REVIEW ARTICLe
Genetics and Molecular Diagnostics
in Retinoblastoma—An Update
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Sameh E. Soliman, MD,*† Hilary Racher, PhD,‡ Chengyue Zhang, MD,§
Heather MacDonald,* and Brenda L. Gallie, MD*¶
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Abstract: Retinoblastoma is the prototype genetic cancer: in one or
both eyes of young children, most retinoblastomas are initiated by bial-
lelic mutation of the retinoblastoma tumor suppressor gene, RB1, in a
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developing retinal cell. All those with bilateral retinoblastoma have heri-
table cancer, although 95% have not inherited the RB1 mutation. Non-
heritable retinoblastoma is always unilateral, with 98% caused by loss of
both RB1 alleles from the tumor, whereas 2% have normal RB1 in tumors
initiated by amplification of the MYCN oncogene. Good understanding
of retinoblastoma genetics supports optimal care for retinoblastoma chil-
dren and their families. Retinoblastoma is the first cancer to officially
acknowledge the seminal role of genetics in cancer, by incorporating “H”
into the eighth edition of cancer staging (2017): those who carry the RB1
cancer-predisposing gene are H1; those proven to not carry the familial
RB1 mutation are H0; and those at unknown risk are HX. We suggest
H0* be used for those with residual <1% risk to carry a RB1 mutation due
to undetectable mosaicism. Loss of RB1 from a susceptible developing
retinal cell initiates the benign precursor, retinoma. Progressive genomic
changes result in retinoblastoma, and cancer progression ensues with in-
creasing genomic disarray. Looking forward, novel therapies are antici-
pated from studies of retinoblastoma and metastatic tumor cells and the
second primary cancers that the carriers of RB1 mutations are at high risk
to develop. Here, we summarize the concepts of retinoblastoma genetics
for ophthalmologists in a question/answer format to assist in the care of
patients and their families.
Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA
sequencing, genetic counseling, prenatal screening
(Asia-Pac J Ophthalmol 2017;6:197–207)
R etinoblastoma is the most common childhood intraocular ma-
lignancy that affects one or both eyes.1 Because of the strong
links between clinical care and genetic causation,2 retinoblastoma
is considered the prototype of heritable cancers.3 Worldwide,
From the *Department of Ophthalmology and Vision Sciences, University of
Toronto, Ontario, Canada; †Department of Ophthalmology, Faculty of
Medicine, University of Alexandria, Alexandria, Egypt; ‡Impact Genetics,
Bowmanville, Ontario, Canada; §Department of Ophthalmology, Beijing
Children’s Hospital, Capital Medical University, Beijing, China; and
¶Departments of Ophthalmology, Molecular Genetics, and Medical Biophysics,
University of Toronto, Toronto, Canada.
Received for publication January 13, 2017; accepted March 9, 2017.
Both S.E.S. and H.R. contributed equally to this review as co-first authors.
H.R. is a paid employee and B.L.G. is an unpaid medical advisor at Impact
Genetics.
The authors have no other funding or conflicts of interest to declare.
Reprints: Brenda L. Gallie, MD, Hospital for Sick Children, 555 University Ave,
Toronto, Ontario, Canada M5G 1X8. E‑mail: brenda@gallie.ca.
Copyright © 2017 by Asia Pacific Academy of Ophthalmology
ISSN: 2162-0989
DOI: 10.22608/APO.201711
Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
Copyright © 2017 Asia-Pacific Academy of Ophthalmology. Unauthorized reproduction of this article is prohibited.
about 8000 children are newly diagnosed with retinoblastoma ev-
ery year (1/16,000 live births)1,4 but most have no access to knowl-
edge of the important role genetics plays in many aspects of retino-
blastoma: clinical presentation, choice of treatment modalities, and
follow-up for both child and family. We now highlight the genetic
etiology of retinoblastoma in the context of individual children and
families, led by the questions commonly asked by parents.
What Is Retinoblastoma?
Retinoblastoma is a cancer that arises because both copies
of the RB1 gene that normally suppresses retinoblastoma are
lost from a developing retinal cell in fetuses, babies, and young
children. Retinoblastoma can affect one (unilateral) or both eyes
(bilateral) and, in 5% of children with heritable retinoblastoma
(H1),5 is associated with a midline brain tumor (trilateral).6 With-
out timely and effective treatment, retinoblastoma may spread
through the optic nerve to the brain, or via blood, particularly to
bone marrow, and result in death.
How Can Cancer Occur
at Such a Young Age?
The cell of origin of retinoblastoma is most likely a develop-
ing cone photoreceptor precursor cell that has lost both alleles of
the RB1 tumor suppressor gene and remains in the inner nuclear
layer of the retina (Fig. 1), perhaps because it is unable to migrate
to the outer retina and function normally.1,7,8 The cell susceptible
to developing cancer is present in the retinas of young children,
from before birth, up to around 7 years of age. Rarely, retinoblas-
toma is first diagnosed in older persons, who likely previously
had an undetected small tumor (retinoma) present from child-
hood that later became active.9,10 In high-income countries, the
mean age at presentation is 12 months in bilateral disease and 24
months in unilateral disease, whereas significant delay in low-in-
come countries impacts negatively on the children.11
What Causes Retinoblastoma?
No one knows what really causes the genomic damage to the
RB1 gene, but retinoblastoma arises at a constant rate in all races
irrespective of local environment. In nearly 50% of patients, the
first copy of the RB1 gene is damaged in most, or all, normal
cells of the patient. A retinal tumor develops when the second
copy of the RB1 gene is also damaged in a developing retinal
cell.1 The RB1 gene resides on chromosome 13q14 and encodes
the retinoblastoma protein (pRB), an important regulator of cell
division cycle in most cell types, and the first tumor suppressor
gene discovered.12 Normally, cell division is inhibited by hypo-
phosphorylated pRB binding to E2F molecules and blocking their
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 Soliman et al Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017

