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Genetics and Molecular Diagnostics in

Retinoblastoma - An Update

Unstructured abstract

Abstract: mmmmmmm

Key Words: retinoblastoma, RB1 gene,

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INTRODUCTION [JEFFRY]

Retinoblastoma is the most common intraocular malignancy in childhood that might affect one or

both eyes. It is initiated by biallelic mutation of the retinoblastoma gene (RB1) in a single precursor

retinal cell. The constitutional RB1 mutation predisposes individuals to developing retinoblastoma that

forms after the somatic mutation. The incidence of retinoblastoma is constant at one case in 15,000-

20,000 livebirths, translating to about 9,000 new cases per year worldwide.

There have the highest mortality about 40-70% of children with retinoblastoma in Asia and Africa,

compared with 3-5% in developed countries. Delayed diagnosis and treatment due to lack of knowledge

pertaining to retinoblastoma of parents and ophthalmologists lead to the low eye salvage rate and high

mortality in developing countries. So the good understanding of retinoblastoma genetics and the

importance of genetic counseling is the optimal way to address above issue in certain extent. In this

review, we highlight the RB1 mutation categories, advanced molecular diagnosis of retinoblastoma and

genetic counseling.

Clinical presentation [Sameh]

Natural History

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

centrifugal growth of the tumor preserving the rounded or oval shape occurs followed by a period of

differential growth period leading toproducing the lobular or nipple growth patternstumor appearance.

Tumor seeding occurs to the subretinal space or the vitreous cavity due to theas a result of poor cohesive

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

tumors, the tumor seeds might migrate to the anterior chamber producing a hypopyon like appearance, the

enlarging tumor might push the iris lens diaphragm causing angle closure glaucoma or rarely the rapid

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necrosis within the tumor can cause an aseptic orbital inflammatory reaction resembling orbital cellulitis.

If untreated, retinoblastoma can spread along the optic nerve and along the visual pathway to the brain.

Retinoblastoma can spread into the choroidal blood vessels and hematogenous spread occurs. Direct

tumor growth through the sclera can cause orbital extension and proptosis.

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.

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

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

glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis. Retinoblastoma (unilateral or

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

retinoblastoma). It might present in a syndromic form (13q deletion syndrome) associated with some

facial features as high and broad forehead, thick and everted ear lobes, short nose, prominent philtrum and

thick everted lower lip, bulbous tip of the noseassociated with various degrees of hypotonea and mental

retardation (Baud 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.

Grouping/staging

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

involved in grouping are size and site of the tumor, amount of subretinal fluid, size and site of tumor

seeds and the presence of high risk features. Multiple grouping systems for the intraocular retinoblastoma

existed with the international intraocular retinoblastoma classification (IIRC) being the most reliable in

the last decade. Recently, it has been replaced by the TNMH classification. The main factors involved in

grouping are size and site of the tumor, 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 staged by genetics in

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addition to the clinical features due to the high impact of genetic status on management. If there is a

positive family history, bilateral disease or documented positive RB1mutation testing, the disease is

staged as H1. Otherwise it is considered as H0. A true H0 is with documented negative RB1 mutation

status.

-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. 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.

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 differentiate…. (Hilary to write details

and choose appropriate site) –Cite Racher paper

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

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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). 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). The clinical presentation

of heritable retinoblastoma consists of 80% bilateral and 15-18% unilateral (cite). 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 (Rushlow et al 2013).

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,

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

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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

(Sanchez-Sanchez et al 2007 PMID: 16988938), (2) mutations in exons 26 and 27 (Mitter et al 2009 PMID:

18509746; ), (3) intronic mutations (Schubert et al 1997 PMID: 9341870; Lefevre et al 2002 PMID:

12011162 ; ) and (4) missense mutations (cite). 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 ; ). Dehainault et al showed that RB1 -/- cells cannot survive in the absence of

MED4.

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). A recent

study by Eloy et al 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 inherited there is

sufficient tumor suppressor activity to prevent 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 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).

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,

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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.

Mosaicism

{FIGURE ON MOSAICISM}

RB1 gene [Hilary]

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

regulator and the first tumor suppressor gene ever discovered (Friend et al 1986 PMID: ). After a cell

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

E2F transcription factor gene, thereby repressing transcription and inhibiting the progression of the cell

cycle from G1 to S phase (Nevins et al 2001 PMID: ; Cobrinik 2005 PMID: ; Sage et al 2012 PMID: ). In

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

of RB to bind to the E2F gene promoter (Knudsen and Knudsen 2008 PMID: ). RB functions to regulate

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proliferation in most cell types (Cobrinik 2005 PMID:). 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).

-?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 RB

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

chromothripsis (Lohmann 1999 PMID: ; McEvoy et al 2014 PMID: ). The majority of RB1 mutations are

de novo, unique to a specific patient or family, however, there are some know recurrent mutations found

across many unrelated individuals. One subset of recurrent mutations involved CpG

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 RB

Greta Bunin

MYCN

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PROGRESSIVE GENOMIC CHANGES

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

DEK, KIF14, E2F3, CDH11

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)

RNA for discovery and VUS functional studies

Protein studies

Genetic Counseling

Importance of high detection rate

Targeted familial testing/prenatal testing, preconception testing

Surveillance for mets and second cancer

Benefits of genetic counselling (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. (Uhlmann et al. (2009), Shugar (2016)). Genetic testing is an integral

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

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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

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

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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.

Screening for Retinoblastoma

The Known RB1 mutation of the proband can be tested in his offspring. This can be performed via

amniocentesis during the second trimester of pregnancy with minimal risks on fetus and mother (prenatal

screening) or it can be performed at birth via umbilical cord blood (postnatal screening). This will help

either eliminate the 50% theoretical risk of the proband’s RB1 mutation heritability or confirm it into

100% risk. Both screening methods are effective in improving visual outcome and eye salvage than non-

screened children, However, prenatal screening allows for planning for earlier delivery in positive

children (late preterm/early term); this was shown to have less number of tumors at birth (20% versus 50

%) with only 15 % visual threatening tumors in prenatatl screening. Prenatal screening with early delivery

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

outcome.

References

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

Blackwell.

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

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

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

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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

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REFERENCES

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Table X:

Subretinal Fluid (RD)

No≤ 5 mm

>5 mm - ≤ 1 quadrant

> 1quadrant

Tum

or

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

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

Se

edin

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

High

risk

feat

ures

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

Extraocular retinoblastoma cT4/??

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

applicable ; RD Retinal detachment

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