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,1-2 Hilary Racher, PhD,3 Chengyue Zhang, MD,4 Hilary Racher, PhDHeather

MacDonald,1 Brenda L. Gallie.1,5

Affiliations:

1Department of Ophthalmology and Vision Sciences, University of Toronto, Ontario, Canada

2Department of Ophthalmology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt.

3Impact Genetics, Bowmanville, Ontario.

4Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University, Beijing, China.

5Departments of Ophthalmology, Molecular Genetics, and Medical Biophysics, University of Toronto,

Toronto, Canada.

Corresponding author:

Brenda L. Gallie: Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8.

Telephone: +1-294-9729

email: brenda@gallie.ca

2

Disclosures:

Both SS and HR contributed equally to this review as first co-first authors.

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

co-authors 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.

Word Count: (/5000)

Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic counselling,

prenatal screening.

3

Unstructured abstract

Abstract: (120/250)

Retinoblastoma is an intraocular malignancy that affects one or both eyes of young children, that is

initiated by biallelic mutation of the retinoblastoma gene (RB1) in a developing retinal cell. A good

understanding of retinoblastoma genetics supports optimal care for retinoblastoma children and their

families. In this scenario the genetics trait description was conducted by the conversation between a

family with a retinoblastoma child and their attending who is mostly the ophthalmologist but can be any

member of the retinoblastoma multidisciplinary team of physicians, nurses and genetic counselors. All the

questions are true and high frequently asked by the parents. This scenario aims to try to simplify the

information around genetics for ophthalmologists to help them improve their patient and family care.

bilateral, unilateral, DNA sequencing, genetic counseling prenatal screening

4

5321/5000 words

INTRODUCTION

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

Because of the strong links between clinical care and genetic causation,2 retinoblastoma is considered the

prototype of heritable cancers.38,000 children are newly diagnosed with retinoblastoma every year

(1/16,000 live births).1,4 Genetics underlies many aspects of retinoblastoma: clinical presentation, choice

of treatment modalities and follow-up for both child and family. We now highlight the genetic etiology of

retinoblastoma in the context of individual children and families.

CASE SCENARIO

A 2-year-old girl presented with left leukocorea (white pupil), noticed by her family in a photograph 5

days earlier. They sought medical advise from their family physician, who suspected retinoblastoma and

referred them urgently to the pediatric ophthalmologist. The family had never before heard of

retinoblastoma, and the mother was 33 weeks pregnant. The child was very uncooperative but the

ophthalmologist was able to visualize a white retinal mass in the left eye. He could see the inferior retina,

intact optic nerve and fovea in the right eye and diagnosed retinoblastoma in the left eye. The following

discussion took place between the pediatric ophthalmologist and the family.

Q1: Father: What is retinoblastoma?

A: (Pediatratric Ophthalmologist) “Retinoblastoma is a cancer that arises from a developing retinal cell

in babies and young children. Retinoblastoma can affect one (unilateral) or both eyes (bilateral) and in 5%

of children is associated with a midline brain tumor (trilateral).5 Without timely and effective treatment,

retinoblastoma may spread through optic nerve to the brain, or via blood particularly to bone marrow,

which will result in death. To be sure of the diagnosis and the best treatment for this rare disease, I will

5

refer your daughter to the Retinoblastoma Centre, where experts treat many of these children. I will phone

now!”

Q2: Father: why this is presenting at such a young age?

A: (Retinoblastoma Ophthalmic Specialist) “The cell of origin of retinoblastoma is most likely a

developing cone photoreceptor precursor cell that has lost both copies of the RB1 tumor suppressor gene,

and remains in the inner nuclear layer of the retina, unable to migrate to the outer retina and function

normally.1,6,7 The susceptible cell that becomes cancer is only present in the retinas of young children,

from before birth, up to around 7 years of age. Rarely, retinoblastoma is first diagnosed in older persons,

but likely there was previously an undetected small tumor (retinoma) present from childhood, that later

became active.8,9 The mean age at presentation is 1 year in bilateral disease and 2 years in unilateral

disease.