transactivation of RB1, E2F, and other promoters of molecules
that support cell division.13‒15 To resume cell division, cyclin-
dependent kinases re-phosphorylate pRB, activating promoters of
key proteins important in cell division.16 Loss of pRB therefore
allows uncontrolled cell division. In many cell types, loss of the
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What Caused These Mutations?
Did I Cause Them?
No one is to blame for the mutations causing retinoblastoma.
Many environmental forces induce DNA damage, including cos-
mic rays, X-rays, DNA viruses, ultraviolet irradiation, and smok-

RB1 gene is compensated by increased expression of other related ing. The DNA damage may be point mutations, small and large
proteins. However, in susceptible cells such as retinal cone cell deletions, promotor methylation shutting down RB1 expression,
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precursors, compensatory mechanisms are insufficient, allowing
uncontrolled cell division to initiate cancer.8
What Causes Retinoblastoma to be
Unilateral versus Bilateral?
In heritable retinoblastoma (also called germline retinoblas-
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and rarely, chromothripsis.24,25 The majority of RB1 mutations
arise de novo, unique to a specific patient or family (Fig. 2B).
However, some recurrent mutations are found in unrelated indi-
viduals, such as those that affect 11 hyper-mutable CpG DNA
sequence sites, which make up 22% of all RB1 mutations.26,27
A de novo RB1 germline mutation may arise either pre- or
post-conception. Pre-conception mutagenesis of RB1 usually oc-

toma), the first RB1 allele is mutated (M1) in nearly all cells, in-
cluding germline reproductive cells, whereas the second allele is
mutated (M2) in the retinal cells that become cancer, usually in
both eyes (Figs. 2A, B). The most common M2 event is loss of
the normal RB1 allele and duplication of the mutated M1 allele, in
that 2 copies of the mutated RB1 gene remain; a mutational event
referred to as loss of heterozygosity (LOH).17,18 Heritable retino-
blastoma encompasses 45% of all reported cases19‒21 with bilateral
(80%), unilateral (15%), or trilateral (5%) tumors.1 Germline RB1
mutations carry the risk of second primary cancers, most com-
monly leiomyosarcoma, osteosarcoma, and melanoma.22 These
patients may benefit from regular surveillance for such cancers
over their lifetime.
Of nonheritable retinoblastoma, 98% have both RB1 M1 and
M2 events within a retinal cell. In the remaining 2%, retinoblas-
toma is induced by somatic amplification of the MYCN oncogene,
in the presence of normal RB1 genes.23
curs during spermatogenesis, perhaps because cell division (an
opportunity for mutation) is very active during spermatogenesis
but not during oogenesis.28,29 Advanced paternal age increases
risk for retinoblastoma,30 suggesting that genomic errors may in-
crease in aging men. The affected child carries the de novo RB1
mutation in every cell, typically presenting with 4‒5 tumors and
bilateral retinoblastoma. In contrast, if mutagenesis occurs post-
conception, during embryogenesis, only a portion of cells will
carry the RB1 mutation (ie, mosaicism).1
Does Only RB1 Mutation Cause
Retinoblastoma?
There are 2 answers to this question: (i) loss of both RB1 al-
leles only causes retinoma, a benign precursor to retinoblastoma,
and other genes are modified to cause progression to cancer;10
and (ii) 2% of unilateral retinoblastoma have normal RB1 and are

A D

B E

C F

figure 1. Early awareness of risk for retinoblastoma optimizes therapy and outcomes. This child was examined because his sibling (triplets) presented
with retinoblastoma. His right eye appeared normal, but optical coherence tomography (OCT) revealed 2 invisible tumors (* and ↓) (A–C), which were
treated with laser therapy only; OCT after laser shows coagulation of tumors, visible in the retinal image (D–F).

