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Format of the review article:
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Genetics and Molecular Diagnostics in
Retinoblastoma - An Update
Authors:
Sameh E. Soliman, MD,1-2 Hilary Racher, PhD,3 Chengyue Zhang, MD,4
Chengyue Zhang, MD.
Hilary Racher, PhDHeather MacDonald,1,5 Brenda L. Gallie.1,6
2Affiliations:
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.
4 Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University.
5 Heather affiliation??
6 Brenda affiliation.
2Impact Genetics, Bowmanville, Ontario.Corresponding author:
Brenda L. Gallie. Address: 525 University Ave, room 806, Toronto, Ontario, Canada. M5G 2L3.
Telephone: +1 xxxxx, email: brenda@gallie.ca
Disclosures:
Both SS and HR contributed equally to this review and would be considered as first co-authers.
We confirm that this manuscript has not been and will not be submitted elsewhere for publication, and
all coauthorsco-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.
No authors have any financial/conflicting interests to disclose.
This paper received no specific grant from any funding agency in the public, commercial or not-for-
profit sectors.
Word Count: (/5000)
Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic
counselingcounselling, prenatal screening.
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Unstructured abstract
Abstract: (120/250)
Retinoblastoma is an intraocular genetic malignancytumor that might affects one or both eyes of
young children, that is and initiated by biallelic mutation of the retinoblastoma gene (RB1) in a single
precursordeveloping retinal cell. that affects the eye(s) of a child; but the physician deals with the whole
family regarding risks and possibilities. Good Uunnderstanding of rRetinoblastoma genetics is crucial in
providing not onlysupports optimalstandard of care for retinoblastoma children and their families. but
also risk foreseeing and genetic counseling for and their families. In this scenario the genetics trait
description was conducted by the based conversation betweenconversation between a family with a
retinoblastoma child and their treating attendingphysician who is mostly the ophthalmologist but can be
any member of the retinoblastoma multidisciplinary team of physicians, nurses and genetic counselors.
All the questions are true and high frequently askedcommon questions that all ocular oncology physiains
face on regular basisby the parents. The main aim of Tthis scenario aimsis to try to simplify the
information around genetics for ophthalmologists to help them improve their patient and family care.
mmmmmmm
Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic counseling
prenatal screening
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5752/56000 words
INTRODUCTION [JEFFRY]
Retinoblastoma is the most common childhood intraocular malignancy in childhood that might affects
one or both eyes.{Dimaras, 2015 #10881} It is considered the prototype of geneticheritable cancers.
{Theriault, 2014 #8591}It Tumors are is initiated by biallelic mutation of the retinoblastoma tumor
suppressor gene (RB1) in a single precursor retinal cell. The first RB1 mutation is present in constitutional
RB1 mutationcells in nearly 50% of patients, who are thereby predisposeds individuals to developing
retinoblastoma that forms after the second RB1 allele is damaged in a somatic mutationcell.{Corson, 2007
#12275;Dimaras, 2012 #8709}. The incidence of retinoblastoma is constant at one case in 165,000-
1820,000 live births, translating to about 89,000 new cases per year worldwide.{Seregard, 2004
#10380;Dimaras, 2015 #10881} UnderstandingThe genetics of retinoblastoma genetics is crucial in
multipleunderlies many aspects of retinoblastoma: such as clinical presentation, choice of treatment
modalitiesy and follow-up for both the child and his/her family. Many Multiple reviews{Dimaras, 2015
#10881;Theriault, 2014 #8591} had described the genetics research advancement of retinoblastoma from
different aspects in depth respectively. In this review We will try tonow highlightaddress the
understanding of retinoblastoma from genetic clinical analysis and research, in the context of individual
children and families. most of the updates on the genetic aspect of retinoblastoma in a clinical scenario
setting that might simplify theseis new advancementsaspects to ophthalmologists all over the world.
Case Scenario: A 2 years old female childgirl presented with left leukocorea (white pupil). The family
noticed the white pupil atin a family photograph 5 days agoearlier. They sought medical advise to their
family physician who suspected retinoblastoma and referred them urgently to the pediatric
ophthalmologist. The family history is irrelevanthad never before heard of retinoblastoma, and the mother
iswas 33 weeks pregnant. The child was extremelyvery uncooperative but the ophthalmologist was able to
visualize a white retinal mass in the left eye. He couldn’t examine exceptcould only see the inferior retina
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, and an intact optic nerve and fovea in the right eye that was apparently free. The diagnosis of
retinoblastoma was made and the following discussion took place between the ophthalmologist and the
family.
Q1: Father: What is retinoblastoma?
