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Format of the review article:
- A word limit of 5,000 words;
- Less than 80 references;
- No strict limit to the number of tables and figures (8-10 recommended);
- An unstructured abstract of ≤ 250 words;
- The maximum number of authors: 6
Genetics and Molecular Diagnostics in
Retinoblastoma - An Update
Authors:
Sameh E. Soliman, MD,1-2 Hilary Racher, PhD,3 Chengyue Zhang, MD,4 Hilary Racher, PhDHeather
MacDonald,1 Brenda L. Gallie.1,5
Affiliations:
1Department of Ophthalmology and Vision Sciences, University of Toronto, Ontario, Canada
2Department of Ophthalmology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt.
3Impact Genetics, Bowmanville, Ontario.
4Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University, Beijing, China.
5Departments of Ophthalmology, Molecular Genetics, and Medical Biophysics, University of Toronto,
Toronto, Canada.
Corresponding author:
Brenda L. Gallie: Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G 1X8.
Telephone: +1-294-9729
Disclosures:
Both SS and HR contributed equally to this review as first co-first authors.
We confirm that this manuscript has not been and will not be submitted elsewhere for publication, and all
co-authors have read the final manuscript within their respective areas of expertise and participated
sufficiently in the review to take responsibility for it and accept its conclusions.
HR is a paid employee and BG is an unpaid medical advisor at Impact Genetics. No other authors have
any financial/conflicting interests to disclose.
This paper received no specific grant from any funding agency in the public, commercial or not-for-profit
sectors.
Word Count: (/5000)
Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic counselling,
prenatal screening.
3
Unstructured abstract
Abstract: (120/250)
Retinoblastoma is an intraocular malignancy that affects one or both eyes of young children, that is
initiated by biallelic mutation of the retinoblastoma gene (RB1) in a developing retinal cell. A good
understanding of retinoblastoma genetics supports optimal care for retinoblastoma children and their
families. In this scenario the genetics trait description was conducted by the conversation between a
family with a retinoblastoma child and their attending who is mostly the ophthalmologist but can be any
member of the retinoblastoma multidisciplinary team of physicians, nurses and genetic counselors. All the
questions are true and high frequently asked by the parents. This scenario aims to try to simplify the
information around genetics for ophthalmologists to help them improve their patient and family care.
bilateral, unilateral, DNA sequencing, genetic counseling prenatal screening
4
5321/5000 words
INTRODUCTION
Retinoblastoma is the most common childhood intraocular malignancy that affects one or both eyes.
{Dimaras, 2015 #10881} Because of the strong links between clinical care and genetic causation,
{Knudson, 1971 #11106} retinoblastoma is considered the prototype of heritable cancers.{Theriault, 2014
#8591} Worldwide, about 8,000 children are newly diagnosed with retinoblastoma every year (1/16,000
live births).{Seregard, 2004 #10380;Dimaras, 2015 #10881} Genetics underlies many aspects of
retinoblastoma: clinical presentation, choice of treatment modalities and follow-up for both child and
family. We now highlight the genetic etiology of retinoblastoma in the context of individual children and
families.
CASE SCENARIO
A 2-year-old girl presented with left leukocorea (white pupil), noticed by her family in a photograph 5
days earlier. They sought medical advise from their family physician, who suspected retinoblastoma and
referred them urgently to the pediatric ophthalmologist. The family had never before heard of
retinoblastoma, and the mother was 33 weeks pregnant. The child was very uncooperative but the
ophthalmologist was able to visualize a white retinal mass in the left eye. He could see the inferior retina,
intact optic nerve and fovea in the right eye and diagnosed retinoblastoma in the left eye. The following
discussion took place between the pediatric ophthalmologist and the family.
Q1: Father: What is retinoblastoma?
A: (Pediatratric Ophthalmologist) “Retinoblastoma is a cancer that arises from a developing retinal cell
in babies and young children. Retinoblastoma can affect one (unilateral) or both eyes (bilateral) and in 5%
of children is associated with a midline brain tumor (trilateral).{de Jong, 2014 #10885} Without timely
and effective treatment, retinoblastoma may spread through optic nerve to the brain, or via blood
5
particularly to bone marrow, which will result in death. To be sure of the diagnosis and the best treatment
for this rare disease, I will refer your daughter to the Retinoblastoma Centre, where experts treat many of
these children. I will phone now!”
Q2: Father: why this is presenting at such a young age?