198 | www.apjo.org © 2017 Asia-Pacific Academy of Ophthalmology

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 Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017 Genetics and Molecular Diagnostics in RB

caused by a different gene.
(i) Retinoma is a premalignant precursor to retinoblastoma
with characteristic clinical features: translucent white mass, re-
active retinal pigment epithelial proliferation, and calcific foci.9
Retinoma may be found retrospectively after a child develops
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landscape of other cancers, retinoblastoma is one of the least mu-
tated, and epigenetic modification of gene expression may play
an important role in retinoblastoma progression.39‒41 SYK is a pos-
tulated methylating proto-oncogene required for retinoblastoma
cell survival. It was found to be upregulated in retinoblastoma

retinoblastoma (Figs. 2C, 3). Pathology of retinoma reveals non- and suggested as a potential target of therapy.40,42 It was present in
proliferative fleurettes.10,31 Comparison of adjacent normal retina, 100% of retinoblastomas and 0% of normal retina histologically
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retinoma, and retinoblastoma showed loss of both RB1 alleles
and early genomic copy number changes in retinoma that were
amplified further in the adjacent retinoblastoma.10 Many retino-
blastomas have underlying elements of retinoma. Retinoma can
transform to retinoblastoma even after many years of stability,
so they need lifelong monitoring. The alternate, confusing term
“retinocytoma” was proposed 1 year later32 and is inappropriate
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by immunohistochemical staining of pathological specimens of
enucleated eyes with retinoblastoma.43
(ii) There is a newly recognized form of retinoblastoma with
normal RB1 genes. Two percent of unilateral patients have RB1+/+
MYCNA tumors, in which the MYCN oncogene is amplified (28‒
121 DNA copies instead of the normal 2 copies).23 These children
are diagnosed at a median age of 4.5 months compared with 24

because it had been used for several different entities, including
active tumors with Flexner-Wintersteiner rosettes.33‒36 In addi-
tion to loss of RB1, specific alterations in copy number of other
genes are common in RB1-/- retinoblastoma. There are gains (4‒
10 copies) in oncogenes MDM4, KIF14 (1q32), MYCN (2p24),
DEK, and E2F3 (6p22) and loss of the tumor suppressor gene
CDH11 (16q22-24).3,37 Other less common genomic alterations in
retinoblastoma tumors include differential expression of specific
microRNAs,38 recurrent single nucleotide variants/insertion-de-
letions in the genes BCOR and CREBBP,39 and upregulation of
spleen tyrosine kinase (SYK).40 In comparison with the genomic
months for nonheritable unilateral RB-/- patients, and the tumors
are histologically distinct with advanced features at diagnosis.
Could We Have Discovered
Retinoblastoma Earlier?
The only way to find retinoblastoma tumor early is to exam-
ine the eye with specific expertise, which we cannot do for every
child. The earliest signs of retinoblastoma detectable by parents
are leukocoria (white pupil), either directly or in photographs
(photo-leukocoria), and strabismus when the macula is involved

figure 2. Pedigrees illustrating inheritance patterns for RB1 mutations. A, Full penetrance and expressivity for null mutation: father (bilaterally
enucleated) and 2/2 bilaterally affected offspring (I-1 unilateral enucleation vision 1.0 remaining eye); all H1 with 11 base pair deletion in exon 12;
DER = 2. B, No family history, new null mutation: triplets1 diagnosed with bilateral retinoblastoma at age 2.5 months due to c.1345G>T (p.Gly449Ter)
RB1 missense mutation resulting in no pRB; parents showed no evidence of the mutation but were considered H0* because there remains a less than
1% risk of mosaicism in either parent; older sibling is negative for the mutation, therefore H0; DER = 2. C, 100% penetrance and variable expressivity:
grandfather (I-1) was diagnosed with bilateral retinoma when his daughter (II-1) was diagnosed with bilateral retinoblastoma due to c.1960G>T
(p.Val654Leu) RB1 missense mutation; she (II-1) later developed meningioma in the radiation field and breast cancer; her brother (II-3) and daughter
(III-2) inherited the mutation and developed unilateral retinoblastoma; DER = 1.5. D, Parent of origin low penetrance62: c.607+1G>T RB1 splice mutation
that shows higher penetrance when inherited from the father (ie, II-1, III-1, III-3; DER = 1) than from the mother (ie, III-1, III-3; DER = 0), likely due to
increased expression from the maternal than the paternal mutant RB1 allele60 (overall DER = 0.7); IV-1 had a small unilateral tumor but died at 11 years
of age due to radiation-induced secondary malignancies; IV-5 has not been tested but developed thyroid cancer.