A: “Retinoblastoma is a malignant tumorcancer that arises from a developing retinal cell. The exact cell
of origin is most likely a unknown but there are many theories suggesting either a conephotoreceptor
photoreceptor precursor cell orthat lost both copies of the RB1 tumor suppressor gene, and remains in the
an 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} cell origin. The visualization of early tumors by
optical coherence tomography (OCT) supports the later but not yet proven. Retinoblastoma can affect one
(unilateral) or both eyes (bilateral) and in rare instances (<51% of children) might beis associated with a
midline brain tumor in the pineal region regardless of the laterality of ocular involvement(trilateral).{de
Jong, 2014 #10885} Without timely and suitableeffective treatment, the aggressive tumorretinoblastoma
maymay spread through optic nerve to the brain, or hematogenousvia blood route into brain orparticularly
to bone marrow, which will result in death of the patient in the end.”
Q2: Father: why it is presenting in such a young age?
A: “Retinoblastoma arises fromThe cell of origin of retinoblastoma is a developinging cell, only present
ins that are present in the retinase of young children, from the intrauterine lifefrom before birth, up to
around 7 years of age. It is believed that all retinal cells are developed by this age. Rarely, retinoblastoma
developsis first diagnosed in older agespersons, 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 around 1 year in bilateral disease and 2 years in unilateral
disease.
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For your daughter, despite we can see tumor in only one eye by clinical examination, we cannot be
sure about the other eye without an examination under anesthetic (EUA) and proper eye examination with
fundus imaging and OCT.”
Q3: Mother: What caused retinoblastoma? What do you mean that it is genetically caused?
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.
“Tumors are initiated by biallelic mutation of the retinoblastoma tumor suppressor gene (RB1) in a
precursor retinal cell. The first RB1 mutation is present in constitutional cells in nearly 50% of patients,
who are thereby predisposed to developing retinoblastoma after the second RB1 allele is damaged in a
somatic cell.{Dimaras, 2015 #10881} The RB1 gene, located on chromosom13q14, encodes the RB
protein (pRB), an important cell cycle regulator and the first tumor suppressor gene discovered.{Friend,
1986 #19025} After a cell completes mitosis, the pRB protein is dephosphorylated, permitting it to bind
to the promoter region of the E2F transcription factor gene, thereby repressing transcription and inhibiting
the progression of the cell cycle from G1 to S phase.{Nevins, 2001 #15292;Cobrinik, 2005 #15298;Sage,
2012 #19061} In order for the cell to enter S phase, cyclin-dependent kinases phosphorylate RB, which
removes the ability of pRB to bind to the E2F gene promoter.{Knudsen, 2008 #15310} pRB functions to
regulate proliferation in most cell types.{Cobrinik, 2005 #15298} Often, loss of RB1 is compensated by
increased expression of its related proteins, however, in certain susceptible cells, such as the retinal cone
cell precursors, compensatory mechanisms are not sufficient and tumorigenesis is initiated.{Xu, 2014
#19253}
Q4: What causes retinoblastoma to be unilateral versus bilateral?
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A: “In most casesThe concept of , retinoblastoma developments whenafter inactivation of both RB1 gene
copies of the RB1 gene are inactivated. This concept was first formulated in 1971, when Knudson used
retinoblastoma as the prototypic cancer to derive the two-hit hypothesis.{Knudson, 1971 #11106} In
heritable retinoblastoma (sometimes called germline retinoblastoma), the first mutational event is
inherited via the germinal cells, while the second event occurs in the somatic cells. In non-heritable
retinoblastoma, both mutation events occur in the somatic cells. Heritable retinoblastoma encompasses
45% of all reported cases.{MacCarthy, 2009 #8367;Moreno, 2014 #18928;Wong, 2014 #15170} The
clinical presentation of heritable retinoblastoma consists of 80% bilateral and 15-18% unilateral.
{Dimaras, 2015 #10881} In non-heritable retinoblastoma (non-germline retinoblastoma) the majority
(98%) of cases have somatic biallelic RB1 loss in the tumor, while the remaining 2% have no mutation in
either copy of RB1 but instead have somatic amplification of the MYCN oncogene.{Rushlow, 2013
#11249} 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: “There are many causes in the environment that can cause thisDNA mutations including cosmic rays,
X-rays, DNA viruses, UV irradiation and irradiation????. This is sporadic and cannot be anticipated
or prevented. There are many ways in which the function of the pRB is impaired including point
mutations, small and large deletions, promotor methylation and chromothripsis.{Lohmann, 1999
#19258;McEvoy, 2014 #19260} The majority of RB1 mutations are de novo, unique to a specific patient
or family, however, there are some known recurrent mutations found across many unrelated individuals.