A: (Retinoblastoma Ophthalmic Specialist) “The cell of origin of retinoblastoma is most likely a
developing cone photoreceptor precursor cell that has lost both copies of the RB1 tumor suppressor gene,
and remains in the inner nuclear layer of the retina, unable to migrate to the outer retina and function
normally.{Dimaras, 2015 #10881;Rootman, 2013 #11096;Xu, 2014 #9924} The susceptible cell that
becomes cancer is only present in the retinas of young children, from before birth, up to around 7 years of
age. Rarely, retinoblastoma is first diagnosed in older persons, but likely there was previously an
undetected small tumor (retinoma) present from childhood, that later became active.{Gallie, 1982
#10343;Dimaras, 2008 #13250} The mean age at presentation is 1 year in bilateral disease and 2 years in
unilateral disease.
Despite the fact that we can see tumor in only one eye by clinical examination of your daughter, we
cannot be sure the other eye is normal until we examine it under anesthetic (EUA).”
Q3: Mother: What caused retinoblastoma? How can a gene cause cancer in a
baby?
A: (Retinoblastoma Ophthalmic Specialist) “No one knows what really causes the damage to the RB1
gene. Maybe a random cosmic ray passes through Planet Earth and hits that large, important gene.
In nearly 50% of patients the first RB1 gene is damaged in most, or all, normal cells, resulting in
predisposition to retinoblastoma. A retinal tumor develops when the second RB1 gene is also damaged in
a developing retinal cell.{Dimaras, 2015 #10881} The RB1 gene on chromosome 13q14 encodes the RB
protein (pRB), an important regulator of the cell division cycle in most cell types, and the first tumor
suppressor gene discovered.{Friend, 1986 #10882} Normally, dephosphorylated pRB represses
6
expression of the E2F gene, thereby blocking cell division.{Nevins, 2001 #15292;Cobrinik, 2005
#15298;Sage, 2012 #7850} To resume cell division, cyclin-dependent kinases re-phosphorylate pRB,
releasing expression of E2F.{Knudsen, 2008 #15310} In many cell types, loss of the RB1 gene is
compensated by increased expression of other related proteins. However, in susceptible cells such as
retinal cone cell precursors, compensatory mechanisms are not sufficient, cell division is uncontrolled,
and cancer is initiated.{Xu, 2014 #9924}
Q4: What causes retinoblastoma to be unilateral versus bilateral?
A: (Retinoblastoma Ophthalmic Specialist) “In heritable retinoblastoma (sometimes called germline
retinoblastoma), the first RB1 allele (M1) is mutant in all cells, including germline reproductive cells,
while the second allele (M2) is mutated in the retina initiating cancer. Often M2 event in the retinal cell is
loss of the normal RB1 allele and duplication of the mutant M1 allele (LOH, loss of heterozygosity).
Heritable retinoblastoma encompasses 45% of all reported cases.{MacCarthy, 2009 #8367;Moreno, 2014
#9935;Wong, 2014 #15170} with either bilateral (80%), unilateral (15%) or trilateral (5%) tumors.
{Dimaras, 2015 #10881} Germline retinoblastoma carries risk of second primary cancers higher than
normal, most commonly osteosarcoma, fibrosarcoma and melanoma. These persons can benefit from
regular surveillance for such cancers for their lifetime.
Of non-heritable retinoblastoma, 98% have RB1 M1 and M2 arise in a retinal cell. The remaining 2%
the retinoblastoma is induced by somatic amplification of the MYCN oncogene, in the presence of normal
RB1 genes.{Rushlow, 2013 #11102}” Germline retinoblastoma carries the risk of development of second
primary cancers, most commonly osteosarcoma and fibrosarcoma due to loss of RB1 gene. This is why
these children should be kept under surveillance for the rest of their lives.
Q5: Mother: What caused these mutations? Did I cause them?
