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.

2 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.

2 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.

No authors have any financial/conflicting interests to disclose.

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

profit sectors.

2

Unstructured abstract

Abstract: (120/250)

Retinoblastoma is an intraocular genetic malignancytumor might affect one or both eyes and

initiated by biallelic mutation of the retinoblastoma gene (RB1) in a single precursor retinal cell. that

affects the eye(s) of a child; but the physician deals with the whole family regarding risks and

possibilities. Good Uunnderstanding Retinoblastoma genetics is crucial in providing not only

optimalstandard of care for retinoblastoma children but also risk foreseeing and genetic counseling

for and their families. In this scenario the genetics trait description was conducted by the based

conversation between a family with a retinoblastoma child and their treating attendingphysician

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

askedcommon questions that all ocular oncology physiains face on regular basisby the parents. The

main aim of Tthis scenario aimsis to try to simplify the information around genetics for

ophthalmologists to help them improve their patient and family care. mmmmmmm

Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, prenatal screening

3

INTRODUCTION [JEFFRY]

Retinoblastoma is the most common childhood intraocular malignancy in childhood that might affects

one or both eyes.1 It is considered the prototype of genetic cancers.2It Tumors are is initiated by biallelic

mutation of the retinoblastoma tumor suppressor gene (RB1) in a single precursor retinal cell. The first

RB1 mutation is present in constitutional RB1 mutationcells in nearly 50% of patients, who are thereby

predisposeds individuals to developing retinoblastoma that forms after the second RB1 allele is damaged

in a somatic mutationcell.{Corson, 2007 #12275;Dimaras, 2012 #8709}. The incidence of retinoblastoma

is constant at one case in 165,000-1820,000 live births, translating to about 89,000 new cases per year

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

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

Multiple reviews had described the genetics research advancement of retinoblastoma from different

aspects in depth respectively. In this review we will try to highlightaddress most of the updates on the

genetic aspect of retinoblastoma in a clinical scenario setting that might simplify theseis new

advancementsaspects 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?

4

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 conephotoreceptor precursor cell or an inner

nuclear layer cell origin. The visualization of early tumors by optical coherence tomography (OCT)

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. Without timely and suitable treatment, the aggressive tumor may spread

through optic nerve or hematogenous route into brain or bone marrow, which will result in death of the

patient in the end.

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

Retinoblastoma arises from developing cells that are present in the retinase 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. The mean age at presentation is around 1 year in bilateral disease

and 2 years in unilateral disease. For your daughter, despite we can see tumor in only one eye by clinical

examination, we cannot be sure about the other eye without an examination under anesthetic (EUA) and

proper eye examination with fundus imaging and OCT.

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

5

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 casesThe concept of , retinoblastoma developments whenafter inactivation of both RB1 gene

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

(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.11-13 The

clinical presentation of heritable retinoblastoma consists of 80% bilateral and 15-18% unilateral.1 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.14 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?

There are many causes in the environment that can cause thisDNA 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.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

6

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?

I just suspect that this professional question can be asked by the parent?????Why not change it as ‘Is

there any new findings about the tumorigenesis?’

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,

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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

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.

8

Q8: Doeos 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

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,mutation; the researchers

discovered that differential methylation of the intron 2 CpG85 skews RB1 expression in favor of the

9

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

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

10

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

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.

Q11: Is retinoblastoma lethal?

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, socioeconomic56 and cultural factors are

major causes of high mortality. .Asia and Africa have the highest mortality, with >70% of affected

11

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 carryretinoblastoma 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.57

Q12: How can we test for retinoblastoma mutations?

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

12

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

13

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

14

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.

68,69 Alternatively, 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.

15

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. 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

16

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

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

Q165: Can we use the known mutation in other benefits?What is the benefit of prenatal mutation

detection versus post natal screening?

ThisRB1 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%

17

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 prenatatlprenatal screening. Prenatal screening with early delivery

showed less tumor and treatment burden with higher treatment success, eye preservation and visual

outcome.73

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

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

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

18

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

Q168: 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.