One subset of recurrent mutations involve 11 CpG sites, which make up ~22% of all RB1 mutations.
{Rushlow, 2009 #10337} The high recurrence of nonsense mutations at these sites is due to the
hypermutabilty and subsequent deamination of 5-methylcytosine.{Richter, 2003 #11998}
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The origin of a de novo RB1 mutation can arise either pre- or post-conception. Most often, pre-
conception mutagenesis occurs during spermatogenesis.{Dryja, 1997 #15586;Munier, 1998 #10955}
Furthermore, advanced paternal age has been shown to increase risk for retinoblastoma.{Toriello, 2008
#15506} This might be due to the larger number of cell divisions during spermatogenesis than oogenesis
or the increased rate for base substitution errors in aging men compared to women. In cases of pre-
conception mutagenesis, the proband carries the de novo RB1 mutation in every cell within their body and
typically presents with bilateral retinoblastoma. In contrast, post-conception RB1 mutagenesis occurs
during embryogenesis. Depending on the embryological stage of development, a few or numerous tissues
may be mosaic for the RB1 mutation. If the mutational event occurs during retinal development, the
presentation is often unilateral retinoblastoma.{Dimaras, 2015 #10881}
Q6: Father: So, only RB1 mutation is sufficient forcauses retinoblastoma to develop?
A: “I just suspect that this professional question can be asked by the parent?????Why not change it as ‘Is
there any new findings about the tumorigenesis?’
Both RB1 mutations are essential but insufficient to develop retinoblastoma evidenced by biallelic RB1
loss in the benign retinoma;{Dimaras, 2008 #11248}.suggesting more genetic or epigenetic changes for
malignant transformation.
In a small subset (2%) of unilateral patients, no RB1 mutation is identified. Instead, striking amplification
(28-121 copies) of the MYCN oncogene is detected.{Rushlow, 2013 #11249} Patients with RB1+/+
MYCN are clinically distinct from RB-/- patients, showing much younger age at diagnosis, distinct
histological features and larger, more invasive tumors. In addition to loss of RB1 or MYCN amplification,
specific somatic copy number alterations commonly occur in the progression of the retinoblastoma.
Commonly seen are gains in 1q32, 2p24, 6p22 and losses at 13q and 16q22-24.{Corson, 2007 #9909}
These regions contain important oncogenes (MDM4, KIF14, MYCN, DEK and E2F3) and tumor
suppressor genes (CDH11), thought to act as drivers promoting the growth of the cancer.{Theriault, 2014
#19306}
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Other less common alterations that have been identified in retinoblastoma tumors include differential
expression of some microRNAs{Huang, 2007 #19315} and recurrent single nucleotide variants/insertion-
deletions in the genes BCOR and CREBBP.{Kooi, 2016 #19325} In comparison to the genomic landscape
of other cancers, retinoblastoma is one of the least mutated.{Kooi, 2016 #19325}
Q7: What is the retinoma that you mentioned and how does it differ from retinoblastoma?
A: “Retinoma is a premalignant precursor with characteristic clinical features: translucent white mass,
reactive retinal pigment epithelial growth and calcific foci.{Gallie, 1982 #10343} Pathology of retinoma
reveals fleurettes structures that are not proliferative. Genetic analysis of retinoma and adjacent normal
retina and retinoblastoma shows loss of both RB1 alleles, and early genomic copy number changes that
are amplified further in the adjacent retinoblastoma.{Dimaras, 2008 #11248} It can transform to
retinoblastoma even after many years of stability.{Theodossiadis, 2005 #5578}
Retinoblastoma starts as a rounded white retinal mass that gradually increases in size. Centrifugal tumor
growth results in small tumors being round; more extensive growth produces lobular growth, likely
related to genomic changes in single (clonal) cells, that provide a proliferative advantage.{Murphree,
2005 #11984;Balmer, 2006 #8323} Tumor seeds float free of the main tumor into the subretinal space or
the vitreous cavity as a result of poor cohesive forces between tumor cells, appearing as dust, spheres or
tumor clouds.{Munier, 2014 #11111} Advanced vitreous tumor seeds can migrate to the anterior chamber
producing a pseudo-hypopyon. Enlarging tumor can push the iris lens diaphragm forward causing angle
closure glaucoma. Rapid necrosis of tumor can cause an aseptic orbital inflammatory reaction resembling
orbital cellulitis, sometimes showing central retinal artery occlusion.{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.
Q8: Doeos all affected individuals with RB1 mutations develop retinoblastoma?
