Format of the review article:

- A word limit of 5,000 words;

- Less than 80 references;

- No strict limit to the number of tables and figures (8-10 recommended);

- 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. HR is a paid employee

and BG is an unpaid medical advisor at Impact Genetics. No other 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, bilateral, unilateral, DNA sequencing,

3

INTRODUCTION

Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.1

It is considered the prototype of genetic cancers.26,000 live births, translating to about 8,000 new cases

per year worldwide.1,3 Understanding retinoblastoma genetics is crucial in multiple aspects as clinical

presentation, choice of treatment modality and follow-up for both the child and his family. Multiple

reviews described the genetics of retinoblastoma in depth. In this review we will try to address most of

the updates on the genetic aspect of retinoblastoma in a clinical scenario setting that might simplify this

aspects to ophthalmologists all over the world.

Case Scenario: a 2 years old female child presented with left leukocorea (white pupil). The family

noticed the white pupil at a family photograph 5 days ago. They sought medical advise to their family

physician who suspected retinoblastoma and referred them urgently to the pediatric ophthalmologist. The

family history is irrelevant and the mother is 33 weeks pregnant. The child was extremely uncooperative

but the ophthalmologist was able to visualize a white retinal mass in the left eye. He couldn’t examine

except the inferior retina, an intact optic and fovea in the right eye that was apparently free. The diagnosis

of retinoblastoma was made and the following discussion took place between the ophthalmologist and the

family.

Q1: Father: What is retinoblastoma?

Retinoblastoma is a malignant tumor that arises from a developing retinal cell. The exact cell of origin is

unknown but there are many theories suggesting either a photoreceptor cell or an inner nuclear layer cell

origin. The visualization of early tumors by optical coherence tomography supports the later but not yet

proven. Retinoblastoma can affect one (unilateral) or both eyes (bilateral) and in rare instances (<1%)

might be associated with a brain tumor in the pineal region regardless of the laterality of ocular

involvement.

4

Q2: Father: why it is presenting in such a young age?

Retinoblastoma arises from developing cells that are present in the retinae of young children from the

intrauterine life up to 7 years of age. It is believed that all retinal cells are developed by this age. Rarely,

retinoblastoma develops in older ages.

Q3: Mother: What caused retinoblastoma?

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.1 The RB1 gene, located on chromosom13q14, encodes the RB protein (pRB), an important

cell cycle regulator and the first tumor suppressor gene discovered.4 After a cell completes mitosis, the

pRB 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.5-7 In order for the cell to enter S phase, cyclin-dependent kinases phosphorylate RB, which

removes the ability of pRB to bind to the E2F gene promoter.8 pRB functions to regulate proliferation in

most cell types.6 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.9

Q4: What causes retinoblastoma to be unilateral versus bilateral?

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.10 In heritable retinoblastoma, the first mutational event is inherited via the

germinal cells, while the second event occurs in the somatic cells. In non-heritable retinoblastoma, both

mutation events occur in the somatic cells. Heritable retinoblastoma encompasses 45% of all reported

cases.11-13 The clinical presentation of heritable retinoblastoma consists of 80% bilateral and 15-18%

5

unilateral.1 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.14

Q5: Mother: What caused these mutations? Did I cause them?

There are many causes in the environment that can cause this including cosmic rays, X-rays, DNA

viruses, UV irradiation and irradiation. This is sporadic and cannot be anticipated or prevented. There are

many ways in which the function of the pRB is impaired including point mutations, small and large

deletions, promotor methylation and chromothripsis.15,16 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.17 The high recurrence of nonsense mutations at these sites is due to the

hypermutabilty and subsequent deamination of 5-methylcytosine.18

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

conception mutagenesis occurs during spermatogenesis.19,20 Furthermore, advanced paternal age has been

shown to increase risk for retinoblastoma.21 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.1

Q6: Father: So, only RB1 mutation is sufficient for retinoblastoma to develop?

6

Both RB1 mutations are essential but insufficient to develop retinoblastoma evidenced by biallelic RB1

loss in the benign retinoma;22.suggesting more genetic or epigenetic changes for malignant

transformation.

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

(28-121 copies) of the MYCN oncogene is detected.14 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 16q22-24.23 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.24

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

expression of some microRNAs25 and recurrent single nucleotide variants/insertion-deletions in the genes

BCOR and CREBBP.26 In comparison to the genomic landscape of other cancers, retinoblastoma is one of

the least mutated.26

Q7: What is the retinoma that you mentioned and how does it differ from retinoblastoma?