A: (Retinoblastoma Expert) “No one is to blame for the mutations causing retinoblastoma. Many
environmental forces induce DNA damage, including cosmic rays, X-rays, DNA viruses, UV irradiation
7
and smoking. The DNA damage may be point mutations, small and large deletions, promotor methylation
shutting down RB1 expression and rarely, chromothripsis.{Lohmann, 1999 #9272;McEvoy, 2014 #8499}
The majority of RB1 mutations arise de novo, unique to a specific patient or family. However, some
recurrent mutations are found in unrelated individuals, such as those that affect 11 sites CpG DNA
sequence sites, which are hyper-mutable and make up 22% of all RB1 mutations.{Rushlow, 2009
#10337;Richter, 2003 #11998}
When there is no family history of retinoblastoma, a de novo RB1 germline mutation may arise either
pre- or post-conception. Pre-conception mutagenesis of RB1 usually occurs during spermatogenesis,
perhaps because cell division (and opportunity for mutation) is very active during spermatogenesis, but
not during oogenesis.{Zhu, 1989 #6514;Dryja, 1997 #15586;Munier, 1998 #10955} Advanced paternal
age increases risk for retinoblastoma,{Toriello, 2008 #15506} suggesting that base substitution errors
may increase in aging men. The affected child carries the de novo RB1 mutation in every cell, typically
presenting with 4-5 tumors and bilateral retinoblastoma. In contrast, if mutagenesis occurs post-
conception, during embryogenesis, only a portion (1-50%) of cells carrying the RB1 mutation and the
person will be mosaic for the RB1 mutation. If the mutation arises during retinal development, the child
will have unilateral retinoblastoma.{Dimaras, 2015 #10881}
Q6: Father: So, only RB1 mutation causes retinoblastoma?
A: (Retinoblastoma Expert) “There are two answers to this question: RB1 mutation only causes a
benign precursor to retinoblastoma, retinoma, and other genes are modified to cause progression to
cancer;{Dimaras, 2008 #13250} and 2% of retinoblastoma have normal RB1 and are caused by a different
gene.
In addition to loss of RB1, specific alterations in copy number of other genes are common in RB1-/-
retinoblastoma. There are gains (4-10 copies) in oncogenes MDM4, KIF14 (1q32), MYCN (2p24), DEK
and E2F3 (6p22), and loss of the tumor suppressor gene CDH11 (16q22-24).{Corson, 2007
#9909;Theriault, 2014 #8591} Other less common genomic alterations in retinoblastoma tumors include
8
differential expression of specific microRNAs{Huang, 2007 #8613} and recurrent single nucleotide
variants/insertion-deletions in the genes BCOR and CREBBP.{Kooi, 2016 #14338} In comparison to the
genomic landscape of other cancers, retinoblastoma is one of the least mutated.{Kooi, 2016 #14338}”
There is a newly recognized form of retinoblastoma with normal RB1 genes. Two percent of unilateral
patients have RB1+/+MYCNA tumors, with the MYCN oncogene is amplified (28-121 instead of the normal
2 DNA copies).{Rushlow, 2013 #11102} These children are diagnosed at median age 4.5 months
compared to 24 months for non-heritable unilateral RB-/- patients, and the tumors are distinct
histologically, with advanced features at diagnosis.
Retinoma is a premalignant precursor to retinoblastoma with characteristic clinical features: translucent
white mass, reactive retinal pigment epithelial proliferation and calcific foci.{Gallie, 1982 #10343}
Pathology of retinoma reveals fleurettes{Tso, 1970 #3456} that are not proliferative.{Dimaras, 2008
#13250} Comparison of adjacent normal retina, retinoma and retinoblastoma shows in retinoma loss of
both RB1 alleles and early genomic copy number changes, that are amplified further in the adjacent
retinoblastoma.{Dimaras, 2008 #13250} Many retinoblastoma have underlying elements of retinoma.
Retinoma can transform to retinoblastoma even after many years of stability.{Theodossiadis, 2005
#5578}
Q6: Father: Could we have discovered retinoblastoma earlier?
The only way to find retinoblastoma tumor early is to look with specific expertise, which we can not
for every child. If we know to look because a relative had retinoblastoma, the smallest visible tumors are
round, white retinal lesions that obscure the underlying choroidal pattern. Centrifugal growth results in
small tumors being round; more extensive growth produces lobular growth, likely related to genomic
changes in single (clonal) cells, that provide a proliferative advantage.{Murphree, 2005 #11984;Balmer,
2006 #8323} Next, tumor seeds spread out of the main tumor a result of poor cohesive forces between
tumor cells into the subretinal space, or the vitreous cavity as appearing as dust, spheres or tumor clouds.
{Munier, 2014 #11111} Advanced vitreous tumor seeds can migrate to the anterior chamber producing a
9
pseudo-hypopyon. Enlarging tumor can push the iris lens diaphragm forward causing angle closure
glaucoma. Advanced tumors may induce iris neovascularization. Rapid necrosis of tumor can cause an
aseptic orbital inflammatory reaction resembling orbital cellulitis, sometimes showing central retinal
artery occlusion.{Balmer, 2007 #8320;Balmer, 2006 #8323;Murphree, 2005 #11984} Untreated,
retinoblastoma spreads into the optic nerve and brain, or hematogenous spread occurs through choroid,
particularly to grow in bone marrow. Direct tumor growth through the sclera can present as orbital
extension and proptosis.