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In heritable retinoblastoma, each offspring of a patient has a 50% risk of inheriting the RB1
pathogenic change. Typically, nonsense and frame-shift germline mutations, which lead to absence of
RB1 expression or truncated dysfunctional RB protein, show nearly complete (90%) penetrance. Often
the second mutational event in the retinal cell is loss of the second RB1 allele (LOH, loss of
heterozygosity). In these families the presentation is typically unilateral multifocal or bilateral
retinoblastoma. In a smaller subset of hereditary retinoblastoma, reduced expressivity and reduced
penetrance is observed. In these families, when retinoblastoma develops, it is often late onset and less
severe, presenting as unilateral, unifocal (reduced expressivity) and in some carrier family member
retinoblastoma never develops (reduced penetrance). The types of reported RB1 mutations that result in
reduced expressivity or penetrance are diverse. Many consist of mutations that reduced RB1 protein
expression. Examples include, (1) mutations in exons 1 and 2,{Sanchez-Sanchez, 2007 #18933} (2)
mutations in exons 26 and 27,{Mitter, 2009 #7347} (3) intronic mutations{Schubert, 1997
#18936;Lefevre, 2002 #18938} and (4) missense mutations.{Scheffer, 2000 #15178;Cowell, 1998
#18940} In addition, large deletions encompassing RB1 gene and MED1 gene cause reduced
expressivity/penetrance.{Dehainault, 2014 #18941;Bunin, 1989 #18950} Dehainault et al showed that
RB1-/- cells cannot survive in the absence of MED4. This can explain why patients with 13q14 deletion
syndrome more often have unilateral tumors, in comparison to patients with gross deletions with one
breakpoint in the RB1 gene whom typically present with bilateral disease.{Mitter, 2011
#15255;Matsunaga, 1980 #19020;Albrecht, 2005 #19022} The severity of risk can be evaluated through
the disease-eye-ratio (DER) calculated by taking the number of eyes affected with tumors divided by the
total number of eyes of carriers within the family.{Lohmann, 1994 #19003}
In some instances of hereditable reduced expressivity/penetrance retinoblastoma, the parental origin
impacts whether or not an individual develops retinoblastoma and subsequently whether their carrier
offspring are at risk to develop retinoblastoma, a phenomenon termed the parent-of-origin effect.{Klutz,
2002 #19004;Schuler, 2005 #19011;Eloy, 2016 #19015} Eloy et al{Eloy, 2016 #19015} proposed a
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potential molecular mechanism to explain the parent-of-origin effect. Using the c.1981C>T
(p.Arg661Trp) reduced penetrance/expressivity missense mutation,mutation; the researchers discovered
that differential methylation of the intron 2 CpG85 skews RB1 expression in favor of the maternal allele.
In other words, when the p.Arg661Trp allele is maternally inherited there is sufficient tumor suppressor
activity to prevent pRB development and 90.3% of carriers remain unaffected. However, when the allele
is paternally transmitted, very little RB1 is expressed, leading to haploinsufficiency and pRB development
in 67.5% of cases. A similar inheritance pattern was also reported for intron 6 c.607+1G>T substitution.
{Klutz, 2002 #19004}
Q9: Mother: could we have discovered it earlier?
I do not think this paragraph answer the above question exactly. Why not combine this question with
Question 16??????
A: “Leukocorea (white pupil) is the main clinical presentation usually detected by parents either directly
or in photographs (photo-leukocorea). Strabismus due early macular involvement is the second most
common.{Balmer, 2007 #8320} In developing countries, buphthalmos and proptosis due to advanced and
extraocular disease respectively represents a higher percentage.{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} that starts early; with the median age of onset 17 months
after retinoblastoma is diagnosed and before the age of 5 years. Retinoblastoma might present in a
syndromic form (13q deletion syndrome) associated with some facial features as high and broad
forehead, thick and everted ear lobes, short nose, prominent philtrum and thick everted lower lip, bulbous
tip of the noseassociated with various degrees of hypotonea and mental retardation.{Baud, 1999
#18925;Bojinova, 2001 #18926;Skrypnyk, 2004 #15166} The main differential diagnosis includes Coats’
disease, persistent hyperplastic primary vitreous and ocular toxicariasis.{Balmer, 2007 #8320}
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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; the International Intraocular
Retinoblastoma Classification (IIRC){Murphree, 2005 #11984} has been recently the most reliable, but
published evidence is confusing because significantly different versions have emerged.{Dimaras, 2015
#10881} The 2017 TNMH classification is based on international consensus and evidence from an
international survey of 1728 eyes, with algorithms evaluating initial features and outcomes by 5 different
eye staging systems.{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 H1; without these features
or testing of blood, HX; and H0 for those relatives who are shown to not carry the proband’s specific RB1
mutation.{Mallipatna, 2017 #14252} We propose H0* for patients with 2 RB1 mutant alleles in blood that
are not detectable in blood, reducing risk of a heritable RB1 mutation to <1%.