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 Soliman et al
by tumor. Facial features and various degrees of hypotony and
mental retardation in 13q deletion syndrome can lead to discov-
ery of retinoblastoma.44,45 If we examine the retina of patients with
positive familial history early, we may discover the smallest visible
tumors that are round, white retinal lesions that obscure the under-
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lying choroidal pattern; invisible definitive tumors can be detected
even earlier by optical coherence tomography (Fig. 1).46,47
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Centrifugal growth results in small tumors being round;
more extensive growth produces lobular growth, likely related to
genomic changes in single (clonal) cells that provide a prolifera-
tive advantage.48 Next, tumor seeds spread out of the main tumor
into the subretinal space or vitreous cavity. Vitreous seeding is
associated with loss of chromosome 16q, including the cadherin
13 gene, a genomic change that may induce poor cohesive forces
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between tumor cells.49,50
Advanced vitreous tumor seeds can migrate to the anterior
chamber producing a pseudohypopyon. Enlarging tumor can push
the iris lens diaphragm forward causing angle closure glaucoma.
Advanced tumors may induce iris neovascularization. Rapid ne-
crosis of tumor can cause an aseptic orbital inflammatory reac-
tion resembling orbital cellulitis, sometimes showing central reti-
nal artery occlusion on pathology.48,51 Untreated, retinoblastoma
spreads into the optic nerve and brain, or hematogenous spread
occurs through choroid, particularly to grow in bone marrow. Tu-
mor extension through sclera can present as orbital extension and
proptosis.
Do All Affected Individuals with RB1
Mutations Develop Retinoblastoma?
Depending on the exact RB1 mutation, most, but not all, car-
riers of an RB1 mutation will develop retinoblastoma and other
cancers throughout life.
Each offspring of a person carrying an RB1 mutated gene
has 50% risk to inherit the RB1 mutated gene (Fig. 2). A measure
of expressivity of a mutant retinoblastoma allele is the disease-
eye ratio (DER) (number of eyes affected with tumor divided
by the number of carriers of the RB1 mutation).52 Nonsense and
frame-shift germline mutations that lead to absent or truncated
dysfunctional pRB result in 90% bilateral retinoblastoma (nearly
figure 3. Retinoma, the benign precursor of retinoblastoma. Bilateral stable translucent retinal masses with calcification and associate retinal pigment
epithelial disturbance were diagnosed to be retinoma in the grandparent I-1 (Fig. 2C), only when his daughter (II-1) was diagnosed with bilateral
retinoblastoma. He never received treatment and was alive and well at age 90 at last follow-up.
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Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
complete penetrance, DER = 2). Partially functional RB1 mutated
alleles show variable penetrance and expressivity (Fig. 2C) with
fewer tumors and later onset,47 and some carriers never develop
retinoblastoma (DER = 1‒1.5). Reduced penetrance mutated RB1
alleles include (i) mutations in exons 1 and 2,53 (ii) mutations near
the 3’ end of the gene in exons 24 to 27,54,55 (iii) splice and intron-
ic mutations,56 and (iv) missense mutations.57 Counterintuitively,
large deletions encompassing the RB1 gene and MED4 gene also
cause reduced expressivity/penetrance because RB1-/- cells can-
not survive in the absence of MED458 and the most common M2
event in retinoblastoma tumors is loss of the normal allele and
reduplication of the mutated allele. In comparison, patients with
large deletions with 1 breakpoint inside the RB1 gene typically
present with bilateral disease.59
There are at least 2 specific RB1 mutant alleles showing a
parent-of-origin effect: c.607+1G>T substitution60,61 (Fig. 2D)
and c.1981C>T (p.Arg661Trp) missense mutation.62 Both may be
explained by differential methylation of intron 2 CpG85, which
skews RB1 expression in favor of the maternal allele.63 When the
mutated allele is maternally inherited, there is sufficient tumor
suppressor activity to limit retinoblastoma development to 10%
of carriers. However, when the mutated RB1 allele is paternally
transmitted, very little pRB is expressed, leading to retinoblas-
toma in 68% of carriers.
What Factors Affect Retinoblastoma
Cancer Staging?
Treatment and prognosis of retinoblastoma 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.5 Multiple staging systems have predicted likelihood to
salvage an eye without using radiation therapy, but published evi-
dence is confusing because significantly different staging versions
have been used.1,5 The 2017 TNMH classification is based on an
international consensus and evidence from an international survey
of 1728 eyes and separates well initial clinical and pathological
features predictive of outcomes to save the eye, in retrospective
comparison with 5 previous eye staging systems.5
© 2017 Asia-Pacific Academy of Ophthalmology
 Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
Does Germline Status Affect
Retinoblastoma Staging?
Retinoblastoma is the first cancer in which staging recog-
nizes the impact of genetic status on outcomes in the 2017 TNMH
staging (Fig. 4): presence of a positive family history, bilateral or
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trilateral disease, or high sensitivity positive RB1 mutation test-
ing is stage H1; without these features before testing blood, HX;
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and H0 for those relatives shown to not carry the proband’s spe-
cific RB1 mutation (Fig. 2).5 We propose H0* for patients tested
and having neither M1 nor M2 RB1 mutated alleles of the tumor