The earliest signs of retinoblastoma detectable by parents are leukocorea (white pupil), either
directly or in photographs (photo-leukocorea) and strabismus when the macula is involvement by tumor.
{Balmer, 2007 #8320} In developing countries, buphthalmos and proptosis due to advanced and
extraocular disease respectively is common.{Canturk, 2010 #13461} Less common presentations include;
heterochromia irides, neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.
{Balmer, 2007 #8320} Retinoblastoma (unilateral or bilateral) might be associated with a brain tumor in
the pineal, suprasellar or parasellar regions (Trilateral retinoblastoma){Popovic, 2007 #9156;Antoneli,
2007 #10877} with the median age of diagnsosis 17 months after retinoblastoma and before the age of 5
years. Retinoblastoma might present as 13q deletion syndrome, with facial features and various degrees
of hypotony and mental retardation.{Baud, 1999 #8118;Bojinova, 2001 #13205;Skrypnyk, 2004 #5276}
The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and
ocular toxicariasis.{Balmer, 2007 #8320}
Q7: Do all affected individuals with RB1 mutations develop retinoblastoma?
Each offspring of a person carrying an RB1 mutant gene has 50% risk to inherit the RB1 mutant gene
[Figure # Pedigree – full penetrance]. Nonsense and frame-shift germline mutations, which lead to absent
or truncated dysfunctional pRB, result in 90% bilateral retinoblastoma (nearly complete penetrance).
Often the second mutational event in the retinal cell is loss of the second RB1 allele (LOH, loss of
heterozygosity). For partially functional RB1 mutant alleles, reduced penetrance and expressivity is
10
observed, with later onset and fewer tumors{Soliman, 2016 #15159}, and some carriers never develop
retinoblastoma. Some reduced penetrance mutations reduce RB1 protein expression: (i) mutations in
exons 1 and 2,{Sanchez-Sanchez, 2007 #6108} (ii) mutations near the 3’ end of the gene in exons 24 to
27,{Bremner, 1997 #12040;Mitter, 2009 #7216} (iii) splice and intronic mutations{Zhang, 2003
#8986;Schubert, 1997 #4830;Lefevre, 2002 #4903} and (iv) missense mutations.{Scheffer, 2000
#15178;Cowell, 1998 #10958} Strangely, large deletions encompassing RB1 gene and MED1 gene also
cause reduced expressivity/penetrance, because RB1-/- cells cannot survive in the absence of MED4.
{Dehainault, 2014 #12140;Bunin, 1989 #4280} In comparison, patients with large deletions with one
breakpoint in the RB1 gene typically present with bilateral disease.{Mitter, 2011 #7339;Matsunaga, 1980
#357;Albrecht, 2005 #10898} A measure of expressivity of a mutant retinoblastoma allele is the disease-
eye-ratio (DER) (number of eyes affected with tumor divided by the total number of eyes in carriers of
the mutation).{Lohmann, 1994 #10954}
There are two specific RB1 mutations showing a parent-of-origin effect: intron 6 c.607+1G>T
substitution{Klutz, 2002 #8593;Schuler, 2005 #5551} [Figure # Pedigree – reduced penetrance family
with parent of origin….] and c.1981C>T (p.Arg661Trp).{Eloy, 2016 #12079} Both may be explained by
at differential methylation of intron 2 CpG85, which skews RB1 expression in favor of the maternal allele.
{Buiting, 2010 #7661;Kanber, 2009 #16381} When the allele is maternally inherited there is sufficient
tumor suppressor activity to prevent retinoblastoma development and 90% of carriers remain unaffected.
However, when the p.Arg661Trp allele is paternally transmitted, very little RB1 is expressed, leading
retinoblastoma in 68% of carriers.
Q10: What are the treatments and what govern the choice?
A: “Treatment and prognosis depend on the stage of disease at initial presentation. Factors predictive of
outcomes include size, location of tumor origin, extent of subretinal fluid, presence of tumor seeds and
the presence of high risk features on pathology.{Mallipatna, 2017 #14252} Multiple staging systems have
predicted likelihood to salvage an eye without using radiation therapy, but published evidence is
11
confusing because significantly different versions have emerged.{Dimaras, 2015 #10881;Mallipatna,
2017 #14252} The 2017 TNMH classification is based on international consensus and evidence from an
international survey of 1728 eyes, and separates more clearly initial clinical and pathological features
relevant to outcomes, in retrospective comparison to 5 previous eye staging systems.{Mallipatna, 2017
#14252} (Table X)
Retinoblastoma is the first cancer in which staging recognizes the impact of genetic status on
outcomes: presence of a positive family history, bilateral or trilateral disease or high sensitivity positive
RB1mutation testing, is stage H1; without these features bfore testing blood, HX; and H0 for those
relatives shown to not carry the proband’s specific RB1 mutation.{Mallipatna, 2017 #14252} We propose
H0* for patients with M1 and M2 RB1 mutant alleles of the tumor not detectable in blood, but with
remaining low risk (<1%) of mosaicism.