Multiple treatments are now available and the choice depends on the laterality of disease and the
grouping of the tumor. Chemotherapy (systemic or intraarterial chemotherapy) to reduce the size of the
tumor followed by consolidation focal therapies (Laser therapy or cryotherapy) is the main stay of
treatment. Enucleation for eyes with advanced tumors or in unilateral disease where the other eye is
normal is more appropriate and definitive. Other therapies include; intravitreal chemotherapy for vitreous
disease, plaque radiotherapy or periocular chemotherapy. External beam radiation therapy has extremely
limited indications nowadays due to its extensive cancer risks and complications.{Dimaras, 2015
#10881}
The main concept of treatment is that life salvage is the main priority during treatment planning
followed by vision salvage and the least important is eye salvage. That’s why we prefer enucleation in
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advanced unilateral intraocular retinoblastoma with low visual potential. The child’s job at this point is to
play and enjoy a healthy life away of all the procedures and their complications that may span over a
couple of years for a 50% chance to save a blind eye and risk of tumor spread.{Soliman, 2015 #10948;
{Soliman, 2016 #18559}}
Q11: Is retinoblastoma lethal?
A: “If untreated, retinoblastoma is lethal. If treated before metastasis occurs, there is a nearly a 100%
chance of life salvage. If metastasis occurs, the treatment options becomes more challenging but there is a
40% chance of mortality related to retinoblastoma. Delayed diagnosis and treatment due to lack of
retinoblastoma knowledge by ophthalmologists and parents, socioeconomic{Soliman, 2015 #10948} and
cultural factors are major causes of high 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} Delayed diagnosis and treatment due to lack of
retinoblastoma knowledge by ophthalmologists and parents, socioeconomic56 and cultural factors are
major causes of high mortality. Broad understanding of retinoblastoma genetics and genetic counseling
can contribute to reducing mortality from retinoblastoma.
Germline retinoblastoma carryretinoblastoma carries the risk of development of second primary
cancers, most commonly osteosarcoma and fibrosarcoma. Sometimes it might be confused with
metastatic retinoblastoma. Fine needle aspiration cytopathology has minimal role in differentiation as
both the metastasis from and second cancers that appear as blue round cell tumors. Mmolecular analysis
might help to distingguishfferentiate.{Racher, 2016 #13990}
Q12: How can we test for retinoblastoma mutations?
A: “The most optimal strategy for retinoblastoma molecular genetic testing is guided by the patient’s
tumor presentation. If the patient is bilaterally affected, the probability of finding a germline mutation in
the RB1 gene is high (example - 97% detection rate in comprehensive laboratory). For this reason, the
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most optimal strategy for testing bilateral patients involves first testing genomic DNA extracted from
peripheral blood lymphocytes (PBL). In rare instances of bilateral retinoblastoma, the predisposing RB1
mutation has occurred sometime during embryonical development. In these cases, the RB1 mutation may
only be present in some cells and may not be detected in DNA from PBL. Therefore, in the event that no
mutation is identified in the blood of a bilaterally affected patient, DNA from tumor should be
investigated.{Canadian Retinoblastoma, 2009 #14251}
In contrast, given that approximately 15% of unilateral patients carry a germline mutation, the most
optimal strategy is to first test DNA extracted from a tumor sample. Upon identification of the tumor
mutations, targeted molecular analysis can be performed on DNA from PBL to determine if the mutation
is present is the patient’s germline. When only the tumor is found to carry the RB1 mutations, this result
dramatically reduces the risk of recurrence in siblings and cousins. In addition, this targeted approach can
allow for a more sensitive assessment of the PBL DNA, which can be useful in the detection of low level
mosaic mutations, more common in unilateral cases.{Canadian Retinoblastoma, 2009 #14251}
Sample preparation impacts the quality of DNA. For best results, fresh or frozen tumor samples
should be collected, as opposed to formalin fixed paraffin embedded tumors, in which DNA is often
highly degraded, making it often too fragmented for use in some molecular diagnostic methods. With
regards to genomic DNA from PBL, it is best to collect whole blood in EDTA or ACD, as these
anticoagulants have minimal impact on downstream molecular methods.{Banfi, 2007 #19549}
Technologies and techniques: Given that there are many ways in which the RB1 gene can be mutated,
several molecular techniques are required to assess for the whole spectrum of oncogenic events.
