/Hilary)

Targeted familial testing/prenatal testing,

Targeted familial testing{Canadian Retinoblastoma, 2009 #18012;Dimaras, 2015 #14249} is used to

determine if a predisposing RB1 mutation has occurred de novo, parental DNA from PBL is investigated.

Even if neither parent is identified to be a carrier, recurrence risk in siblings is still increased due to the

risk of germline mosaicism. DNA from PBL for all siblings of affected patients should be tested for the

proband’s mutation. As well, DNA from PBL for children of all affected patient’s should also be tested

for the predisposing mutation.

If the proband’s mutation was identified to be mosaic (ie postzygotic in origin) in DNA from PBL,

parents and siblings of the proband are not at risk to carry the predisposing mutation. However, the

children of mosaic proband should be tested, as their risk of inheriting the predisposing RB1 mutation can

be as high as 50% depending on the mutation burden in the probands germline.

When a RB1 mutation has been identified in a family, The Known RB1 mutation of the proband can

be tested in his offspring. Couples may consider multiple options with respect to planning a pregnancy.

Genetic testing performed early in the course of the pregnancy is available in many countries around the

world. Two early procedures are available: 1) chorionic villus sampling (CVS) and 2) amniocentesis.

CVS is a test typically performed between 11-14 weeks gestation during which as sample of the placenta

is obtained either by transvaginal or transabdominal approach. Amniocentesis is a test performed after 16

weeks of gestation whereby as sample of the amniotic fluid is gathered with a transabdominal approach.

CVS has a procedure-associated risk of miscarriage of ~1%. Amniocentesis has a procedure-associated

risk of miscarriage between 0.1-0.5%. Though uncommon, there is a risk for maternal cell contamination

that occurs more frequently with CVS.{Akolekar, 2015 #19427}

Genetic testing results can be used by the family and health care team to manage the pregnancy. If a

mutation is not identified, the pregnancy can proceed with no further intervention, as there is no increased

14

risk for retinoblastoma beyond the general population risk. If the mutation is identified, some couples

may consider deciding to stop the pregnancy; other couples will decide to continue with the pregnancy

and appropriate intervention, such as early delivery, will be put into place to improve outcomes.{Soliman,

2016 #18924}

Some couples know that they wish to continue their pregnancy regardless of the genetic testing results

and are concerned by the risk of miscarriage associated with early invasive prenatal testing. Where

available, couples can also consider the option of late amniocentesis, performed between 30-34 weeks

gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early

delivery rather than miscarriage.{Akolekar, 2015 #19427} The risk for procedure-associated significant

preterm delivery is low (<3%). Results of genetic testing will be available with enough time to plan for

early delivery when a mutation has been inherited.

In many countries around the world, the option for prenatal genetic testing is not available. Even

where available, some couples may elect to do no invasive testing during the course of the pregnancy.

For these conceptions, if the pregnancy is at 50% risk for inheriting a RB1 mutation, it is crucial that the

pregnancy does not go post-dates. Induction of labour should be seriously considered if natural delivery

has not occurred by the due date.{Soliman, 2016 #18924;Canadian Retinoblastoma, 2009 #18012}

Preconception testing

In some countries around the world, there is an in vitro fertilization option available to couples called

preimplantation genetic diagnosis (PGD).{Dhanjal, 2007 #19428;Dommering, 2004 #19429;Xu, 2004

#19430;Girardet, 2003 #19431} For PGD, a couple undergoes in vitro fertilization. Conceptions are

tested at an early stage of development (typically 8-cell) for the presence of the familial mutation. Only

those conceptions that do not carry the mutation will be used for fertilization. The procedure is costly,

ranging from $10,000-$15,000 per cycle. In some countries, there may be full or partial coverage of the

costs associated with procedure. In addition to cost, couples must consider the medical and time impact of

undergoing in vitro fertilization. Couples also need to be aware that the full medical implications of PGD

15

are not yet understood; there is emerging evidence that there may be a low risk for epigenetic changes in

the conception as a result of the procedure. For couples that undergo PGD, it is recommended that typical

prenatal testing be pursued during the course of the pregnancy to confirm the results.{Dhanjal, 2007

#19428;Dommering, 2004 #19429;Girardet, 2003 #19431;Xu, 2004 #19430}

Molecular Screening for Retinoblastoma

This can be performed either prenatal or it can be performed at birth via umbilical cord blood

(postnatal screening). This will help either eliminate the 50% theoretical risk of the proband’s RB1

mutation heritability or confirm it into 100% risk. Both screening methods are effective in improving

visual outcome and eye salvage than non-screened children, However, prenatal screening allows for

planning for earlier delivery in positive children (late preterm/early term); this was shown to have less

number of tumors at birth (20% versus 50 %) with only 15 % visual threatening tumors in prenatatl

screening. Prenatal screening with early delivery showed less tumor and treatment burden with higher

treatment success, eye preservation and visual outcome.{Soliman, 2016 #18924}

Surveillance for mets and second cancer

Benefits of genetic counseling (Table of risk% [skalet etc] [impact new data?] ie: siblings, offspring,

cousins, faroff relatives, stats below population risk]

Genetic counseling is both a psychosocial and educational process for patients and their families with

the aim of helping families better adapt to the genetic risk, the genetic condition, and the process of

informed decision-making.{Uhlmann, 2009 #19436;Shugar, 2016 #19461;Shugar, 2016 #19471}.