Choice of treatment depends on the laterality of disease, tumor stage and genetic status. Focal therapy
only can control cT1a eyes, but visually threatening or large cT1b tumors and cT2 eyes need
chemotherapy (systemic or intra-arterial chemotherapy) to reduce the size of the tumor followed by
consolidation focal therapies (laser therapy or cryotherapy) as initial treatment. Enucleation of eyes with
advanced tumors in unilateral disease where the other eye is normal is a definitive cure.{Dimaras, 2015
#10881} Ancillary therapies for specific indications include plaque radiotherapy and periocular
chemotherapy. Intravitreal chemotherapy for vitreous disease has recently dramatically improved safe eye
salvage.{Munier, 2012 #8588;Munier, 2012 #8587} For persons carrying RB1 mutations, external beam
radiation therapy is rarely indicated due to the high risk of inducing later second cancers.{Dimaras, 2015
#10881}
Saving life is the priority of retinoblastoma treatment, followed by vision salvage; the least important
is eye salvage. The child’s job is to play and develop in a healthy life; the many procedures and their
complications that may span years for at best a 50% chance to save a blind eye with risk of tumor spread,
are not justified, especially when the other eye is normal.{Soliman, 2015 #10948;Soliman, 2016 #14269}
12
Q11: Is retinoblastoma lethal?
A: “If untreated, retinoblastoma is lethal. If treated before metastasis occurs, cure is nearly 100%. If
metastasis occurs, the treatment becomes challenging and there is around 40% chance of mortality.
Delayed diagnosis and treatment due to lack of knowledge by ophthalmologists and parents,
socioeconomic{Soliman, 2015 #10948} and cultural factors are major causes of mortality. Asia and
Africa have the highest mortality, with >70% of affected children dying of retinoblastoma, compared
with <5% in developed countries.{Chantada, 2011 #13420;Canturk, 2010 #13461} ibute to reducing
mortality from retinoblastoma.
Germline retinoblastoma carries the risk of development of second primary cancers, most commonly
leiomyosarcoma, osteosarcoma, and melanoma.{MacCarthy, 2013 #11093} Occasionally metastatic
retinoblastoma may confused with a second cancer; blue round cell tumors on cytopathology may not
differentiate from retinoblastoma, but molecular demonstration of the same RB1 mutations as the
intraocular retinoblastoma will confirm metastases.{Racher, 2016 #13990}
Q12: How can we test for retinoblastoma mutations?
A: “If the patient is bilaterally affected, the probability of finding a germline mutation in the RB1 gene in
DNA extracted from blood is high (97% in a comprehensive RB1 laboratory). In 3% of bilateral
retinoblastoma patients, the predisposing RB1 mutation may be mosaic at a low level. Identification of
M1 and M2 RB1 mutations in DNA from tumor lead to identification of a germline mutation.{Astudillo,
2014 #10893;Rushlow, 2009 #10337;Canadian Retinoblastoma, 2009 #14251}
Similarly, to detect the 15% of unilateral patients carrying a germline mutation, optimal strategy is to
first tumor DNA, then check for those mutations in blood. If the blood is not found to carry one of the
tumor RB1 mutations, risk of germline status is reduced to <1% (Table) for parents, siblings and cousins.
{Canadian Retinoblastoma, 2009 #14251}
13
Quality of genetic results depends on quality of DNA. Fresh or frozen tumor samples are fine, but
formalin fixed paraffin embedded tumors generally produce highly degraded DNA. For blood genomic
whole blood in EDTA or ACD, provide high quality DNA.{Banfi, 2007 #15789}
The RB1 gene can be mutated in many ways, best identified by a series of techniques. Single
nucleotide variants (SNVs) and small insertions/deletions can be identified by DNA sequencing (Sanger
dideoxy-sequencing or next-generation sequencing (NGS) methods.{Singh, 2016 #19381;Li, 2016
#19404;Chen, 2014 #19419} The most appropriate technology depends on the clinical question being
asked. NGS may be the most effective screening strategy to investigate for an unknown de novo mutation
in an affected proband, and may have a lower limit of detection (analytic sensitivity) for mosaic
mutations.{Chen, 2014 #14457} To screen family members for a known sequencing-detectable RB1
mutation, targeted Sanger sequencing is 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), quantitative
multiplex PCR (QM-PCR) or array comparative genomic hybridization (aCGH) are used identify RB1
deletions and duplications, and other genomic copy number alterations, such as MYCN amplification.