DNA sequencing: Single nucleotide variants (SNVs) and small insertions/deletions can be identified
using DNA sequencing strategies including Sanger dideoxy-sequencing or massively parallel next-
generation sequencing (NGS) methods.{Singh, 2016 #19381;Li, 2016 #19404;Chen, 2014 #19419}
While both strategies function to produce DNA sequences, NGS has the added advantage of producing
millions of DNA sequences in a single run, in contrast to one sequence per reaction with Sanger.
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Deciding on which technology to use depends on the clinical question being asked. When screening
family members for a known sequencing-detectable RB1 mutation, targeted Sanger sequencing is a more
cost and time effective strategy. In contrast, NGS may be the most effective screening strategy to
investigate for an unknown de novo mutation in an affected proband. Another added advantage to NGS is
the ability to perform deep sequencing, which allows for a much lower limit of detection (analytic
sensitivity) for identify low level mosaic mutations in comparison to Sanger sequencing.{Chen, 2014
#19419}
Copy number analysis: Large RB1 deletions or duplications that span whole exons or multiple exons
typically cannot be easily detected by DNA sequencing. Instead, techniques including multiplex ligation-
dependent probe amplification (MLPA), quantitative multiplex PCR (QM-PCR) or array comparative
genomic hybridization (aCGH) areare often used to interrogate for large deletions (ex. 13q14 deletion
syndrome) and duplications. In addition, these techniques can also be used to identify other genomic
copy number alterations seen in retinoblastoma tumors, such as MYCN amplification. Recently, new
developments in bioinformatics analysis have created ways in which NGS data can be interrogated for
copy number variants.{Devarajan, 2015 #15675;Li, 2016 #19404} While the data is promising; the
current limitation of targeted NGS is that capture efficiency is uneven, which reduces the sensitivity of
detecting CNVs in comparison to conventional methods.
Low-level mosaic detection: Somatic mosaicism can arise in either the presenting patient or their
parent. Detecting a mosaic mutation can be difficult depending on the individual’s level of mosaicism.
NGS can be used detect low-level mosaicism (see above). In addition, allele-specific PCR (AS-PCR) is
an another strategy that can be used in situations where the RB1 mutation is known.{Rushlow, 2009
#10337} This strategy involves the generation of a unique set of primers specific to the mutation of
interest and can detect mosaicism levels as low as 1%.
Microsatellite analysis: The second mutational event in the majority of retinoblastoma tumors
consists of loss of heterozygosity (LOH). LOH is common event in many cancers and is strongly
15
associated with loss of the wild-type allele in 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 useful in identity testing
and in determining the presence of maternal cell contamination in prenatal diagnostic testing.
Methylation analysis: In addition to genetic changes, epigenetic changes have been recognized as
another mechanism of retinoblastoma development.{Ohtani-Fujita, 1993 #2258} Hypermethylation of the
RB1 promoter CpG island results in transcription inhibition of the RB1 gene and has been identified 10-
12% of retinoblastoma tumors.{Richter, 2003 #11998;Zeschnigk, 1999 #15496} This epigenetic event
primarily occurs somatically, however, rare instance of heritable mutations in the RB1 promoter and
translocations disrupting RB1 regulator sites have been reported to also cause RB1 promoter
hypermethylation.{Quinonez-Silva, 2016 #19594}
RNA analysis: In rare instance, no RB1 mutation is identified in the coding, promoter or flanking
intronic sequence in blood from a bilateral patient. Conventional molecular methods do not interrogate
all RB1 intronic nucleotides due to the large amount of sequence and repetitive nature of intronic DNA.
However, deep intronic sequencing alterations have been identified to disrupt RB1 transcription in
patients with retinoblastoma. {Zhang, 2008 #7502;Dehainault, 2007 #5872} In order to investigate for
deep intronic changes, analysis of the RB1 transcript by reverse-transcriptase PCR (RT-PCR) is
performed. RNA studies are also useful in clarifying the pathogenicity of intronic sequencing alterations
detected by conventional DNA sequencing. {Zhang, 2008 #7502;Dehainault, 2007 #5872}Alternatively,
as sequencing costs continue to decrease; whole genome sequence (WGS) may become the method of
choice to uncover deep intronic changes.
Protein studies
Cytogenetic strategies: Karyotype, fluorescent in situ hybridization (FISH) or array comparative
genomic hybridization (aCGH) of peripheral blood lymphocytes can be used to identify large deletions
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and rearrangements in patient’s suspected of 13q14 deletion syndrome.{Caselli, 2007 #19622;Mitter,
2011 #15255} In parents of 13q14 deletion patients, karyotype analysis can be used to assess for balanced
translocations, which increases the risk of recurrence in subsequent offspring.{Baud, 1999 #19768}
Q13: Are these tests available worldwide?