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

reactive retinal pigment epithelial growth and calcific foci.27 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.22 It can transform to retinoblastoma even after many

years of stability.28

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

7

related to genomic changes in single (clonal) cells, that provide a proliferative advantage.29,30 Tumor seeds

float free of the main tumor into the subretinal space or the vitreous cavity as a result of poor cohesive

forces between tumor cells, appearing as dust, spheres or tumor clouds.31 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.29,30,32 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.

Q8: Do all affected individuals with RB1 mutations develop retinoblastoma?

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 RB 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 2,33 (2) mutations in exons 26 and 27,34 (3)

intronic mutations35,36 and (4) missense mutations.37,38 In addition, large deletions encompassing RB1 gene

and MED1 gene cause reduced expressivity/penetrance.39,40 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

8

the RB1 gene whom typically present with bilateral disease.41-43 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.44

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.45-47 Eloy

et al47 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 pRB development and 90.3% of carriers remain unaffected. However, when the allele

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

in 67.5% of cases. A similar inheritance pattern was also reported for intron 6 c.607+1G>T substitution.45

Q9: Mother: could we have discovered it earlier?

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.32 In developing countries, buphthalmos and proptosis due to advanced and extraocular disease

respectively represents a higher percentage.48 Less common presentations include; heterochromia irides,

neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.32 Retinoblastoma

(unilateral or bilateral) might be associated with a brain tumor in the pineal, suprasellar or parasellar

regions (Trilateral retinoblastoma)49,50 that starts early; with the median age of onset 17 months after

retinoblastoma is diagnosed and before the age of 5 years. Retinoblastoma might present in a syndromic

form (13q deletion 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

9

noseassociated with various degrees of hypotonea and mental retardation.51-53 The main differential

diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and ocular toxicariasis.32

Q10: What are the treatments and what govern the choice?

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.54 Multiple staging systems have predicted likelihood to

salvage an eye without using radiation therapy; the International Intraocular Retinoblastoma

Classification (IIRC)29 has been recently the most reliable, but published evidence is confusing because

significantly different versions have emerged.1 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.54 (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.54 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%.

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

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.1

Q11: Is retinoblastoma lethal?

10

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

compared with <5% in developed countries.48,55 Delayed diagnosis and treatment due to lack of

retinoblastoma knowledge by ophthalmologists and parents, socioeconomic56 and cultural factors are

major causes of high mortality. Broad understanding of retinoblastoma genetics and genetic counseling

can contribute to reducing mortality from retinoblastoma.

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.57

Q12: How can we test for retinoblastoma?

The most optimal strategy for retinoblastoma molecular genetic testing is guided by the patient’s

tumor presentation. 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 first testing genomic DNA extracted from

peripheral blood lymphocytes (PBL). In rare instances of 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.58

In contrast, given that approximately 15% of unilateral patients carry a germline mutation, the most

optimal strategy 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 RB1 mutations, this result

dramatically reduces the risk of recurrence in siblings and cousins. In addition, this targeted approach can

11

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.58

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

should be collected, 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 or ACD, as these

anticoagulants have minimal impact on downstream molecular methods.59

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.60-62 While both strategies function to produce DNA sequences,

NGS has the added 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 is a more cost and time effective strategy. In contrast, NGS may be the most effective

screening strategy to investigate for an unknown de novo mutation in an affected proband. Another added

advantage to NGS is the ability to perform deep sequencing, which allows for a much lower limit of

detection (analytic sensitivity) for identify low level mosaic mutations in comparison to Sanger

sequencing.62

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.61,63 While the data is promising; the current limitation of targeted NGS is that

capture efficiency is uneven, which reduces the sensitivity of detecting CNVs in comparison to

conventional methods.

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.17 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: The second mutational event in the majority of retinoblastoma tumors

consists of loss of heterozygosity (LOH). LOH is common event in many cancers and is strongly

associated with loss of the wild-type allele in individuals with an inherited cancer predisposition

syndrome.64 Polymorphic microsatellite markers distributed throughout chromosome 13 can be used to

detect a change from a heterozygous state in blood compared to the homozygous state in a tumor with

LOH. Microsatellite marker analysis is also useful in identity testing and in determining the presence of

maternal cell contamination in prenatal diagnostic testing.