New developments in bioinformatics analysis suggest that NGS data can be interrogated for copy number
variants,{Devarajan, 2015 #15675;Li, 2016 #19404} but sensitivity is not yet optimized.
Somatic mosaicism can arise in either the presenting patient or their parent. Allele-specific PCR (AS-
PCR) has excellent sensitivity when the RB1 mutation is known,{Rushlow, 2009 #10337} and with
primers specific to the mutation can detect as low as 1% mosaicism.
The second mutational event in 70% of retinoblastoma tumors is loss of heterozygosity (LOH), a
common event associated with loss of the normal allele in tumor from individuals with an inherited
cancer predisposition syndrome.{Cavenee, 1983 #9210} Microsatellite marker analysis is also important
in identity testing and in maternal cell contamination in prenatal diagnostic tests.
14
Epigenetic changes can also initiate retinoblastoma development.{Ohtani-Fujita, 1993 #2258}
Hypermethylation of the RB1 promoter CpG island results in inhibition of RB1 gene transcription in 10-
12% of retinoblastoma tumors, commonly involving both alleles.{Richter, 2003 #11998;Zeschnigk, 1999
#15496} This epigenetic gene silencing event primarily occurs in somatic cells, but heritable RB1
promoter mutations and translocations disrupting RB1 regulatory sites may cause RB1 promoter
hypermethylation.{Quinonez-Silva, 2016 #12111} Occasionally hypermethylation of the RB1 promotor
is found in blood, strongly suggestion the presence of a translocation, for example to the X Chromosome.
In rare instance, no RB1 mutation is identified in the coding, promoter or flanking intronic sequence
in blood from a bilateral patient. Deep intronic sequencing alterations that disrupt RB1 transcription by
interfering with correct splicing in patients with retinoblastomaare best detected by analysis of the RB1
transcript by reverse-transcriptase PCR (RT-PCR).{Zhang, 2008 #7502;Dehainault, 2007 #5872} RNA
studies also clarify pathogenicity of intronic sequence alterations.{Zhang, 2008 #7502;Dehainault, 2007
#5872} As NGS costs continue to decrease, whole genome sequencing (WGS) may 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 rearrangements in
patient’s suspected of 13q14 deletion syndrome.{Caselli, 2007 #15862;Mitter, 2011 #7339} In parents of
13q14 deletion patients, karyotype analysis discover balanced translocations in carriers, informing about
risk of retinoblastoma in subsequent generations.{Baud, 1999 #8118}
Q13: Are these tests available worldwide?
A: “High sensitivity RB1 mutation testing is established in core labs mainly in high-income countries.{,
2015 #10824} In low- and middle-income countries, genetic counseling as a specialty does not exist, so
families may not understand the benefits of genetic testing and counseling in retinoblastoma treatment
and follow-up.{Dimaras, 2015 #10881} In many places, existing health insurance does cover genetic
testing testing. The new Chinese government policy allowing one more child per family will make
15
genetic testing and counseling more important. A novel collaboration between Impact Genetics and
Geneseeq in China is optimizing expertise to achieve high quality RB1 testing for families.
In Egypt,{Soliman, 2016 #14713} Genetic testing for retinoblastoma is not available and genetic
counseling is the only way to address the issues. Ophthalmologists with insufficient training in
retinoblastoma genetics and counseling are burdened with the task. Genetic counseling was found to
increase knowledge in families retinoblastoma genetics, but gaps remained in translation of knowledge
into action.{Soliman, 2016 #14713}
Q14: What is done after finding the RB1 mutation?
A: “Targeted familial testing{Canadian Retinoblastoma, 2009 #14251;Dimaras, 2015 #10881} is used to
determine if a predisposing RB1 mutation has occurred de novo, through investigation of parental DNA
from PBL. Even if neither parent is identified to be a carrier, recurrence risk in siblings is still increased
due to the risk of germline mosaicism. DNA from PBL for all siblings of affected patients should be
tested for the proband’s mutation. As well, DNA from PBL for children of all affected patients should
also be tested for the predisposing mutation. Table Y shows the risk of having retinoblastoma in different
family relatives.