detectable in blood and parents showing no evidence of the M1
allele of their offspring, but with remaining low risk (<1%) of
undetectable mosaicism. Offspring of H0* persons who do not
show their family’s RB1 mutation are H0; if they test H1 for the
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inherited mutation they need intensive surveillance for eye tu-
mors from birth.
What Are the Main Goals of
Retinoblastoma Management?
Saving life is the top priority of retinoblastoma treatment,
followed by vision salvage; least important is eye salvage. The
child’s job is to play and develop over a healthy life; many pro-
cedures and their complications that may span years for at best
a 50% chance to save a blind eye with risk of tumor spread are
not justified, especially when the other eye is normal.64,65 Mul-
tiple treatment modalities are currently present with promising
tumor control in the short term (5 years) with minimal data on
their long-term effects. Only after 30 years of being used on every
child, the enormous impact of external beam irradiation (EBRT)
was recognized: more children with bilateral retinoblastoma who
were treated with radiation die of their second (or third, and so
on) cancer than of retinoblastoma.66,67
However, often missing from choices in the complex care of
children with retinoblastoma are truly informed parents. Essen-
tially, the doctors decide what treatment they “feel” is best, based
on little substantial evidence. There currently exists no easy way to
show the parents prospectively the true “costs” of each treatment:
the burden of invasive therapies and potential complications;
A B
figure 4. Eighth edition TNMH cancer staging for intraocular retinoblastoma.54 Clinical staging for eyes with retinoblastoma, compared with IIRC
(A).38 Heritability stage for persons at risk to carry an RB1 germline mutation (B). H0* is our proposal to clarify the remaining small risk of undetectable
mosaicism in parents of H1 patients who test negative for the RB1 pathogenic variant in their H1 child and in unilateral patients with no RB1 pathogenic
variant detected in blood.
© 2017 Asia-Pacific Academy of Ophthalmology
Copyright © 2017 Asia-Pacific Academy of Ophthalmology. Unauthorized reproduction of this article is prohibited.
Genetics and Molecular Diagnostics in RB
the imposition of hours and days in hospitals and feeling ill on
the child, whose real job in those critical, irreplaceable years is
to play; the true costs including time off work and uncertainties;
and the burden of “false hope” in the absence of real evidence.
There are imminent solutions on the horizon: DePICTRB (depic-
trb.technainstitute.com) collects retinoblastoma-specific details
including genetic results and makes them viewable online by the
family and patient68 and the burgeoning field of patient-reported
outcomes for research with accurate patient details. These new
attitudes and tools may empower, in the future, well-informed
choices by parents for their child and family.
What Are the Treatments and
What Governs the Choice?
Choice of treatment depends on the tumor stage, genetic
status, and laterality of disease. Focal therapy can only control
cT1a eyes, but visually threatening or large cT1b tumors and
cT2 eyes need chemotherapy (systemic or intra-arterial chemo-
therapy) to reduce the size of the tumor followed by consolida-
tion focal therapies (laser therapy or cryotherapy) as initial treat-
ment. Enucleation of eyes with advanced tumors (cT3) is usually
a definitive cure, and if high risk features are found, appropriate
treatment response has a chance for cure.1 Ancillary therapies for
specific indications include plaque radiotherapy and periocular
chemotherapy. Intravitreal chemotherapy for vitreous disease has
recently dramatically improved safe eye salvage.69 Retinoblasto-
ma with extraocular spread (cT4) requires more aggressive treat-
ments with high dose systemic chemotherapy, surgery, and EBRT
with or without autologous stem cell transplantation. For persons
carrying RB1 mutations, EBRT is rarely indicated due to the high
risk of inducing later second cancers.1
Laterality at presentation also directs treatment choices. In
unilateral retinoblastoma, enucleation is the preferred definitive
treatment modality if there is anticipated poor visual outcome of
the affected eye.1 If useful vision is anticipated, chemotherapy
(systemic or intra-arterial) combined with focal therapy might be
utilized. In bilateral retinoblastoma, systemic chemotherapy fol-
lowed by consolidation focal therapy is preferred when both eyes
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 Soliman et al
are salvageable (eighth edition TNMH cancer staging5 cT1b, cT2)
[International Intraocular Retinoblastoma Classification (IIRC)48
B, C, D eyes]. When one eye is cT1a and the other is advanced,
enucleation of the advanced eye and focal therapy for the cT1a
eye might be more appropriate. When both eyes are nonsalvage-
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able, bilateral enucleation is the definitive treatment to save life
and minimize morbidity.
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Is Retinoblastoma Lethal?
Untreated, retinoblastoma is lethal. If treated before metasta-
sis occurs, the cure rate is nearly 100%. Occasionally, metastatic
retinoblastoma may be confused with a second cancer; molecular
demonstration of the RB1 mutations of the intraocular retinoblas-
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toma will confirm metastases.70 Globally, the chance for cure is
remote, and lack of knowledge of genetics results too often in
the death of children who could have been saved if they had sur-
veillance and definitive treatment when tumors were small. We
look forward to the cancer genomic revolution focusing on meta-
static retinoblastoma, leading to breakthrough drugs that make
