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 Heather MacDonald, 1 Brenda L. Gallie, MD. 1,5 Affiliations: 1 Department of Ophthalmology and Vision Sciences, University of Toronto, Ontario, Canada 2 Department of Ophthalmology, Faculty of Medicine, University of Alexandria, Alexandria, Egypt. 3 Impact Genetics, Bowmanville, Ontario.
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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 Heather MacDonald,1 Brenda L.
Gallie, MD.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: +1-416-294-9729
Gallie Brenda, 01/05/17,
Organizing Text: Number the pages of the manuscript consecutively, beginning with the introduction as page 1. The text of an original article should not exceed 4,000 words with up to 8 images and tables and 50 references while that of a review article should not exceed 6,000 words with up to 8 images and tables and 100 references. The text of an annual review should not exceed 15,000 words with up to 200 references.
Gallie Brenda, 01/05/17,
Title page: Include on the title page (a) complete manuscript title; (b) authors’ full names, highest academic degrees, and affiliations; (c) name and address for correspondence, including fax number, telephone number and email address; (d) address for reprints if different from that of corresponding author; and (e) sources of support that require acknowledgement.
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. …..highlight the new things….The goal of this article is to simplify the concepts of
retinoblastoma genetics for ophthalmologists to assist in the care of patients and their families.
4
Gallie Brenda, 01/05/17,
Review articles should emphasize new developments and areas of controversy in clinical or laboratory ophthalmology. An unstructured abstract of no more than 250 words should be submitted on a separate page.
4134/5000 words
94/80 references
table x cTNMH
table x risk assessment derived from impact
table or discussion for surveillance for eye and lifetime
figure: small tumors and OCT image vs large tumors without surveillance
figure: pedigree: F445 parent of origin and lp
figure: pedigree potentially siotas?
Figure: potentially update to second cancers for Ramsey?
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)
{Seregard, 2004 #10380;Dimaras, 2015 #10881} but most have no access to knowledge of the important
role genetics plays in 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, lead 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, is lost from a developing retinal cell in babies and young children. Retinoblastoma can
5
Gallie Brenda, 01/09/17,
We have not included floolowup for the eyes OR surveillance for second cancers. TABLES etc and genetic counselingSameh: I added a question.
Gallie Brenda, 01/05/17,
unfinished
Sameh Gaballah, 01/09/17,
I added a question regarding follow up.
Gallie Brenda, 01/05/17,
Delete redundant or not so important ones
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, particularly to bone marrow, which will result in
death.
“How can this cancer show up at such a young age?”
The cell of origin of retinoblastoma is most likely a developing cone photoreceptor precursor cell that has
lost both copies alleles of the RB1 tumor suppressor gene, and remains in the inner nuclear layer of the
retina, , perhaps because it is unable to migrate to the outer retina and function normally.{Dimaras, 2015
#10881;Rootman, 2013 #11096;Xu, 2014 #9924} The susceptible cell susceptible to become 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 who likely there was previously had an
undetected small tumor (retinoma) present from childhood, that later became active.{Gallie, 1982
#10343;Dimaras, 2008 #13250} The mean age at presentation is 12 months in bilateral disease and 24
months in unilateral disease, in high-income countries, but is significantly delayed in low-income
countries.{Nyamori, 2012 #8389}
“What causesd 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 RB1 gene is
damaged in most, or all, normal cells of the patient. 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 retinoblastoma protein (pRB), an important regulator of cell division cycle in most cell
types, and the first tumor suppressor gene discovered.{Friend, 1986 #10882} Normally, dephosphorylated
pRB represses expression of the E2F gene, thereby blocking cell division.{Nevins, 2001
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}
However, often missing from choices in the complex care of children with retinoblastoma are the
truly informed parents. Essentially, the doctors decide, based on very little evidence, what treatment they
“feel” is best. There exists no easy way to show the parents prospectively the true “costs” of each
treatment: the burden of invasive therapies and potential complications; 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, uncertainties; and the burden of “false hope” in the absence of real
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evidence. There are imminent solutions on the horizon, such as eCancerCare encompassing the whole
medical record for a lifetime with retinoblastoma, viewable on line by the family and patient, and the
burgeoning field of patient reported outcomes. These new attitudes and tools may empower in the future
good choices by parents for their child and family.
“Is retinoblastoma lethal?”
If Untreated, retinoblastoma is lethal. If treated before metastasis occurs, cure is nearly 100%. Globally,
the chance for cure is even more remote, and lack of knowledge of genetics results too often result in
death of many children who could have been saved if they had surveillance and definitive treatment when
tumors were small. If metastasis occurs, the treatment becomes challenging and there is around 40%
chance of mortality despite best current therapies. 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} Broad understanding of retinoblastoma genetics and genetic counseling can will contribute
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} When Only after 30 years of
being used on every child, the enormous impact of external beam irradiation was recognized: after 30
years of being used on every child, it was discovered that more children with bilateral retinoblastoma who
were treated with radiation, (H1) were dyingdie of their second (third, etc) cancer than of retinoblastoma.
{Eng, 1993 #10933;}{Temming, 2016 #14267}
Occasionally metastatic retinoblastoma may be 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}
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Gallie Brenda, 01/09/17,
This whole nice little “editorial” does not really belong in this review of genetics…..?????Sameh: I agree. What about writing a simple paragraph saying that unilateral non germline advanced is better enucleated for cure and tumor sample. And bilateral (germline) require more treatments to preserve as much vision as we can. I can draft that if you all agree.