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

another mechanism of retinoblastoma development.65 Hypermethylation of the RB1 promoter CpG island

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

tumors.18,66 This epigenetic event primarily occurs somatically, however, rare instance of heritable

mutations in the RB1 promoter and translocations disrupting RB1 regulator sites have been reported to

also cause RB1 promoter hypermethylation.67

13

RNA analysis: In rare instance, no RB1 mutation is identified in the coding, promoter or flanking

intronic sequence in blood from a bilateral patient. Conventional molecular methods do not interrogate

all RB1 intronic nucleotides due to the large amount of sequence and repetitive nature of intronic DNA.

However, deep intronic sequencing alterations have been identified to disrupt RB1 transcription in

patients with retinoblastoma. 68,69 In order to investigate for deep intronic changes, analysis of the RB1

transcript by reverse-transcriptase PCR (RT-PCR) is performed. RNA studies are also useful in clarifying

the pathogenicity of intronic sequencing alterations detected by conventional DNA sequencing.

68,69Alternatively, as sequencing costs continue to decrease; whole genome sequence (WGS) may become

the method of choice to uncover deep intronic changes.

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.41,70 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.51

Q13: Are these tests available worldwide?

No, They are mainly present in developed countries. 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.

14

In Egypt,71 Genetic testing for retinoblastoma is not available and genetic counseling is the only way for

addressing retinoblastoma genetics. This counseling is performed through ophthalmologists mainly with

defective training in this aspect. Genetic counseling was found to increase the level of knowledge

regarding familial retinoblastoma genetics but the proper translation of this knowledge into appropriate

screening action was deficient.71

Q14: What after finding the RB1 mutation?

Targeted familial testing1,58 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.72

15

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

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.73

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.72 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.58,73

Q15: Can we use the known mutation in other benefits?

Preconception testing

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

preimplantation genetic diagnosis (PGD).74-77 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

16

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 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.74-77

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.73

Q16: what is genetic counseling?

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.78-80. 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.

Q17: what are the risks for the relatives in the family?

17

Q18: What are the long term risks for germ line RB1 mutation?

Genetic Counseling (Heather/Hilary)

Targeted familial testing/prenatal testing,

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]

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

18

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

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

19

Conclusions

20

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

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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

1. Dimaras H, Corson TW, Cobrinik D, et al. Retinoblastoma. Nature Reviews Disease Primers. 2015:15021.

2. Theriault BL, Dimaras H, Gallie BL, Corson TW. The genomic landscape of retinoblastoma: a review. Clin Experiment Ophthalmol. 2014;42(1):33-52.

3. Seregard S, Lundell G, Svedberg H, Kivela T. Incidence of retinoblastoma from 1958 to 1998 in Northern Europe: advantages of birth cohort analysis. Ophthalmology. 2004;111(6):1228-1232.

4. Friend SH, Bernards R, Rogelj S, et al. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature. 1986;323(6089):643-646.

5. Nevins JR. The Rb/E2F pathway and cancer. Hum Mol Genet. 2001;10(7):699-703.6. Cobrinik D. Pocket proteins and cell cycle control. Oncogene. 2005;24(17):2796-2809.7. Sage J, Cleary ML. Genomics: The path to retinoblastoma. Nature. 2012;481(7381):269-270.

22

8. Knudsen ES, Knudsen KE. Tailoring to RB: tumour suppressor status and therapeutic response. Nat Rev Cancer. 2008;8(9):714-724.

9. Xu XL, Singh HP, Wang L, et al. Rb suppresses human cone-precursor-derived retinoblastoma tumours. Nature. 2014;514(7522):385-388.

10. Knudson AG. Mutation and cancer: statistical study of retinoblastoma. Proceedings of the National Academy of Science, USA. 1971;68(4):820-823.

11. MacCarthy A, Birch JM, Draper GJ, et al. Retinoblastoma: treatment and survival in Great Britain 1963 to 2002. Br J Ophthalmol. 2009;93(1):38-39.

12. Moreno F, Sinaki B, Fandino A, Dussel V, Orellana L, Chantada G. A population-based study of retinoblastoma incidence and survival in Argentine children. Pediatr Blood Cancer. 2014;61(9):1610-1615.

13. Wong JR, Tucker MA, Kleinerman RA, Devesa SS. Retinoblastoma incidence patterns in the US Surveillance, Epidemiology, and End Results program. JAMA ophthalmology. 2014;132(4):478-483.

14. Rushlow DE, Mol BM, Kennett JY, et al. Characterisation of retinoblastomas without RB1 mutations: genomic, gene expression, and clinical studies. The lancet oncology. 2013;14(4):327-334.