A: “No, They are mainly present in developed countries. In China, many families with retinoblastoma
children do not understand the benefits of genetic testing and genetic counseling in treatment and follow-
up. Meanwhile, the health insurance can’t cover the cost for it. So all the obstacles mentioned above
result in the limited application of genetic testing and genetic counseling nationwide, which also lead to
the redundant economic burden on the affected families. The Chinese government started new policy that
allowed every family to have one more child nowadays. Therefore, genetic testing and genetic
counseling should be put into good use especially for the families carrying the germline RB1 mutation.
In Egypt,{Soliman, 2016 #14713} Genetic testing for retinoblastoma is not available and genetic
counseling is the only way for addressing retinoblastoma genetics. This counseling is performed through
ophthalmologists mainly with defective training in this aspect. Genetic counseling was found to increase
the level of knowledge regarding familial retinoblastoma genetics but the proper translation of this
knowledge into appropriate screening action was deficient.{Soliman, 2016 #14713}
Q14: What 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, parental DNA from PBL is investigated.
Even if neither parent is identified to be a carrier, recurrence risk in siblings is still increased due to the
risk of germline mosaicism. DNA from PBL for all siblings of affected patients should be tested for the
proband’s mutation. As well, DNA from PBL for children of all affected patient’s should also be tested
for the predisposing mutation. Table Y shows the risk of having retinoblastoma in different family
relatives.
17
If the proband’s mutation was identified to be mosaic (ie postzygotic in origin) in DNA from PBL,
parents and siblings of the proband are not at risk to carry the predisposing mutation. However, the
children of mosaic proband should be tested, as their risk of inheriting the predisposing RB1 mutation can
be as high as 50% depending on the mutation burden in the probands germline.
When a RB1 mutation has been identified in a family, The Known RB1 mutation of the proband can be
tested in his offspring. Couples may consider multiple options with respect to planning a pregnancy.
Q15: Can we use the known mutation to test my coming child? I am 33 weeks pregnant
Genetic testing is usually performed early in the course of the pregnancy is available in many
countries around the world. Two early procedures are available: 1) chorionic villus sampling (CVS) and
2) amniocentesis. CVS is a test typically performed between 11-14 weeks gestation during which as
sample of the placenta is obtained either by transvaginal or transabdominal approach. Amniocentesis is a
test performed after 16 weeks of gestation whereby as sample of the amniotic fluid is gathered with a
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 #19427}
Genetic testing results can be used by the family and health care team to manage the pregnancy. If a
mutation is not identified, the pregnancy can proceed with no further intervention, as there is no increased
risk for retinoblastoma beyond the general population risk. If the mutation is identified, some couples
may consider deciding to stop the pregnancy; other couples will decide to continue with the pregnancy
and appropriate intervention, such as early delivery, will be put into place to improve outcomes.{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
18
gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early
delivery rather than miscarriage.{Akolekar, 2015 #19427} The risk for procedure-associated significant
preterm delivery is low (<3%). Results of genetic testing will be available with enough time to plan for
early delivery when a mutation has been inherited.
In many countries around the world, the option for prenatal genetic testing is not available. Even
where available, some couples may elect to do no invasive testing during the course of the pregnancy.
For these conceptions, if the pregnancy is at 50% risk for inheriting a RB1 mutation, it is crucial that the
pregnancy does not go post-dates. Induction of labour should be seriously considered if natural delivery
has not occurred by the due date.{Soliman, 2016 #15159;Canadian Retinoblastoma, 2009 #14251}
Q165: Can we use the known mutation in other benefits?What is the benefit of prenatal mutation
detection versus post natalpostnatal screening?