Despite the fact that we can see tumor in only one eye by clinical examination of your daughter, we

cannot be sure the other eye is normal until we examine it under anesthetic (EUA).”

Q3: Mother: What caused retinoblastoma? How can a gene cause cancer in a

baby?

A: (Retinoblastoma Ophthalmic Specialist) “No one knows what really causes the damage to the RB1

gene. Maybe a random cosmic ray passes through Planet Earth and hits that large, important gene.

In nearly 50% of patients the first RB1 gene is damaged in most, or all, normal cells, resulting in

predisposition to retinoblastoma. A retinal tumor develops when the second RB1 gene is also damaged in

a developing retinal cell.1 The RB1 gene on chromosome 13q14 encodes the RB protein (pRB), an

important regulator of the cell division cycle in most cell types, and the first tumor suppressor gene

discovered.10 Normally, dephosphorylated pRB represses expression of the E2F gene, thereby blocking

cell division.11-13 To resume cell division, cyclin-dependent kinases re-phosphorylate pRB, releasing

expression of E2F.14 In many cell types, loss of the RB1 gene is compensated by increased expression of

6

other related proteins. However, in susceptible cells such as retinal cone cell precursors, compensatory

mechanisms are not sufficient, cell division is uncontrolled, and cancer is initiated.7

Q4: What causes retinoblastoma to be unilateral versus bilateral?

A: (Retinoblastoma Ophthalmic Specialist) “In heritable retinoblastoma (sometimes called germline

retinoblastoma), the first RB1 allele (M1) is mutant in all cells, including germline reproductive cells,

while the second allele (M2) is mutated in the retina initiating cancer. Often M2 event in the retinal cell is

loss of the normal RB1 allele and duplication of the mutant M1 allele (LOH, loss of heterozygosity).

Heritable retinoblastoma encompasses 45% of all reported cases.15-17 with either bilateral (80%), unilateral

(15%) or trilateral (5%) tumors.1 Germline retinoblastoma carries risk of second primary cancers higher

than normal, most commonly osteosarcoma, fibrosarcoma and melanoma. These persons can benefit from

regular surveillance for such cancers for their lifetime.

Of non-heritable retinoblastoma, 98% have RB1 M1 and M2 arise in a retinal cell. The remaining 2%

the retinoblastoma is induced by somatic amplification of the MYCN oncogene, in the presence of normal

RB1 genes.18” Germline retinoblastoma carries the risk of development of second primary cancers, most

commonly osteosarcoma and fibrosarcoma due to loss of RB1 gene. This is why these children should be

kept under surveillance for the rest of their lives.

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

A: (Retinoblastoma Expert) “No one is to blame for the mutations causing retinoblastoma. Many

environmental forces induce DNA damage, including cosmic rays, X-rays, DNA viruses, UV irradiation

and smoking. The DNA damage may be point mutations, small and large deletions, promotor methylation

shutting down RB1 expression and rarely, chromothripsis.19,20 The majority of RB1 mutations arise de

novo, unique to a specific patient or family. However, some recurrent mutations are found in unrelated

individuals, such as those that affect 11 sites CpG DNA sequence sites, which are hyper-mutable and

make up 22% of all RB1 mutations.21,22

7

When there is no family history of retinoblastoma, a de novo RB1 germline mutation may arise either

pre- or post-conception. Pre-conception mutagenesis of RB1 usually occurs during spermatogenesis,

perhaps because cell division (and opportunity for mutation) is very active during spermatogenesis, but

not during oogenesis.23-25 Advanced paternal age increases risk for retinoblastoma,26 suggesting that base

substitution errors may increase in aging men. The affected child carries the de novo RB1 mutation in

every cell, typically presenting with 4-5 tumors and bilateral retinoblastoma. In contrast, if mutagenesis

occurs post-conception, during embryogenesis, only a portion (1-50%) of cells carrying the RB1 mutation

and the person will be mosaic for the RB1 mutation. If the mutation arises during retinal development,

the child will have unilateral retinoblastoma.1

Q6: Father: So, only RB1 mutation causes retinoblastoma?