If the proband’s mutation was identified to be mosaic (ie postzygotic in origin) in DNA from PBL,
parents and siblings of the proband are not at risk to carry the predisposing mutation. However, the
children of mosaic proband should be tested, as their risk of inheriting the predisposing RB1 mutation can
be as high as 50% depending on the mutation burden in the probands germline.
When a RB1 mutation has been identified in a family, the known RB1 mutation of the proband can be
tested in his offspring. Couples may consider multiple options with respect to planning a pregnancy.
16
Q15: Can we use the known mutation to test my upcoming child? I am 33 weeks
pregnant
Prenatal genetic testing is usually performed early in the course of the pregnancy and is available in
many countries worldwide. Two early procedures are available: 1) chorionic villus sampling (CVS) and
2) amniocentesis. CVS is a test typically performed between 11-14 weeks gestation during which as
sample of the placenta is obtained either by transvaginal or transabdominal approach. Amniocentesis is a
test performed after 16 weeks of gestation whereby as sample of the amniotic fluid is gathered with a
transabdominal approach. CVS has a procedure-associated risk of miscarriage of ~1%. Amniocentesis
has a procedure-associated risk of miscarriage between 0.1-0.5%. Though uncommon, there is a risk for
maternal cell contamination that occurs more frequently with CVS.{Akolekar, 2015 #9479}
Genetic testing results can be used by the family and health care team to manage the pregnancy. If a
mutation is not identified, the pregnancy can proceed with no further intervention, as there is no increased
risk for retinoblastoma beyond the general population risk. If the mutation is identified, some couples
may decide to stop the pregnancy, while other couples may decide to continue with the pregnancy and
apply appropriate interventions, such as early delivery.{Soliman, 2016 #15159}
Some couples know that they wish to continue their pregnancy regardless of the genetic testing results
and are concerned by the risk of miscarriage associated with early invasive prenatal testing. Where
available, couples can also consider the option of late amniocentesis, performed between 30-34 weeks
gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early
delivery rather than miscarriage.{Akolekar, 2015 #9479} The risk for procedure-associated significant
preterm delivery is low (<3%). Results of genetic testing will be available with enough time to plan for
early delivery when a mutation has been inherited.
In many countries around the world, the option for prenatal genetic testing is not available. Even
where available, some couples may elect to not do invasive testing during the course of the pregnancy.
For these conceptions, if the pregnancy is at 50% risk for inheriting a RB1 mutation, it is crucial that the
17
pregnancy does not go post-dates. Induction of labour should be seriously considered if natural delivery
has not occurred by the due date.{Soliman, 2016 #15159;Canadian Retinoblastoma, 2009 #14251}
Q16: What is the benefit of prenatal mutation detection versus postnatal
screening?
A: “RB1 mutation detection can be performed either prenatal, as discussed previously, or it can be
performed at birth via umbilical cord blood (postnatal screening). This will help either eliminate the 50%
theoretical risk of the proband’s RB1 mutation heritability or confirm it to be 100% risk. Both screening
methods are effective in improving visual outcome and eye salvage compared to non-screened children.
However, prenatal screening allows for planning for earlier delivery in positive children (late
preterm/early term); this was shown to have less number of tumors at birth (20% versus 50%) with only
15% visual threatening tumors in prenatal screening. Prenatal screening with early delivery showed less
tumor and treatment burden with higher treatment success, eye preservation and visual outcome.
{Soliman, 2016 #15159}
Q17: Can we plan our next pregnancy to avoid having this RB1 mutation?
A: “In some countries around the world, there is an in vitro fertilization option available to couples called
preimplantation genetic diagnosis (PGD).{Dhanjal, 2007 #9216;Dommering, 2004 #10248;Xu, 2004
#9246;Girardet, 2003 #9219} 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
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prenatal testing be pursued during the course of the pregnancy to confirm the results.{Dhanjal, 2007
#9216;Dommering, 2004 #10248;Girardet, 2003 #9219;Xu, 2004 #9246}
Q18: what is genetic counseling?
A: “Genetic counseling is both a psychosocial and educational process for patients and their families with
the aim of helping families better adapt to the genetic risk, the genetic condition, and the process of
informed decision-making.{Uhlmann, 2009 #15690;Shugar, 2016 #15715;Shugar, 2016 #15725}. Genetic
testing is an integral component of genetic counseling that results in more informed and precise genetic
counseling. Concrete knowledge of the genetic test outcomes results in specificity, reducing the need for
other possible scenarios to be discussed with the family. This enhances the educational component of
genetic counseling and also provides further time for psychosocial support to be provided to the family.