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

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. Q17: what

are the risks for the relatives in the family?

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

19

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

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 allow the

20

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?

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

There have the highest mortality, with >70about 40-70% of affected children with dying of

retinoblastoma in Asia and Africa, compared with <53-5% in developed countries.48,55 Delayed diagnosis

and treatment due to lack of knowledge pertaining to retinoblastoma of parents56 and ophthalmologists is

one of the major causes leading to the low eye salvage rateof and high mortality in developing countries.

So theBroad good understanding of retinoblastoma genetics and the importance of genetic counseling is a

suitablethe optimal waycan contribute to reducing mortality from retinoblastoma. to address above issue

in certain extent. In this review, we highlight the RB1 mutation categoriestypes, advanced molecular

diagnosis of retinoblastoma and genetic counseling.

Clinical presentation [Sameh]

Natural History

71 Start with retinoma and molecular features…..

452246Retinoblastoma starts as a rounded white retinal mass that gradually increases in size. At first,

equal Centrifugal tumor growth of the tumor preserving the rounded or oval shaperesults in small tumors

being round; occurs followed bymore extensive growth a period of differential growth period leading

toproducesing the lobular or nipplegrowth growth patternstumo, likely related to genomic changes in

21

single (clonal) cells, that provide a proliferative advantager appearance.47,48 Tumor seeds float free of the

main tumor intoing occurs to the subretinal space or the vitreous cavity due to theas a result of poor

cohesive forces between tumor cells,49, this can be into the subretinal space or the vitreous cavity. In

Advanced vitreous tumors, the tumor seeds might can migrate to the anterior chamber producing a

pseudo-hypopyon like appearance., the Enlarging tumor might can push the iris lens diaphragm forward

causing angle closure glaucoma. or rarely the Rapid necrosis within of the tumor can cause an aseptic

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

occlusions.47,48,50 If Untreated, retinoblastoma can spreads along into the optic nerve and along the visual

pathway to the brain, or hematogenous spread occurs . Retinoblastoma can spread into thethrough

choroidal blood vessels and, particularly to grow in bone marrow hematogenous spread occurs. Direct

tumor growth through the sclera can cause present as orbital extension and proptosis. {Gallie, In Press

#15554}is a precursor with characteristic clinical features: translucent white mass,{Gallie, 1982 #5686}

Pathology of retinoma reveals fleurettes structures that are not proliferative. Genetic analysis of retinoma

and adjacent normal retina and retinoblastoma shows loss of both RB1 alleles, and early genomic copy

number changes that are amplified further in the adjacent retinoblastoma. {Gallie, 1982 #5686}

{Theodossiadis, 2005 #5649}

Clinical Features

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

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

common.50 In developing countries, buphthalmos and proptosis due to advanced and extraocular disease

respectively represents a higher percentage.43 Less common presentations include; heterochromia irides,

neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.50 Retinoblastoma

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

regions (Trilateral retinoblastoma)51,52.{Popovic, 2007 #11607;Antoneli, 2007 #14202;de Jong, 2015

#14413} It might present in a syndromic form (13q deletion syndrome) associated with some facial

22

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

retardation53-55 (Baud et al 1999 PMID: ; Bojinova et al 2001 PMID: ; Skrypnyk and Bartsch 2004 PMID:)

The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and

ocular toxicariasis.50

Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a

brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma

(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median age

of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014

PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,

from very few to nearly half of all patients and is dependent on early detection and small tumor size (de

Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy

and autologous stem-cell rescue.