A: (Retinoblastoma Expert) “There are two answers to this question: RB1 mutation only causes a

benign precursor to retinoblastoma, retinoma, and other genes are modified to cause progression to

cancer;9 and 2% of retinoblastoma have normal RB1 and are caused by a different gene.

In addition to loss of RB1, specific alterations in copy number of other genes are common in RB1-/-

retinoblastoma. There are gains (4-10 copies) in oncogenes MDM4, KIF14 (1q32), MYCN (2p24), DEK

and E2F3 (6p22), and loss of the tumor suppressor gene CDH11 (16q22-24).3,27 Other less common

genomic alterations in retinoblastoma tumors include differential expression of specific microRNAs28 and

recurrent single nucleotide variants/insertion-deletions in the genes BCOR and CREBBP.29 In comparison

to the genomic landscape of other cancers, retinoblastoma is one of the least mutated.29”

There is a newly recognized form of retinoblastoma with normal RB1 genes. Two percent of unilateral

patients have RB1+/+MYCNA tumors, with the MYCN oncogene is amplified (28-121 instead of the normal

2 DNA copies).18 These children are diagnosed at median age 4.5 months compared to 24 months for non-

heritable unilateral RB-/- patients, and the tumors are distinct histologically, with advanced features at

diagnosis.

8

Retinoma is a premalignant precursor to retinoblastoma with characteristic clinical features: translucent

white mass, reactive retinal pigment epithelial proliferation and calcific foci.8 Pathology of retinoma

reveals fleurettes30 that are not proliferative.9RB1 alleles and early genomic copy number changes, that are

amplified further in the adjacent retinoblastoma.9 Many retinoblastoma have underlying elements of

retinoma. Retinoma can transform to retinoblastoma even after many years of stability.31

Q6: Father: Could we have discovered retinoblastoma earlier?

The only way to find retinoblastoma tumor early is to look with specific expertise, which we can not

for every child. If we know to look because a relative had retinoblastoma, the smallest visible tumors are

round, white retinal lesions that obscure the underlying choroidal pattern. Centrifugal 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.32,33 Next, tumor seeds spread out of

the main tumor a result of poor cohesive forces between tumor cells into the subretinal space, or the

vitreous cavity as appearing as dust, spheres or tumor clouds.34 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. Advanced tumors may induce iris

neovascularization. Rapid necrosis of tumor can cause an aseptic orbital inflammatory reaction

resembling orbital cellulitis, sometimes showing central retinal artery occlusion.32,33,35 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.

The earliest signs of retinoblastoma detectable by parents are leukocorea (white pupil), either

directly or in photographs (photo-leukocorea) and strabismus when the macula is involvement by tumor.35

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

is common.36 Less common presentations include; heterochromia irides, neovascular glaucoma, vitreous

hemorrhage, hypopyon or aseptic orbital cellulitis.35 Retinoblastoma (unilateral or bilateral) might be

9

associated with a brain tumor in the pineal, suprasellar or parasellar regions (Trilateral retinoblastoma)37,38

with the median age of diagnsosis 17 months after retinoblastoma and before the age of 5 years.

Retinoblastoma might present as 13q deletion syndrome, with facial features and various degrees of

hypotony and mental retardation.39-41 The main differential diagnosis includes Coats’ disease, persistent

hyperplastic primary vitreous and ocular toxicariasis.35

Q7: Do all affected individuals with RB1 mutations develop retinoblastoma?

Each offspring of a person carrying an RB1 mutant gene has 50% risk to inherit the RB1 mutant gene

[Figure # Pedigree – full penetrance]. Nonsense and frame-shift germline mutations, which lead to absent

or truncated dysfunctional pRB, result in 90% bilateral retinoblastoma (nearly complete penetrance).

