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,1,5 Brenda L. Gallie.1,6

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.

5Heather affiliation??

6Brenda affiliation.

Corresponding author:

Brenda L. Gallie. Address: 525 University Ave, room 806, Toronto, Ontario, Canada. M5G 2L3.

Telephone: +1 xxxxx, email: brenda@gallie.ca

Disclosures:

Both SS and HR contributed equally to this review and would be considered as first co-authers.

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.

2

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. 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

3

5752/5000 words

INTRODUCTION

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

{Dimaras, 2015 #10881} It is considered the prototype of heritable cancers.{Theriault, 2014 #8591} The

incidence of retinoblastoma is constant at one case in 16,000 live births, translating to about 8,000 new

cases per year worldwide.{Seregard, 2004 #10380;Dimaras, 2015 #10881} Genetics underlies many

aspects of retinoblastoma: clinical presentation, choice of treatment modalities and follow-up for both the

child and family. Recent reviews{Dimaras, 2015 #10881;Theriault, 2014 #8591} describe different

aspects in depth. We now highlight understanding of retinoblastoma from genetic clinical analysis and

research, in the context of individual children and families.

Case Scenario: A 2 year old girl presented with left leukocorea (white pupil). The family noticed the

white pupil in a family photograph 5 days earlier. They sought medical advise to 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

only see the inferior retina and an intact optic nerve and fovea in the right eye. The diagnosis of

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

family.

Q1: Father: What is retinoblastoma?

A: “Retinoblastoma is a cancer that arises from a developing retinal cell. The cell of origin is most likely

a cone photoreceptor precursor cell that 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.

{Dimaras, 2015 #10881;Rootman, 2013 #11096;Xu, 2014 #9924} Retinoblastoma can affect one

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(unilateral) or both eyes (bilateral) and in 5% of children is associated with a midline brain tumor

(trilateral).{de Jong, 2014 #10885} 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.”

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

A: “The cell of origin of retinoblastoma is a developing cell, 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.{Gallie, 1982 #10343;Dimaras, 2008 #13250} The mean age at presentation is around 1

year in bilateral disease and 2 years in unilateral disease.

For your daughter, we see tumor in only one eye by clinical examination. We must check the other

eye under anesthetic (EUA) and proper eye examination with fundus imaging and OCT, to carefully

check if there is also a tumor in the right eye.”

Q3: Mother: What caused retinoblastoma? What do you mean that it is genetically caused?

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.

Retinoblastoma tumors are initiated by 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.{Dimaras, 2015 #10881} The RB1 gene, located on chromosom13q14, encodes the RB

protein (pRB), an important cell cycle regulator and the first tumor suppressor gene discovered.{Friend,

1986 #19025} After a cell completes cell division (mitosis), pRB is dephosphorylated, permitting it to

5

bind to the promoter region of the E2F transcription factor gene, thereby repressing transcription and

inhibiting the progression of the cell cycle from G1 to S phase.{Nevins, 2001 #15292;Cobrinik, 2005

#15298;Sage, 2012 #19061} 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.{Knudsen, 2008

#15310} pRB functions to regulate proliferation in most cell types.{Cobrinik, 2005 #15298} Often, loss

of RB1 is compensated by increased expression of its related proteins, however, in certain susceptible

cells, such as the retinal cone cell precursors, compensatory mechanisms are not sufficient and

tumorigenesis is initiated.{Xu, 2014 #19253}

Q4: What causes retinoblastoma to be unilateral versus bilateral?

A: “The concept of retinoblastoma development after inactivation of both RB1 gene copies was first

formulated in 1971, when Knudson used retinoblastoma as the prototypic cancer to derive the two-hit

hypothesis.{Knudson, 1971 #11106} In heritable retinoblastoma (sometimes called germline

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.{MacCarthy, 2009 #8367;Moreno,

2014 #18928;Wong, 2014 #15170} The clinical presentation of heritable retinoblastoma consists of 80%

bilateral and 15-18% unilateral.{Dimaras, 2015 #10881} In non-heritable retinoblastoma (non-germline

retinoblastoma) the majority (98%) of cases have somatic biallelic RB1 loss in the tumor, while the

remaining 2% have no mutation in either copy of RB1 but instead have somatic amplification of the

MYCN oncogene.{Rushlow, 2013 #11249} 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?