15. Lohmann DR. RB1 gene mutations in retinoblastoma. Hum Mutat. 1999;14(4):283-288.16. McEvoy J, Nagahawatte P, Finkelstein D, et al. RB1 gene inactivation by chromothripsis in

human retinoblastoma. Oncotarget. 2014;5(2):438-450.17. Rushlow D, Piovesan B, Zhang K, et al. Detection of mosaic RB1 mutations in families with

retinoblastoma. Hum Mutat. 2009;30(5):842-851.18. Richter S, Vandezande K, Chen N, et al. Sensitive and efficient detection of RB1 gene mutations

enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72(2):253-269.19. Dryja TP, Morrow JF, Rapaport JM. Quantification of the paternal allele bias for new germline

mutations in the retinoblastoma gene. Hum Genet. 1997;100(3-4):446-449.20. Munier FL, Thonney F, Girardet A, et al. Evidence of somatic and germinal mosaicism in

pseudo-low-penetrant hereditary retinoblastoma, by constitutional and single-sperm mutation analysis. Am J Hum Genet. 1998;63(6):1903-1908.

21. Toriello HV, Meck JM, Professional P, Guidelines C. Statement on guidance for genetic counseling in advanced paternal age. Genet Med. 2008;10(6):457-460.

22. Dimaras H, Khetan V, Halliday W, et al. Loss of RB1 induces non-proliferative retinoma: increasing genomic instability correlates with progression to retinoblastoma. Hum Mol Genet. 2008;17(10):1363-1372.

23. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes Cancer. 2007;46(7):617-634.

24. Theriault BL, Dimaras H, Gallie BL, Corson TW. The genomic landscape of retinoblastoma: a review. Clin Exp Ophthalmol. 2014;42(1):33-52.

25. Huang JC, Babak T, Corson TW, et al. Using expression profiling data to identify human microRNA targets. Nat Methods. 2007;4(12):1045-1049.

26. Kooi IE, Mol BM, Massink MP, et al. Somatic genomic alterations in retinoblastoma beyond RB1 are rare and limited to copy number changes. Sci Rep. 2016;6:25264.

27. Gallie BL, Ellsworth RM, Abramson DH, Phillips RA. Retinoma: spontaneous regression of retinoblastoma or benign manifestation of the mutation? Br J Cancer. 1982;45(4):513-521.

28. Theodossiadis P, Emfietzoglou I, Grigoropoulos V, Moschos M, Theodossiadis GP. Evolution of a retinoma case in 21 years. Ophthalmic Surg Lasers Imaging. 2005;36(2):155-157.

29. Murphree AL. Intraocular retinoblastoma: the case for a new group classification. Ophthalmology clinics of North America. 2005;18:41-53.

30. Balmer A, Zografos L, Munier F. Diagnosis and current management of retinoblastoma. Oncogene. 2006;25(38):5341-5349.

23

31. Munier FL. Classification and management of seeds in retinoblastoma. Ellsworth Lecture Ghent August 24th 2013. Ophthalmic Genet. 2014;35(4):193-207.

32. Balmer A, Munier F. Differential diagnosis of leukocoria and strabismus, first presenting signs of retinoblastoma. Clin Ophthalmol. 2007;1(4):431-439.

33. Sanchez-Sanchez F, Ramirez-Castillejo C, Weekes DB, et al. Attenuation of disease phenotype through alternative translation initiation in low-penetrance retinoblastoma. Hum Mutat. 2007;28(2):159-167.

34. Mitter D, Rushlow D, Nowak I, Ansperger-Rescher B, Gallie BL, Lohmann DR. Identification of a mutation in exon 27 of the RB1 gene associated with incomplete penetrance retinoblastoma. Fam Cancer. 2009;8(1):55-58.

35. Schubert EL, Strong LC, Hansen MF. A splicing mutation in RB1 in low penetrance retinoblastoma. Hum Genet. 1997;100(5-6):557-563.

36. Lefevre SH, Chauveinc L, Stoppa-Lyonnet D, et al. A T to C mutation in the polypyrimidine tract of the exon 9 splicing site of the RB1 gene responsible for low penetrance hereditary retinoblastoma. J Med Genet. 2002;39(5):E21.

37. Scheffer H, Van Der Vlies P, Burton M, et al. Two novel germline mutations of the retinoblastoma gene (RB1) that show incomplete penetrance, one splice site and one missense. J Med Genet. 2000;37(7):E6.

38. Cowell JK, Bia B. A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor phenotype. Oncogene. 1998;16(24):3211-3213.