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

heterozygosity). For partially functional RB1 mutant alleles, reduced penetrance and expressivity is

observed, with later onset and fewer tumors42, and some carriers never develop retinoblastoma. Some

reduced penetrance mutations reduce RB1 protein expression: (i) mutations in exons 1 and 2,43 (ii)

mutations in exons 26 and 27,44 (iii) intronic mutations45,46 and (iv) missense mutations.47,48 Strangely,

large deletions encompassing RB1 gene and MED1 gene also cause reduced expressivity/penetrance,

because RB1-/- cells cannot survive in the absence of MED4.49,50 In comparison, patients with large

deletions with one breakpoint in the RB1 gene typically present with bilateral disease.51-53 A measure of

expressivity of a mutant retinoblastoma allele is the disease-eye-ratio (DER) (number of eyes affected

with tumor divided by the total number of eyes in carriers of the mutation).54

There are two specific RB1 mutations showing a parent-of-origin effect: intron 6 c.607+1G>T

substitution55,56 [Figure # Pedigree – reduced penetrance family] and c.1981C>T (p.Arg661Trp).57 Both

may be explained by at differential methylation of intron 2 CpG85, which skews RB1 expression in favor

of the maternal allele. When the allele is maternally inherited there is sufficient tumor suppressor activity

to prevent retinoblastoma development and 90% of carriers remain unaffected. However, when the

10

p.Arg661Trp allele is paternally transmitted, very little RB1 is expressed, leading retinoblastoma in 68%

of carriers.

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

A: “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.58 Multiple staging systems have predicted likelihood to

salvage an eye without using radiation therapy, but published evidence is confusing because significantly

different versions have emerged.1,58 The 2017 TNMH classification is based on international consensus

and evidence from an international survey of 1728 eyes, and separates more clearly initial clinical and

pathological features relevant to outcomes, in retrospective comparison to 5 previous eye staging

systems.58 (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 stage H1; without these features bfore testing blood, HX; and H0 for those

relatives shown to not carry the proband’s specific RB1 mutation.58 We propose H0* for patients with M1

and M2 RB1 mutant alleles of the tumor not detectable in blood, but with remaining low risk (<1%) of

mosaicism.

Choice of treatment depends on the laterality of disease, tumor stage and genetic status. Focal therapy

only can control cT1a eyes, but visually threatening or large cT1b tumors and cT2 eyes need

chemotherapy (systemic or intra-arterial chemotherapy) to reduce the size of the tumor followed by

consolidation focal therapies (laser therapy or cryotherapy) as initial treatment. Enucleation of eyes with

advanced tumors in unilateral disease where the other eye is normal is a definitive cure.1adiotherapy and

periocular chemotherapy. Intravitreal chemotherapy for vitreous disease has recently dramatically

improved safe eye salvage.59,60 For persons carrying RB1 mutations, external beam radiation therapy is

rarely indicated due to the high risk of inducing later second cancers.1

11

Life is is the main priority of retinoblastoma treatment, followed by vision salvage; the least

important is eye salvage. The child’s job is to play and develop in a healthy life; the many procedures and

their complications that may span years for at best a 50% chance to save a blind eye with risk of tumor

spread, are not justified, especially when the other eye is normal.61,62

Q11: Is retinoblastoma lethal?

A: “If untreated, retinoblastoma is lethal. If treated before metastasis occurs, cure is nearly 100%. If

metastasis occurs, the treatment becomes challenging and there is around 40% chance of mortality.

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

socioeconomic61 and cultural factors are major causes of mortality. Asia and Africa have the highest

mortality, with >70% of affected children dying of retinoblastoma, compared with <5% in developed

countries.36,63

Germline retinoblastoma carries 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 the metastasis from second cancers

that appear as blue round cell tumors. Molecular analysis might help to distingguish.64

Q12: How can we test for retinoblastoma mutations?