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A: “There are many causes in the environment that can cause DNA mutations 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.{Lohmann, 1999 #19258;McEvoy,

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

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

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

#10337} The high recurrence of nonsense mutations at these sites is due to the hypermutabilty and

subsequent deamination of 5-methylcytosine.{Richter, 2003 #11998}

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

conception mutagenesis occurs during spermatogenesis.{Dryja, 1997 #15586;Munier, 1998 #10955}

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

#15506} This might be due to the larger number of cell divisions during spermatogenesis than oogenesis

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

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

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

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

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

presentation is often unilateral retinoblastoma.{Dimaras, 2015 #10881}

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

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

RB1 loss in the benign retinoma;{Dimaras, 2008 #11248}.suggesting more genetic or epigenetic changes

for malignant transformation.

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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.{Rushlow, 2013 #11249} Patients with RB1+/+

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

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

specific somatic copy number alterations commonly occur in the progression of the retinoblastoma.

Commonly seen are gains in 1q32, 2p24, 6p22 and losses at 13q and 16q22-24.{Corson, 2007 #9909}

These regions contain important oncogenes (MDM4, KIF14, MYCN, DEK and E2F3) and tumor

suppressor genes (CDH11), thought to act as drivers promoting the growth of the cancer.{Theriault, 2014

#19306}

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

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

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

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

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

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

reactive retinal pigment epithelial growth and calcific foci.{Gallie, 1982 #10343} 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.{Dimaras, 2008 #11248} It can transform to

retinoblastoma even after many years of stability.{Theodossiadis, 2005 #5578}

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

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

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

2005 #11984;Balmer, 2006 #8323} 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

8

tumor clouds.{Munier, 2014 #11111} Advanced vitreous tumor seeds can migrate to the anterior chamber

producing a pseudo-hypopyon. Enlarging tumor can push the iris lens diaphragm forward causing angle

closure glaucoma. Rapid necrosis of tumor can cause an aseptic orbital inflammatory reaction resembling

orbital cellulitis, sometimes showing central retinal artery occlusion.{Balmer, 2007 #8320;Balmer, 2006

#8323;Murphree, 2005 #11984} 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,{Sanchez-Sanchez, 2007 #18933} (2)

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

#18936;Lefevre, 2002 #18938} and (4) missense mutations.{Scheffer, 2000 #15178;Cowell, 1998

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

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

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

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

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

#15255;Matsunaga, 1980 #19020;Albrecht, 2005 #19022} The severity of risk can be evaluated through

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

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

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

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

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

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

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

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

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

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

activity to prevent 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.

{Klutz, 2002 #19004}

Q9: Mother: could we have discovered it earlier?

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

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

{Balmer, 2007 #8320} In developing countries, buphthalmos and proptosis due to advanced and

extraocular disease respectively represents a higher percentage.{Canturk, 2010 #13461} Less common

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

aseptic orbital cellulitis.{Balmer, 2007 #8320} Retinoblastoma (unilateral or bilateral) might be

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

{Popovic, 2007 #9156;Antoneli, 2007 #10877} that starts early; with the median age of onset 17 months

10

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 noseassociated with various degrees of hypotonea and mental retardation.{Baud, 1999

#18925;Bojinova, 2001 #18926;Skrypnyk, 2004 #15166} The main differential diagnosis includes Coats’

disease, persistent hyperplastic primary vitreous and ocular toxicariasis.{Balmer, 2007 #8320}

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.{Mallipatna, 2017 #14252} Multiple staging systems have

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

Retinoblastoma Classification (IIRC){Murphree, 2005 #11984} has been recently the most reliable, but

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

#10881} The 2017 TNMH classification is based on international consensus and evidence from an

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

eye staging systems.{Mallipatna, 2017 #14252} (Table X) Retinoblastoma is the first cancer in which

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

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

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

mutation.{Mallipatna, 2017 #14252} 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

11

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

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

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

#10881}

The main concept of treatment is that life salvage is the main priority during treatment planning

followed by vision salvage and the least important is eye salvage. That’s why we prefer enucleation in

advanced unilateral intraocular retinoblastoma with low visual potential. The child’s job at this point is to

play and enjoy a healthy life away of all the procedures and their complications that may span over a

couple of years for a 50% chance to save a blind eye and risk of tumor spread.{Soliman, 2015

#10948;Soliman, 2016 #18559}

Q11: Is retinoblastoma lethal?