retinoblastoma a zero death cancer. However, delayed diagnosis
and treatment due to lack of knowledge by ophthalmologists and
parents, along with socioeconomic64 and cultural factors are the
major causes of mortality. Asia and Africa have more than 70%
mortality from retinoblastoma compared with less than 5% in de-
veloped countries.71
How Can We Test for
Retinoblastoma Mutations?
Genetic counseling is the psychosocial and educational pro-
cess to help patients and families adapt to the genetic risk, the ge-
netic condition, and the process of informed decision-making.72,73
Especially when genetic testing is not available or unaffordable,
genetic counseling is very important. Concrete knowledge of ge-
netic test outcomes supports informed and precise genetic coun-
seling. Genetic testing supports precision medicine for those who
need it (carry the RB1 mutated allele) and is more cost effective
than examining all at-risk family members.27,74
If the proband is bilaterally affected, the probability of find-
ing a germline mutation in the RB1 gene in DNA extracted from
blood is high (97% in a comprehensive RB1-specialized labora-
tory). In 3% of bilateral retinoblastoma patients, the predispos-
ing RB1 mutation cannot be detected in blood. In these instances,
identification of M1 and M2 RB1 mutations in DNA from tumor
can assist in the identification of a germline mutation, including
low-level mosaic mutations.26,75,76
Similarly, to detect the 15% of unilateral patients carrying a
germline mutation, the optimal strategy first tests tumor DNA and
then looks for the mutations in blood. If the blood is not found
to carry 1 of the tumor RB1 mutations, risk of germline status is
reduced to less than 1% (Table 1) for parents, siblings, and cous-
ins.76 Quality of genetic results depends on quality of DNA. Fresh
or frozen tumor samples are ideal, whereas DNA from forma-
lin fixed paraffin embedded tumors is suboptimal. Whole blood
in EDTA or ACD anticoagulant typically provides high quality
DNA.
The RB1 gene can be mutated in many ways, best identi-
fied by a series of techniques. The most appropriate technology
depends on the clinical question being asked. Next-generation
202 | www.apjo.org
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Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
sequencing (NGS) may be the most effective screening strategy
to find an unknown de novo mutation in an affected proband
and may detect lower level mosaic mutations.77,78 To screen fam-
ily members for a known sequencing-detectable RB1 mutation,
targeted Sanger dideoxy sequencing is still more cost and time
effective.
Large RB1 deletions or duplications that span whole exons or
multiple exons typically cannot be detected by DNA sequencing.
Multiplex ligation-dependent probe amplification (MLPA), quan-
titative multiplex polymerase chain reaction (QM-PCR), or array
comparative genomic hybridization (aCGH) are used to identify
RB1 deletions and duplications and other genomic copy number
alterations, such as MYCN amplification. New developments in
bioinformatic analysis methods suggest that NGS data can be in-
terrogated for copy number variants,77,79 but sensitivity is not yet
optimal. Somatic mosaicism can arise in either the presenting pa-
tient or their parent. Allele-specific PCR (AS-PCR) has excellent
sensitivity when the RB1 mutation is known26 and can detect as
low as 1% mosaicism.
Polymorphic microsatellite markers distributed throughout
chromosome 13 can be used to detect a change from a hetero-
zygous state in blood compared with the homozygous state in a
tumor with LOH. Microsatellite marker analysis is also important
in identity testing and in identifying maternal cell contamination
in prenatal samples.
Epigenetic changes can also initiate retinoblastoma develop-
ment.80 Hypermethylation of the RB1 promoter CpG island re-
sults in inhibition of RB1 gene transcription in 10‒12% of reti-
noblastoma tumors, commonly involving both alleles.27,81 This
epigenetic gene-silencing event primarily occurs in somatic cells,
but heritable RB1 promoter mutations and translocations disrupt-
ing RB1 regulatory sites or translocations involving the X chro-
mosome have been shown to cause constitutional RB1 promoter
hypermethylation.82,83
Rarely, no RB1 mutation is identified in the coding, promot-
er, or flanking intronic sequence in blood from a bilateral patient.
Deep intronic sequencing alterations that disrupt RB1 transcrip-
tion by interfering with correct splicing in patients with retino-
blastoma can be detected by analysis of the RB1 transcript by
reverse transcriptase PCR (RT-PCR).56,84 RNA studies can also
clarify pathogenicity of intronic sequence alterations. As costs
decrease, whole genome sequencing may become the method of
choice to uncover deep intronic changes.
Karyotype, fluorescent in situ hybridization (FISH), or array
comparative genomic hybridization (aCGH) of peripheral blood
lymphocytes can be used to identify large deletions and rearrange-
ments in retinoblastoma patients, including patients suspected of
13q14 deletion syndrome.59 In parents of 13q14 deletion patients,
karyotype analysis can be used to investigate for balanced trans-
locations, which increase the risk for retinoblastoma in subse-
quent generations.44
What Is Done After Finding
the RB1 Mutation?
Family members at risk to also carry the identified RB1 mu-
tation are offered testing on blood samples (Fig. 2, Table 1).1,76,85
If the proband’s mutation is not found in either parent, a risk for
low level mosaicism still exists. Offspring of any family mem-
ber carrying the RB1 mutation can be tested during pregnancy
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Table 1. Risks for Probands and Relatives to Develop Retinoblastoma and Second Cancers and Clinical Surveillance Plans85