Grouping/Retinoblastoma Cancer Staging

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

involvedpredictive of outcomes include in grouping are size, and site of thelocation of tumor origin,

amountextent of subretinal fluid, size and sitepresence of tumor seeds and the presence of high risk

features on pathology.56 Multiple grouping staging systems have predicted likelihood to salvage an eye

without using radiation therapy; for the intraocular retinoblastoma existed with thethe International

Intraocular Retinoblastoma Classification (IIRC)47 being has been the recently the most reliable, but

published evidence is in the last decadebecause significantly different versions have emerged.1 Recently,

it has been replaced by tThe 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.56 The main factors involved in grouping are size and site of the tumor,

23

amount of subretinal fluid, size and site of tumor seeds and the presence of high risk features. (Table X)

Retinoblastoma is the first cancer to be stagedin which staging recognizes the impact of by genetics in

addition to the clinical features due to the high impact of genetic status on managementon outcomes: . If

there ispresence of a positive family history, bilateral or trilateral disease or documentedhigh sensitivity

positive RB1mutation testing, the disease is staged as is H1; without these features or testing of blood,

HX; and H0 for those relatives who are shown to not carry the. Otherwise it is considered as H0. A true

H0 is with documented negative specific RB1 mutation status.56 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%.

-Pedigree defining H0 (*define a true H0 vs most likely H0), H1, HX

Treatments

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

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

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

treatment.1 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

Metastasis and Second Cancers

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

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

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

appear as blue round cell tumors. Genetic analysis might help to differentiate57…. (Hilary to write details

and choose appropriate site) –Cite Racher paper

24

Add differential diagnosis? NO, ELSEWHERE IN JOURNAL ISSUE; BUT ONE SENTENCE

ONLY….MERGE THE ABOVE HEADINGS INTO TWO PARAS…AT MOST.

Add retinoblastoma/retinoma? ONLY THE GENETICS OF IT

Inheritance pattern [Hilary]

Knudson two-hit hypothesis: In most cases, retinoblastoma develops when both copies of the RB1

gene are inactivated. This concept was first formulated in 1971, when Knudson used retinoblastoma as

the prototypic cancer to derive the two-hit hypothesis (Knudson, 1971).31 In heritable retinoblastoma, the

first mutational event is inherited via the germinal cells, while the second event occurs in the somatic

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

retinoblastoma encompasses 45% of all reported cases (MacCarthy et al 2009; Moreno et al 2014; Wong

et al {risk of subse malig neoplasms in long term hereditary rb surviv…}2014).32-34 The clinical

presentation of heritable retinoblastoma consists of 80% bilateral and 15-18% unilateral (cite).1 In

nonheritable 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 35

Heritable Retinoblastoma and Penetrance

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

pathogenic change. Whether the individual for whom inherited the RB1 mutation develops

retinoblastoma depends on the RB1 DNA alteration. Typically, nonsense and frameshift germline

mutations, which lead to absence of RB1 expression or truncated dysfunctional RB1 protein, show nearly

complete (90%) penetrance. Often the second mutational event in the retinal cell is loss of the second

RB1 allele (LOH, loss of heterozygosity). In these families the presentation is typically unilateral,

25

multifocal or bilateral retinoblastoma. In a smaller subset of hereditary retinoblastoma, reduced

expressivity and reduced penetrance is observed (citations). 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

RB1 mutations reported that result in reduced expressivity/penetrance are diverse. Many consist of

mutations which reduced the expression of the RB1 protein. Examples include, (1) mutations in exons 1

and 2 25,36 (2) mutations in exons 26 and 2726,37{Mitter, 2009 #18935;Mitter, 2009 #7347} (3) intronic

mutations38,39 (Schubert et al 1997 PMID: 9341870; Lefevre et al 2002 PMID: 12011162 ; ) and (4)

missense mutations (cite).40,41 In addition, large deletions that encompass the RB1 gene and the MED1

gene cause reduced expressivity/penetrance (Dehainault et al 2014 PMID: 24858910; Bunin et al 1989

PMID: 2915374 ; ).42,43 Dehainault et al showed that RB1 -/- cells cannot survive in the absence of MED4.

Patients with 13q14 deletion syndrome more often have unilateral tumors only, in comparison to patients

with gross deletions with one breakpoint in the RB1 gene whom typically present with bilateral 44-46Rb

(Mitter et al 2011 PMID: ; Matsunaga et al 1980 PMID: ; Baud et al 1999; Albrecht et al 2002 PMID: ).