39. Dehainault C, Garancher A, Castera L, et al. The survival gene MED4 explains low penetrance retinoblastoma in patients with large RB1 deletion. Hum Mol Genet. 2014;23(19):5243-5250.

40. Bunin GR, Emanuel BS, Meadows AT, Buckley JD, Woods WG, Hammond GD. Frequency of 13q abnormalities among 203 patients with retinoblastoma. J Natl Cancer Inst. 1989;81(5):370-374.

41. Mitter D, Ullmann R, Muradyan A, et al. Genotype-phenotype correlations in patients with retinoblastoma and interstitial 13q deletions. Eur J Hum Genet. 2011;19(9):947-958.

42. Matsunaga E. Retinoblastoma: host resistance and 13q- chromosomal deletion. Hum Genet. 1980;56(1):53-58.

43. Albrecht P, Ansperger-Rescher B, Schuler A, Zeschnigk M, Gallie B, Lohmann DR. Spectrum of gross deletions and insertions in the RB1 gene in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2005;26(5):437-445.

44. Lohmann DR, Brandt B, Hopping W, Passarge E, Horsthemke B. Distinct RB1 gene mutations with low penetrance in hereditary retinoblastoma. Hum Genet. 1994;94(4):349-354.

45. Klutz M, Brockmann D, Lohmann DR. A parent-of-origin effect in two families with retinoblastoma is associated with a distinct splice mutation in the RB1 gene. Am J Hum Genet. 2002;71(1):174-179.

46. Schuler A, Weber S, Neuhauser M, et al. Age at diagnosis of isolated unilateral retinoblastoma does not distinguish patients with and without a constitutional RB1 gene mutation but is influenced by a parent-of-origin effect. European Journal Of Cancer. 2005;41(5):735-740.

47. Eloy P, Dehainault C, Sefta M, et al. A Parent-of-Origin Effect Impacts the Phenotype in Low Penetrance Retinoblastoma Families Segregating the c.1981C>T/p.Arg661Trp Mutation of RB1. PLoS Genet. 2016;12(2):e1005888.

48. Canturk S, Qaddoumi I, Khetan V, et al. Survival of retinoblastoma in less-developed countries impact of socioeconomic and health-related indicators. Br J Ophthalmol. 2010;94(11):1432-1436.

49. Popovic MB, Diezi M, Kuchler H, et al. Trilateral retinoblastoma with suprasellar tumor and associated pineal cyst. J Pediatr Hematol Oncol. 2007;29(1):53-56.

50. Antoneli CB, Ribeiro Kde C, Sakamoto LH, Chojniak MM, Novaes PE, Arias VE. Trilateral retinoblastoma. Pediatr Blood Cancer. 2007;48(3):306-310.

24

51. Baud O, Cormier-Daire V, Lyonnet S, Desjardins L, Turleau C, Doz F. Dysmorphic phenotype and neurological impairment in 22 retinoblastoma patients with constitutional cytogenetic 13q deletion. Clin Genet. 1999;55(6):478-482.

52. Bojinova RI, Schorderet DF, Addor MC, et al. Further delineation of the facial 13q14 deletion syndrome in 13 retinoblastoma patients. Ophthalmic Genet. 2001;22(1):11-18.

53. Skrypnyk C, Bartsch O. Retinoblastoma, pinealoma, and mild overgrowth in a boy with a deletion of RB1 and neighbor genes on chromosome 13q14. American journal of medical genetics. 2004;124A(4):397-401.

54. Mallipatna A, Gallie BL, Chévez-Barrios P, et al. Retinoblastoma. In: Amin MB, Edge SB, Greene FL, eds. AJCC Cancer Staging Manual. Vol 8th Edition. New York, NY: Springer; 2017:819-831.

55. Chantada GL, Qaddoumi I, Canturk S, et al. Strategies to manage retinoblastoma in developing countries. Pediatric blood & cancer. 2011;56(3):341-348.

56. Soliman SE, Dimaras H, Souka AA, Ashry MH, Gallie BL. Socioeconomic and psychological impact of treatment for unilateral intraocular retinoblastoma. Journal Francais D Ophtalmologie. 2015;38:550—558.

57. Racher H, Soliman S, Argiropoulos B, et al. Molecular analysis distinguishes metastatic disease from second cancers in patients with retinoblastoma. Cancer Genet. 2016.

58. Canadian Retinoblastoma S. National Retinoblastoma Strategy Canadian Guidelines for Care: Strategie therapeutique du retinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44 Suppl 2:S1-88.