No Molecular Testing Molecular Testing Perfomed on Blood

Positive Blood Test for Negative Blood Test for Proband RB1 Mutation
Known Mutation* Known Mutation Not Found*

Risk for RB1 Surveillance Risk for RB1 Surveillance Risk for RB1 Surveillance Risk for RB1 Surveillance
Proband Presentation Relative Mutation Plan Mutation Plan Mutation Plan Mutation Plan

Bilateral*‡ Proband 100% EUAs 100% EUAs, cancer surveillance NA NA 100% EUAs
Offspring 50% EUAs 100% Early delivery, EUAs, 0.006% No clinic exams 50% EUAs
cancer surveillance
Unaffected parent† 5% Retinal exam 100% Retinal exam for 0.20% Retinal exam 5% Retinal exam

© 2017 Asia-Pacific Academy of Ophthalmology

for retinoma retinoma, cancer for retinoma for retinoma
surveillance
Siblings 2.50% EUAs 100% Early delivery, EUAs, 0.006%§ No clinic exams 2.50% EUAs
cancer surveillance
Unilateral blood only; Proband 15% EUAs 100% EUAs, cancer surveillance NA NA 0.45% Clinic exams
no tumor available
Offspring 7.50% EUAs 100% Early delivery, EUAs, 0.006% No clinic exams 0.22% Clinic exams
cancer surveillance
Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017

Unaffected parent† 0.75% Retinal exam 100% Retinal exam for 0.20% Retinal exam 0.02% Retinal exam
for retinoma retinoma, cancer for retinoma for retinoma
surveillance
Siblings 0.38% EUAs 100% Early delivery, EUAs, 0.006%§ No clinic exams 0.01% Clinic exams
cancer surveillance
Unilateral tumor; both RB1 Proband 15% EUAs 100% EUAs, cancer surveillance NA NA
mutations known*‡
Offspring 7.50% EUAs 100% Early delivery, EUAs, 0.006% No clinic exams
cancer surveillance
Unaffected parent† 0.75% Retinal exam 100% Retinal exam for 0.20% Retinal exam
for retinoma retinoma, cancer for retinoma
surveillance
Siblings 0.38% EUAs 100% Early delivery, EUAs, 0.006%§ No clinic exams
cancer surveillance
Unilateral tumor; Proband 0.006% Clinical care
RB1+/+MYCNamp

Copyright © 2017 Asia-Pacific Academy of Ophthalmology. Unauthorized reproduction of this article is prohibited.
Offspring 0.006% No clinic exams 0.006% No clinic exams
Unaffected parent†
Siblings

Population risk 1:16,000 per live birth = 0.006%; risk for offspring of RB1 mutant parent to inherit mutation = 50%.

www.apjo.org |
*Risk to miss RB1 mutant allele in bilateral blood or retinoblastoma tumor = 3%; †Unaffected parental carrier rate (6/120 parents of bilaterally affected children, Impact Genetics) = 5%; ‡Estimated risk to miss mosaicism;
§Negative for proband’s tumor mutation.