One way in which the severity of risk can be evaluated is through the disease-eye-ratio (DER) (Lohmann

et al 1994). 47 The DER is calculated by taking the number of eyes affected divided by the total number of

eyes of carriers within the family.

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 et

al 2002 PMID: 12016586; Schuler et al 2004 PMID: 15763650; Eloy et al 2016 PMID: 26925970).48-50 A

recent study by Eloy et al50 helped shed light on 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 favour of the maternal allele. In other words, when the p.Arg661Trp allele is maternally

26

inherited there is sufficient tumor suppressor activity to prevent pRB RB development; 90.3% of carriers

of maternally inherited p.Arg661Trp remain unaffected. However, when the mutation is paternally

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

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

(Klutz et al 2002 PMID: 12016586).48

Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a

brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma

(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median age

of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014

PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,

from very few to nearly half of all patients and is dependent on early detection and small tumor size (de

Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy

and autologous stem-cell rescue.

13q deletion syndrome

In patients with large interstitial 13q14 deletions that include the RB1 gene, variable clinical features

are present in addition to retinoblastoma, termed 13q14 deletion syndrome. Common facial features

includes high and broad forehead, thick and everted ear lobes, short nose, prominent philtrum and thick

everted lower lip, bulbous tip of the nose and mental retardation (Baud et al 1999 PMID: ; Bojinova et al

2001 PMID: ; Skrypnyk and Bartsch 2004 PMID: ). Patients with 13q14 deletion syndrome more often

have unilateral tumors only, in comparison to patients with gross deletions with one breakpoint in the RB1

gene whom typically present with bilateral Rb (Mitter et al 2011 PMID: ; Matsunaga et al 1980 PMID: ;

Baud et al 1999; Albrecht et al 2002 PMID: ).

?mechanism ?non-allelic homologous recombination.

27

Mosaicism

{FIGURE ON MOSAICISM}

RB1 gene [Hilary]

Function: The RB1 gene, located on 13q14, encodes the pRB RB protein (pRB), which is an

important cell cycle regulator and the first tumor suppressor gene ever discovered (Friend et al 1986

PMID: ).41 After a cell completes mitosis, the pRB RB protein is dephosphorylated, permitting it to bind

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

the progression of the cell cycle from G1 to S phase (Nevins et al 2001 PMID: ; Cobrinik 2005 PMID: ;

Sage et al 2012 PMID: ).42-44 In order for the cell to enter S phase, cyclin-dependent kinases

phosphorylate RB, which removes the ability of pRB RB to bind to the E2F gene promoter (Knudsen and

Knudsen 2008 PMID: ).45 pRB RB functions to regulate proliferation in most cell types (Cobrinik 2005

PMID:).43 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 et al 2014 – Nature – Rb suppresses human cone-precur

PMID).46

-?A and B pockets

-Also describe the role in genomic instability (Demaris. Rushlow)

RB1 Mutations

Different ways in which RB1 can be disrupted: There are many ways in which the function of the

pRB RB protein is impaired including point mutations, small and large deletions, promotor methylation

and chromothripsis (Lohmann 1999 PMID: ; McEvoy et al 2014 PMID: ).47,48 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 involved CpGOne

28

subset of recurrent mutations involve 11 CpG sites, which make up ~22% of all RB1 mutations (Rushlow

et al 2009 PMID: 19280657).49 The high recurrence of nonsense mutations at these sites is due to the

hypermutabilty and subsequent deamination of 5-methylcytosine (Richter et al 2003).50

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

conception mutagenesis occur during spermatogenesis (Munier et al 1998 PMID: 9837842; Dryja et al 1997

PMID: 9272170)51,52.51,52 Furthermore, advanced paternal age has been shown to increase risk for

retinoblastoma.53 This is thought to be due to the large number of cell divisions during spermatogenesis

and 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

Coding sequencing mutations

Promoter methylation

Hot-spot mutations – CpG transition

Non-coding/regulatory changes

?in genetic counselling?? Origin of new mutations

Xu et al. new mutations are on fathers chromosome

Older fathers, but not older mothers for RB50

Greta Bunin

MYCN

29

PROGRESSIVE OTHER GENOMIC CHANGES IN ADDITION TO RB1

Other genomic changes in addition to alterations in RB1 [Hilary]