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

chance of life salvage. If metastasis occurs, the treatment options becomes more challenging but there is a

40% chance of mortality related to retinoblastoma. Delayed diagnosis and treatment due to lack of

retinoblastoma knowledge by ophthalmologists and parents, socioeconomic{Soliman, 2015 #10948} and

cultural factors are major causes of high mortality. .Asia and Africa have the highest mortality, with

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

{Chantada, 2011 #13420;Canturk, 2010 #13461}

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 as both metastasis and second cancers

appear as blue round cell tumors. molecular analysis might help to differentiate.{Racher, 2016 #13990}

Q12: How can we test for retinoblastoma mutations?

12

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 embryonal development. In these cases, the RB1 mutation may

only be present in some cells and may not be detected in DNA from PBL. Therefore, in the event that no

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

investigated.{Canadian Retinoblastoma, 2009 #14251}

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.{Canadian Retinoblastoma, 2009 #14251}

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-

13

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

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.{Chen, 2014

#19419}

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

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

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

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

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

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

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

14

#10337} 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.{Cavenee, 1983 #9210} 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.{Ohtani-Fujita, 1993 #2258} Hypermethylation of the

RB1 promoter CpG island results in transcription inhibition of the RB1 gene and has been identified 10-

12% of retinoblastoma tumors.{Richter, 2003 #11998;Zeschnigk, 1999 #15496} 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.{Quinonez-Silva, 2016 #19594}

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. {Zhang, 2008 #7502;Dehainault, 2007 #5872} 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. {Zhang, 2008 #7502;Dehainault, 2007 #5872}Alternatively,

15

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.{Caselli, 2007 #19622;Mitter,

2011 #15255} 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.{Baud, 1999 #19768}

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. 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.

In Egypt,{Soliman, 2016 #14713} 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.{Soliman, 2016 #14713}

Q14: What after finding the RB1 mutation?

A: “Targeted familial testing{Canadian Retinoblastoma, 2009 #14251;Dimaras, 2015 #10881} is used to

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

16

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. 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 coming child? I am 33 weeks pregnant

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

countries around the world. Two early procedures are available: 1) chorionic villus sampling (CVS) and

2) amniocentesis. CVS is a test typically performed between 11-14 weeks gestation during which as

sample of the placenta is obtained either by transvaginal or transabdominal approach. Amniocentesis is a

test performed after 16 weeks of gestation whereby as sample of the amniotic fluid is gathered with a

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

has a procedure-associated risk of miscarriage between 0.1-0.5%. Though uncommon, there is a risk for

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

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

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

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

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

2016 #15159}

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

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

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

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

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

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

early delivery when a mutation has been inherited.

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

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

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

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

has not occurred by the due date.{Soliman, 2016 #15159;Canadian Retinoblastoma, 2009 #14251}

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

A: “RB1 mutation detection can be performed either prenatal as discussed earlier 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 prenatal screening. Prenatal screening with early delivery showed less tumor and treatment

burden with higher treatment success, eye preservation and visual outcome.{Soliman, 2016 #15159}

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

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

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

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

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

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

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

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

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

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

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

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

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

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.{Uhlmann, 2009 #15690;Shugar, 2016 #15715;Shugar, 2016 #15725}. 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 all have heritable retinoblastoma and a RB1 mutation

(H1 in the TNMH classification). Genetic testing provides (1) more accurate information about the type

19

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

place and data accumulate, surveillance of the proband may one day be matched more precisely to the

level of risk for new tumours for individuals with low penetrance mutations.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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.

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

occurring?

A: “

Conclusions

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.

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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

23