A: “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 embryonic 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

12

mutation is identified in the blood of a bilaterally affected patient, DNA from tumor should be

investigated.65

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

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.65

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.{Banfi, 2007 #19549}

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 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

13

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.66

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

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.{Devarajan, 2015 #15675;Li, 2016 #19404} 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 to 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.21 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.67 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.

14

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

another mechanism of retinoblastoma development.68 Hypermethylation of the RB1 promoter CpG island

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

tumors.22,69 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.70

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. 71,72 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.

71,72Alternatively, as sequencing costs continue to decrease; whole genome sequencing (WGS) may

become the method of choice to uncover deep intronic changes.

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.51,73 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.39

Q13: Are these tests available worldwide?

A: “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. In addition, the health insurance cannot cover the cost for testing. Given all the obstacles, there is

limited application of genetic testing and genetic counseling nationwide, which also lead to the redundant

15

economic burden on the affected families. Recently, the Chinese government started a new policy that

allows every family the ability to have one more child. Therefore, genetic testing and genetic counseling

should be put into good use, especially for the families carrying a germline RB1 mutation.

In Egypt,74 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 insufficient 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.74

Q14: What is done after finding the RB1 mutation?

A: “Targeted familial testing1,65 is used to determine if a predisposing RB1 mutation has occurred de novo,

through investigation of parental DNA from PBL. 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 patients should also be tested for the predisposing mutation. Table Y shows the

risk of having retinoblastoma in different family relatives.

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.

Q15: Can we use the known mutation to test my upcoming child? I am 33 weeks pregnant

Prenatal genetic testing is usually performed early in the course of the pregnancy and is available in

many countries worldwide. Two early procedures are available: 1) chorionic villus sampling (CVS) and

16

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.75

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 decide to stop the pregnancy, while other couples may decide to continue with the pregnancy and

apply appropriate interventions, such as early delivery.42

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.75 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 not do 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.42,65

17

Q16: What is the benefit of prenatal mutation detection versus postnatal

screening?

A: “RB1 mutation detection can be performed either prenatal, as discussed previously, 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 to be 100% risk. Both screening

methods are effective in improving visual outcome and eye salvage compared to 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 prenatal screening. Prenatal screening with early delivery showed less

tumor and treatment burden with higher treatment success, eye preservation and visual outcome.42

Q17: Can we plan our next pregnancy to avoid having this RB1 mutation?

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

preimplantation genetic diagnosis (PGD).76-79 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 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.76-79

18

Q18: what is genetic counseling?

A: “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.80-82. 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.

Q19: Can genetic counseling suffice alone? If yes, what are the benefits of

genetic testing?

A: “In countries where genetic testing is not available or unaffordable, genetic counseling is the option. It

was found that genetic testing is more cost effective than examining all the at-risk family members.

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

RB1 mutation (H1 in the TNMH classification). Genetic testing provides (1) more accurate information

about the type of heritable retinoblastoma and allows for straightforward testing to determine if additional

family members are at risk. (2) 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. (3) 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. (4) 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

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.

19

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 laboratories 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

increased lifelong risk for second primary 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.

Q20: When is the appropriate timing for collecting samples for genetic testing?

For blood samples, they can be collected at any time but preferably when the child is under EUA where

there is no fear from the needle prick. For tumor samples, they would be collected from the enucleated

eye just after enucleation. Tumor cells will be preserved in a specific transport medium that allows the

cells to grow. We can also freeze some tumor cells (cryopreservation) for future necessity or for research

purposes.

20

Q21: If we know the mutation prenatally, is there any treatment to prevent

retinoblastoma from occurring?

A: “

Retinoblastoma genetics is challenging to understand, but once understood It largely affect the level

of care presented to retinoblastoma patients and their families. It helps alleviate the psychological burden

of the families regarding moving forward with their life choices regarding the affected child and future

siblings. It also helps the family to understand the risks of different family members giving them the

chance of the level of disclosure they wish.

21

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

22

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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