“How can we test for retinoblastoma mutations?”
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 cannot be detected. 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.{Astudillo, 2014 #10893;Rushlow, 2009
#10337;Canadian Retinoblastoma, 2009 #14251}
Similarly, to detect the 15% of unilateral patients carrying a germline mutation, the optimal strategy is
to first test tumor DNA, and then look for these 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}
Quality of genetic results depends on quality of DNA. Fresh or frozen tumor samples are ideal, 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 forfind 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 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), quantitative
multiplex PCR (QM-PCR) or array comparative genomic hybridization (aCGH) are used to identify RB1
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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 #9654;Li, 2016 #14646} 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 can detect
mutations 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} Polymorphic microsatellite markers distributed
throughout chromosome 13 can be used to detect a change from a heterozygous state in blood compared
to the homozygous state in a tumor with LOH. Microsatellite marker analysis is also important in identity
testing and in maternal cell contamination in prenatal diagnostic tests.
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 or translocations involving the X
chromosome, have been shown to cause constitutional RB1 promoter hypermethylation.{Jones, 1997
#16167;{Quinonez-Silva, 2016 #12111}{Jones, 1997 #16167} (Jones et al 1997 PMID: 9199583)
Rarely, 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 retinoblastoma can be 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
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NGS costs continue to decrease, whole genome sequencing (WGS) 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 rearrangements in
retinoblastoma patients, including patient’s suspected of 13q14 deletion syndrome.{Caselli, 2007
#15862;Mitter, 2011 #7339} In parents of 13q14 deletion patients, karyotype analysis can be used to
investigate for balanced translocations, which increases the risk for retinoblastoma in subsequent
generations.{Baud, 1999 #8118}
“What is done after finding the RB1 mutation?”
Family members at risk to also carry the identified RB1 mutation are offered testing on blood samples
(Table from impact…).{Canadian Retinoblastoma, 2009 #14251;Dimaras, 2015 #10881} If the mutation
is found in neither parent, a small risk for low level mosaicism still exists, leaving a low level risk for
siblings. Offspring of any family member carrying the RB1 mutation can be tested during pregnancy or
immediately after birth (see below). If the proband is mosaic for the RB1 mutation, parents and siblings of
the proband are not at risk, since mosaicism cannot be inherited. However, the children of a mosaic
proband needs to be tested as early as possible, as their risk to inherit the predisposing RB1 mutation is up
to 50%; if they do carry the mutation, they are at population risk for bilateral retinoblastoma.
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“How does the RB1 status affect follow up of the child?”
If the child doesn’t carry an RB1 germline mutation, no EUAs isare required for the other eye with
fFollow-up only in clinic. If the child carries a germline mutation or is mosaic for the RB1 mutation, the
possibility of new tumor s development renders frequent EUAs essential for ocular follow up to at least 3
years of age.{Valenzuela, 2010 #10009} The frequency depends on degree of ocular involvement and
duration of stability after last treatment. The germline status also predisposes to other second malignant
neoplasms, (SMNs), requiring frequent annual surveillance with the pediatric oncologist. The use of
whole body MRI as a method for surveillance is controversial as a high false positive rate it might still
involveprovoke moreunnecessary invasive procedures due to a higher false positive rate. Development of
surveillance programs beyond the pediatric age is essential for early detection of these often lethal second
RB1 induced cancersSMNs.{Temming, 2016 #14267}
“Can we use the known mutation to test my future children?”
Prenatal genetic testing is can be performed in the course of the pregnancy. Two early procedures are
available: i) chorionic villus sampling (CVS) performed between 11-14 weeks gestation, which involves
obtaining a sample of the placenta either trans-vaginally or trans-abdominally; and ii) amniocentesis after
16 weeks gestation, which involving amniotic fluid trans-abdominally. The procedure-associated risk of
miscarriage of CVS is 1%, while amniocentesis is 0.1-0.5%. Maternal cell contamination is more
frequently with CVS,{Akolekar, 2015 #9479} and is assessed for by the clinical molecular lab.
If the fetus does not carry the mutation, the pregnancy can proceed with no further intervention. If the
fetus carries the familial mutation, the parents have several choices. Some may decide to stop the
pregnancy, while others may know they wish 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.{Akolekar, 2015
#9479}. Prenatal or postnatal RB1 mutation testing will either show the baby to be “H0” (for the family
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RB1 mutation) or “H1”, confirmed to carry the mutation. If the fetus has the familial RB1 mutation, early
pre-term delivery achieves smaller tumors and with higher treatment success, eye preservation and visual
outcome than delivery at full term.{Soliman, 2016 #15159}
In many countries, the option for prenatal genetic testing is not available, and some parents may
choose to not do prenatal invasive testing. If the risk for retinoblastoma in the fetus is 50% it is important
that the pregnancy does not go past 40 weeks.{Soliman, 2016 #15159;Canadian Retinoblastoma, 2009
#14251}
Can we plan our next pregnancy to passing on this RB1 mutation?
In some countries, preimplantation genetic diagnosis (PGD) with in vitro fertilization is an option.