Q19: Can genetic counseling suffice alone? If yes, what are the benefits of
genetic testing?
A: “In countries where genetic testing is not available or unaffordable, genetic counseling is the option. It
was found that genetic testing is more cost effective than examining all the at-risk family members.
Patients with bilateral retinoblastoma at presentation are presumed to have heritable retinoblastoma and a
RB1 mutation (H1 in the TNMH classification). Genetic testing provides (1) more accurate information
about the type of heritable retinoblastoma and allows for straightforward testing to determine if additional
family members are at risk. (2) Through genetic testing, a patient may be found to have a large deletion
extending beyond the RB1 gene as part of the 13q deletion spectrum. Individuals with 13q deletion
syndrome are at risk for additional health concerns requiring appropriate medical management and
intervention. (3) Results may reveal a mosaic mutation which indicates that the mutation is definitively de
novo; only the individual’s own children are at risk and no further surveillance or genetic testing is needed
for other family members. (4) The results may find a low-penetrance mutation which indicates the patient
is at reduced risk to develop future tumours. As genetic testing for retinoblastoma becomes more common
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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 laboratories focused on enhanced detection of RB1 mutations are able to identify
nearly 97% of all retinoblastoma mutations. Genetic testing of the patient’s blood is sensitive enough
when thorough methods are used that not finding a mutation results in a residual risk of heritable
retinoblastoma low enough to remove the need for examinations under anesthesia. This reduces the health
risk for the patient and the cost to the health care system. Testing is even more accurate when a tumour
sample is collected and tested when available. When mutations are identified in the tumour and are
negative in blood, the results can eliminate the need for screening of family members and provide
accurate testing for the patient’s future children. Whether or not a tumour sample is available, finding a
RB1 mutation in a patient’s blood confirms that this patient has heritable retinoblastoma. This patient now
benefits from increased surveillance designed to detect tumours at the earliest stages and awareness of an
increased lifelong risk for second primary cancers. Members of the patient’s family can have appropriate
genetic testing to accurately determine who is at risk. As with patients with bilateral retinoblastoma,
knowing the specific type of mutation provides the most detailed provision of medical management and
counselling.
Q20: When is the appropriate timing for collecting samples for genetic testing?
For blood samples, they can be collected at any time but preferably when the child is under EUA where
there is no fear from the needle prick. For tumor samples, they would be collected from the enucleated
eye just after enucleation. Tumor cells will be preserved in a specific transport medium that allows the
cells to grow. We can also freeze some tumor cells (cryopreservation) for future necessity or for research
purposes.
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Q21: If we know the mutation prenatally, is there any treatment to prevent
retinoblastoma from occurring?
A: “
Retinoblastoma genetics is challenging to understand, but once understood It largely affect the level
of care presented to retinoblastoma patients and their families. It helps alleviate the psychological burden
of the families regarding moving forward with their life choices regarding the affected child and future
siblings. It also helps the family to understand the risks of different family members giving them the
chance of the level of disclosure they wish.
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REFERENCES
Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-
Blackwell.
Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling
Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of print.
PMID: 27891554 DOI: 10.1007/s10897-016-0042-y
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Table X:
Subretinal Fluid (RD)
No≤ 5 mm
>5 mm - ≤ 1 quadrant
> 1quadrant
Tum
or
Tumors ≤ 3 mm and further than 1.5 mm from the disc and fovea cT1a/A cT1a/B cT2a/C cT2a/D
Tumors > 3 mm or closer than 1.5 mm to the disc and fovea cT1b/B cT1b/B cT2a/C cT2a/D
Se
edin
g Localized vitreous/ subretinal seeding cT2b/C cT2b/C cT2b/C cT2b/Ddiffuse vitreous/subretinal seeding cT2b/D
High
risk
feat
ures
Phthisis or pre-phthisis bulbi cT3a/ETumor invasion of the pars plana, ciliary body, lens, zonules, iris or anterior chamber cT3b/ERaised intraocular pressure with neovascularization and/or buphthalmos cT3c/EHyphema and/or massive vitreous hemorrhage cT3d/EAseptic orbital cellulitis cT3e/EDiffuse infiltrating retinoblastoma ??/E
Extraocular retinoblastoma cT4/??
clinical T (cT) versus International Intraocular retinoblastoma Classification (IIRC) (cT/IIRC); ?? Not
applicable ; RD Retinal detachment
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