DEK, KIF14, E2F3, CDH11

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

amplification (28-121 copies) of the MYCN oncogene is detected (Rushlow et al 2013 PMID: 23498719).35

Patients with RB1+/+ MYCNA 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 and Gallie 2007 PMID: 17437278).54 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 et al 2014 PMID: 24433356).55

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

expression of some microRNAs56 (Huang et al 2007 PMID: 18026111) and recurrent single nucleotide

variants/insertion-deletions in the genes BCOR and CREBBP (Kooi et al 2016 PMID: 27126562).57 In

comparison to the genomic landscape of other cancers, retinoblastoma is one of the least mutated57 (Kooi

et al 2016 PMID: 27126562)

Molecular diagnosis [Hilary]

Strategic testing - Tumor testing first for unilateral/PBL for bilateral

Technologies and techniques

NGS [flow chart of molecular techniques]

Cytogenetic strategies (FISH/microarray)

30

RNA for discovery and VUS functional studies

Protein studies

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

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 (cite).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 (Banfi et al 2007

PMID:17484616).59

31

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 et al 2016 PMID: 27582626; Li et al 2016 PMID: 27155049;

Chen et al 2014 PMID: 24282159).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 (Chen et al 2014 PMID: 24282159)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

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

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

copy number variants59 (Devarajan et al 2015; Li et al 2016 PMID: 27155049).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.

32

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.

As described in the DNA sequencing section, NGS is one tool that can be used detect low level

mosaicism. In addition, allele-specific PCR (AS-PCR) is an another strategy that can be used in

situations where the RB1 mutation is known (Rushlow et al 2009 PMID: 19280657).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 (Canvenee et al 1983 PMID: 6633649).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 (Ohtani-Fujita et al 1993 PMID: 8455933). 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 (Richter et al 2003).18,66(Zeshnigk et al 1999 PMID:

10528863) 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 et al 2016 PMID: 26753011). 67

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

33

patients with retinoblastoma (Zhang et al PMID: 18181215; Dehainault et al., 2007 PMID:17299438). 68,69

Inorder 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 et al PMID: 18181215;

Dehainault et al., 2007 PMID: 17299438). Alternatively, 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 et al 2007 PMID:

17502991; Mitter et al 2011 PMID: 21505449). 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 (Baud et al 1999 PMID: 10450867).51

Genetic Counseling (Heather/Hilary)

Importance of high detection rate

Targeted familial testing/prenatal testing, preconception testing

Targeted familial testing1,58: 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

34

children of mosaic affecteds 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 a number of 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 11-14wks 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

which occurs more frequently with CVS.71

Results of genetic testing 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.72

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 30-34wks

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

delivery rather than miscarriage.71 The risk for procedure-associated significant preterm delivery is low

35

(<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.58,72

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

preimplantation genetic diagnosis (PGD).73-76 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 results73-76

72Surveillance for mets and second cancer

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

offspring, cousins, faroff relatives, stats below population risk]

Genetic counselling 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.77-79 (Uhlmann et al. (2009), Shugar (2016)). Genetic testing is an integral

36

component of genetic counselling that results in more informed and precise genetic counselling. 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

counselling and also provides further time for psychosocial support to be provided to the family.

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

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

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

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

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

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

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

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

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

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

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

for individuals with low penetrance mutations.

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

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

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

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

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

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

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

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

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

37

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.

63

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

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

Compare/contrast Canada vs China vs Jordon

Societal/cultural challenges to GC

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

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

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

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

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

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

carrying the germline RB1 mutation.

8080References

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

Blackwell.

38

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

Benefits of genetic testing for the proband and family members [Heather]

Prenatal vs Postnatal [Sameh]

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

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.

39

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

40

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.

41

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.

42

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.

43

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.

44

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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