59. Banfi G, Salvagno GL, Lippi G. The role of ethylenediamine tetraacetic acid (EDTA) as in vitro anticoagulant for diagnostic purposes. Clinical chemistry and laboratory medicine : CCLM / FESCC. 2007;45(5):565-576.

60. Singh J, Mishra A, Pandian AJ, et al. Next-generation sequencing-based method shows increased mutation detection sensitivity in an Indian retinoblastoma cohort. Mol Vis. 2016;22:1036-1047.

61. Li WL, Buckley J, Sanchez-Lara PA, et al. A Rapid and Sensitive Next-Generation Sequencing Method to Detect RB1 Mutations Improves Care for Retinoblastoma Patients and Their Families. J Mol Diagn. 2016;18(4):480-493.

62. Chen Z, Moran K, Richards-Yutz J, et al. Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing. Hum Mutat. 2014;35(3):384-391.

63. Devarajan B, Prakash L, Kannan TR, et al. Targeted next generation sequencing of RB1 gene for the molecular diagnosis of Retinoblastoma. BMC Cancer. 2015;15:320.

64. Cavenee WK, Dryja TP, Phillips RA, et al. Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature. 1983;305(5937):779-784.

65. Ohtani-Fujita N, Fujita T, Aoike A, Osifchin NE, Robbins PD, Sakai T. CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene. 1993;8(4):1063-1067.

66. Zeschnigk M, Lohmann D, Horsthemke B. A PCR test for the detection of hypermethylated alleles at the retinoblastoma locus. J Med Genet. 1999;36(10):793-794.

67. Quinonez-Silva G, Davalos-Salas M, Recillas-Targa F, Ostrosky-Wegman P, Aranda DA, Benitez-Bribiesca L. "Monoallelic germline methylation and sequence variant in the promoter of the RB1 gene: a possible constitutive epimutation in hereditary retinoblastoma". Clin Epigenetics. 2016;8:1.

68. Zhang K, Nowak I, Rushlow D, Gallie BL, Lohmann DR. Patterns of missplicing caused by RB1 gene mutations in patients with retinoblastoma and association with phenotypic expression. Hum Mutat. 2008;29(4):475-484.

69. Dehainault C, Michaux D, Pages-Berhouet S, et al. A deep intronic mutation in the RB1 gene leads to intronic sequence exonisation. Eur J Hum Genet. 2007;15(4):473-477.

25

70. Caselli R, Speciale C, Pescucci C, et al. Retinoblastoma and mental retardation microdeletion syndrome: clinical characterization and molecular dissection using array CGH. J Hum Genet. 2007;52(6):535-542.

71. Soliman SE, ElManhaly M, Dimaras H. Knowledge of genetics in familial retinoblastoma. Ophthalmic Genet. 2016:1-7.

72. Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 2015;45(1):16-26.

73. Soliman SE, Dimaras H, Khetan V, et al. Prenatal versus Postnatal Screening for Familial Retinoblastoma. Ophthalmology. 2016;123(12):2610-2617.

74. Dhanjal S, Kakourou G, Mamas T, et al. Preimplantation genetic diagnosis for retinoblastoma predisposition. Br J Ophthalmol. 2007;91(8):1090-1091.

75. Dommering CJ, Moll AC, Imhof SM, de Die-Smulders CE, Coonen E. Another liveborn after preimplantation genetic diagnosis for retinoblastoma. Am J Ophthalmol. 2004;138(6):1088-1089.

76. Xu K, Rosenwaks Z, Beaverson K, Cholst I, Veeck L, Abramson DH. Preimplantation genetic diagnosis for retinoblastoma: the first reported liveborn. Am J Ophthalmol. 2004;137(1):18-23.

77. Girardet A, Hamamah S, Anahory T, et al. First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers. Mol Hum Reprod. 2003;9(2):111-116.

78. Uhlmann WR. Response to Robert G. Resta commentary (Unprepared, understaffed, and unplanned: thoughts on the practical implications of discovering new breast and ovarian cancer causing genes). J Genet Couns. 2009;18(6):524-526.

79. Shugar A. Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling Adaptation Continuum Model to Address Psychosocial Complexity. J Genet Couns. 2016.

80. Shugar AL, Quercia N, Trevors C, Rabideau MM, Ahmed S. Risk for Patient Harm in Canadian Genetic Counseling Practice: It's Time to Consider Regulation. J Genet Couns. 2016.

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