Genetic testing is an integral component of genetic counseling that results in more informed and precise

genetic counseling. Concrete knowledge of the genetic test outcomes results in specificity, reducing the

need for other possible scenarios to be discussed with the family. This enhances the educational

component of genetic counseling and also provides further time for psychosocial support to be provided

to the family.

16

Patients with bilateral retinoblastoma at presentation are presumed to have heritable retinoblastoma

and a RB1 mutation. Genetic testing provides more accurate information about the type of heritable

retinoblastoma and allows for straightforward testing to determine if additional family members are at

risk. Through genetic testing, a patient may be found to have a large deletion extending beyond the RB1

gene as part of the 13q deletion spectrum. Individuals with 13q deletion syndrome are at risk for

additional health concerns requiring appropriate medical management and intervention. Results may

reveal a mosaic mutation which indicates that the mutation is definitively de novo; only the individual’s

own children are at risk and no further surveillance or genetic testing is needed for other family members.

The results may find a low-penetrance mutation which indicates the patient is at reduced risk to develop

future tumours. As genetic testing for retinoblastoma becomes more common place and data accumulate,

surveillance of the proband may one day be matched more precisely to the level of risk for new tumours

for individuals with low penetrance mutations.

Patients with unilateral retinoblastoma greatly benefit from genetic testing and counselling.

Approximately 15% of patients with unilateral retinoblastoma will be found to have heritable

retinoblastoma. Correctly identifying these patients can be lifesaving, for both the patients and their

families. Genetic testing companies focused on enhanced detection of RB1 mutations are able to identify

nearly 97% of all retinoblastoma mutations. Genetic testing of the patient’s blood is sensitive enough

when thorough methods are used that not finding a mutation results in a residual risk of heritable

retinoblastoma low enough to remove the need for examinations under anesthesia. This reduces the health

risk for the patient and the cost to the health care system. Testing is even more accurate when a tumour

sample is collected and tested when available. When mutations are identified in the tumour and are

negative in blood, the results can eliminate the need for screening of family members and provide

accurate testing for the patient’s future children. Whether or not a tumour sample is available, finding a

RB1 mutation in a patient’s blood confirms that this patient has heritable retinoblastoma. This patient now

benefits from increased surveillance designed to detect tumours at the earliest stages and awareness of an

17

increased lifelong risk for second cancers. Members of the patient’s family can have appropriate genetic

testing to accurately determine who is at risk. As with patients with bilateral retinoblastoma, knowing the

specific type of mutation provides the most detailed provision of medical management and counselling.

Cost-effectiveness [Brenda/Crystal] FIGURE/FLOW CHART

Difficulties and opportunities across different jurisdictions/countries [Jeffry/Sameh]

Compare/contrast Canada vs China vs Jordon

Societal/cultural challenges to GC

In China, many families with retinoblastoma children do not understand the benefits of genetic testing

and genetic counseling in treatment and follow-up. Meanwhile, the health insurance can’t cover the cost

for it. So all the obstacles mentioned above result in the limited application of genetic testing and genetic

counseling nationwide, which also lead to the redundant economic burden on the affected families. The

Chinese government started new policy that allowed every family to have one more child nowadays.

Therefore, genetic testing and genetic counseling should be put into good use especially for the families

carrying the germline RB1 mutation.

18

Conclusions

19

REFERENCES

Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-

Blackwell.

Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling

Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of

print. PMID: 27891554 DOI: 10.1007/s10897-016-0042-y

20

Table X:

Subretinal Fluid (RD)

No≤ 5 mm

>5 mm - ≤ 1 quadrant

> 1quadrant

Tum

or

Tumors ≤ 3 mm and further than 1.5 mm from the disc and fovea cT1a/A cT1a/B cT2a/C cT2a/D

Tumors > 3 mm or closer than 1.5 mm to the disc and fovea cT1b/B cT1b/B cT2a/C cT2a/D

Se

edin

g Localized vitreous/ subretinal seeding cT2b/C cT2b/C cT2b/C cT2b/Ddiffuse vitreous/subretinal seeding cT2b/D

High

risk

feat

ures

Phthisis or pre-phthisis bulbi cT3a/ETumor invasion of the pars plana, ciliary body, lens, zonules, iris or anterior chamber cT3b/ERaised intraocular pressure with neovascularization and/or buphthalmos cT3c/EHyphema and/or massive vitreous hemorrhage cT3d/EAseptic orbital cellulitis cT3e/EDiffuse infiltrating retinoblastoma ??/E

Extraocular retinoblastoma cT4/??

clinical T (cT) versus International Intraocular retinoblastoma Classification (IIRC) (cT/IIRC); ?? Not

applicable ; RD Retinal detachment

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