203
Genetics and Molecular Diagnostics in RB
 Soliman et al
or immediately after birth. If the proband is mosaic for the RB1
mutation, parents and siblings are not at risk, as mosaicism can-
not be inherited. The children of a mosaic proband need to be
tested as early as possible because if they do inherit the RB1 mu-
tated allele, they will usually be at high risk of bilateral disease.
EJrmFlTk2CdA2fkvq0vLF5iHVgJKZNE9U4OF4ZKX80CtS4r27li93c7kdjsuUMSF0Kx8DNNsy48J3C6tLlOwUFWRuPcVpZjlKVZD
If they do not carry the mutation, they are at population risk for
retinoblastoma.
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How Does the RB1 Status Affect
Follow-Up of the Child?
If the child does not carry an RB1 germline mutation, no ex-
aminations under anesthesia (EUAs) are required for the unaf-
fected eye with follow-up only in the clinic (Table 1). If the child
z8+cW/hhcZl2stsmntwn4NWWmiJXHFQ== on 03/08/2023
carries a germline mutation or is mosaic for the RB1 mutation,
the possibility of new tumors renders frequent EUAs essential
up to at least 3 years of age.86 The germline status also predis-
poses to other second malignant neoplasms, requiring frequent
annual surveillance. The use of whole body magnetic resonance
imaging as a method for surveillance is controversial, as a high
false positive rate may provoke unnecessary invasive procedures.
Development of surveillance programs beyond the pediatric age
is a high priority for early detection of these often-lethal second
RB1-induced cancers.67
Can We Use the Known Mutation
to Test My Future Children?
Prenatal genetic testing can be performed in the course of the
pregnancy. Two early procedures are available: (i) chorionic vil-
lus sampling (CVS), performed between 11‒14 weeks gestation,
involves obtaining a sample of the placenta either transvaginally
or transabdominally; and (ii) amniocentesis after 16 weeks gesta-
tion involves sampling amniotic fluid transabdominally. The pro-
cedure-associated risk of miscarriage with CVS is 1%, whereas
figure 5. Postnatal diagnosis of familial retinoblastoma too late to save good vision. Bilateral inherited retinoblastoma in a neonate with positive family
history born full term (39 weeks) and examined at 5 days after birth: right eye stage cT2b (IIRC group C), left eye stage cT1b (IIRC group B) involving both
foveas. Treatment with multiple periocular and systemic chemotherapies and 3 years of focal therapies salvaged both eyes with legal blindness. The
child has been previously published (patient #2).47
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Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
amniocentesis is 0.1‒0.5%. Maternal cell contamination is more
frequent with CVS87 and is routinely assessed by the clinical mo-
lecular lab.
If the fetus does not carry the mutation, the pregnancy can
proceed with no further intervention. If the fetus carries the famil-
ial mutation, the parents have several choices. Some may decide
to stop the pregnancy, whereas others may decide to continue the
pregnancy regardless of test results. If the parents are concerned
by the risk of miscarriage, they can consider late amniocentesis
between 30‒34 weeks gestation when the major complication is
early delivery rather than miscarriage.87 Prenatal or postnatal RB1
mutation testing will either show the baby to be H0 (no family
RB1 mutation) or H1 (confirmed to carry the mutation). Early
preterm delivery of an H1 baby achieves smaller tumors with
higher treatment success, eye preservation, and visual outcome
than delivery at full term (Fig. 1).47
In many countries, the option for prenatal genetic testing is
not available, and some parents may choose not to do prenatal
invasive testing. All offspring of an H1 parent have a 50% risk for
retinoblastoma, so it is important that the pregnancy does not go
past 40 weeks (Table 1, Fig. 5).47,76
Can We Plan Our Next Pregnancy To Avoid
Passing on This RB1 Mutation?
In some countries, preimplantation genetic diagnosis (PGD)
with in vitro fertilization is an option.88 Conceptions are tested for
the familial mutation at an early stage of embryonal development
(typically at the 8 cell stage). Those that do not carry the RB1
mutation are implanted. The procedure is costly, ranging from
$10,000‒$15,000 Canadian dollars per cycle; in some countries,
there may be full or partial coverage of the costs. The full medical
implications of PGD are not yet understood; there is emerging
evidence of a low risk for epigenetic changes in the conception as
a result of the procedure. It is recommended that typical prenatal
© 2017 Asia-Pacific Academy of Ophthalmology
 Asia-Pacific Journal of Ophthalmology • Volume 6, Number 2, March/April 2017
testing be pursued during the course of the pregnancy to confirm
the results.88
Are These Tests Available Worldwide?
EJrmFlTk2CdA2fkvq0vLF5iHVgJKZNE9U4OF4ZKX80CtS4r27li93c7kdjsuUMSF0Kx8DNNsy48J3C6tLlOwUFWRuPcVpZjlKVZD
High-sensitivity RB1 mutation testing is established in core
labs mainly in high-income countries.89 In low- and middle-
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income countries, genetic counseling as a specialty does not ex-
ist, so many families with retinoblastoma children do not have
the opportunity to understand the benefits of genetic testing and
counseling in retinoblastoma treatment and follow-up.1 In many
places, existing health insurance does not cover costs of genetic
testing. Given all these obstacles, there is limited application of
genetic testing and genetic counseling worldwide, which leads
z8+cW/hhcZl2stsmntwn4NWWmiJXHFQ== on 03/08/2023
to increased health care to deal with late diagnosis, along with
increased economic burden on affected families. As an example,
the Chinese government’s new policy to allow families to have
additional children will make the need for genetic testing and
counseling even more important.
In Egypt, genetic testing for retinoblastoma is not available,
therefore, genetic counseling is the only way to address the issues
facing retinoblastoma families.90 Ophthalmologists with insuffi-
cient training in retinoblastoma genetics are burdened with this
task. Genetic counseling was found to decrease knowledge gaps
remaining in the translation of this knowledge into appropriate
screening action.
Conclusions
Genetics is a core element in care of retinoblastoma pa-
tients and their families. Action based on knowledge of genet-
ics in retinoblastoma improves outcomes for the eye and points
to life and treatment strategies that alleviate suffering and may
reduce early mortality.90 The whole family benefits economi-
cally in health by precision in diagnosis of risk, based on genetic
testing. Treatments accommodate the risks of therapies, and sur-
veillance protocols can achieve earlier diagnosis of retinoblas-
toma and second cancers, leading to better outcomes. Knowl-
edge of test results alleviates psychological burden on families
moving forward with their life choices for the affected child and
future siblings and exposure to environmental carcinogens for
the whole family.
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