A: “ThisRB1 mutation detection can be performed either prenatal as discussed earlier or it can be
performed at birth via umbilical cord blood (postnatal screening). This will help either eliminate the 50%
theoretical risk of the proband’s RB1 mutation heritability or confirm it into 100% risk. Both screening
methods are effective in improving visual outcome and eye salvage than non-screened children.,
However, prenatal screening allows for planning for earlier delivery in positive children (late
preterm/early term); this was shown to have less number of tumors at birth (20% versus 50 %) with only
15 % visual threatening tumors in 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}
Preconception testing 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 #19428;Dommering, 2004 #19429;Xu, 2004
#19430;Girardet, 2003 #19431} For PGD, a couple undergoes in vitro fertilization. Conceptions are tested
19
at an early stage of development (typically 8-cell) for the presence of the familial mutation. Only those
conceptions that do not carry the mutation will be used for fertilization. The procedure is costly, ranging
from $10,000-$15,000 per cycle. In some countries, there may be full or partial coverage of the costs
associated with procedure. In addition to cost, couples must consider the medical and time impact of
undergoing in vitro fertilization. Couples also need to be aware that the full medical implications of PGD
are not yet understood; there is emerging evidence that there may be a low risk for epigenetic changes in
the conception as a result of the procedure. For couples that undergo PGD, it is recommended that typical
prenatal testing be pursued during the course of the pregnancy to confirm the results.{Dhanjal, 2007
#19428;Dommering, 2004 #19429;Girardet, 2003 #19431;Xu, 2004 #19430}
Molecular Screening for Retinoblastoma
Q168: 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. Q17:
what are the risks for the relatives in the family?
20
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
place and data accumulate, surveillance of the proband may one day be matched more precisely to the
level of risk for new tumours for individuals with low penetrance mutations.
Patients with unilateral retinoblastoma greatly benefit from genetic testing and counselling.
Approximately 15% of patients with unilateral retinoblastoma will be found to have heritable
retinoblastoma. Correctly identifying these patients can be lifesaving, for both the patients and their
families. Genetic testing companies focused on enhanced detection of RB1 mutations are able to identify
nearly 97% of all retinoblastoma mutations. Genetic testing of the patient’s blood is sensitive enough
when thorough methods are used that not finding a mutation results in a residual risk of heritable
retinoblastoma low enough to remove the need for examinations under anesthesia. This reduces the health
risk for the patient and the cost to the health care system. Testing is even more accurate when a tumour
sample is collected and tested when available. When mutations are identified in the tumour and are
negative in blood, the results can eliminate the need for screening of family members and provide
accurate testing for the patient’s future children. Whether or not a tumour sample is available, finding a
RB1 mutation in a patient’s blood confirms that this patient has heritable retinoblastoma. This patient now
21
benefits from increased surveillance designed to detect tumourss 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 allowa
specific transport medium that allows the cells to grow. We can also freeze some tumor cells
(cryopreservation) for future necessity or for research purposes.
Q21: If we know the mutation prenatally, is there any treatment to prevent retinoblastoma from
occurring?
A: “
Q18: What are the long term risks for germ line RB1 mutation?
There have the highest mortality, with >70about 40-70% of affected children with dying of
retinoblastoma in Asia and Africa, compared with <53-5% in developed countries.48,55 Delayed diagnosis
and treatment due to lack of knowledge pertaining to retinoblastoma of parents56 and ophthalmologists is
one of the major causes leading to the low eye salvage rateof and high mortality in developing countries.
So theBroad good understanding of retinoblastoma genetics and the importance of genetic counseling is a
suitablethe optimal waycan contribute to reducing mortality from retinoblastoma. to address above issue
22
in certain extent. In this review, we highlight the RB1 mutation categoriestypes, advanced molecular
diagnosis of retinoblastoma and genetic counseling.
Clinical presentation [Sameh]
Natural History
71 Start with retinoma and molecular features…..
452246Retinoblastoma starts as a rounded white retinal mass that gradually increases in size. At first,
equal Centrifugal tumor growth of the tumor preserving the rounded or oval shaperesults in small tumors
being round; occurs followed bymore extensive growth a period of differential growth period leading
toproducesing the lobular or nipplegrowth growth patternstumo, likely related to genomic changes in
single (clonal) cells, that provide a proliferative advantager appearance.47,48 Tumor seeds float free of the
main tumor intoing occurs to the subretinal space or the vitreous cavity due to theas a result of poor
cohesive forces between tumor cells,49, this can be into the subretinal space or the vitreous cavity. In
Advanced vitreous tumors, the tumor seeds might can migrate to the anterior chamber producing a
pseudo-hypopyon like appearance., the Enlarging tumor might can push the iris lens diaphragm forward
causing angle closure glaucoma. or rarely the Rapid necrosis within of the tumor can cause an aseptic
orbital inflammatory reaction resembling orbital cellulitis, sometimes showing central retinal artery
occlusions.47,48,50 If Untreated, retinoblastoma can spreads along into the optic nerve and along the visual
pathway to the brain, or hematogenous spread occurs . Retinoblastoma can spread into thethrough
choroidal blood vessels and, particularly to grow in bone marrow hematogenous spread occurs. Direct
tumor growth through the sclera can cause present as orbital extension and proptosis. {Gallie, In Press
#15554}is a precursor with characteristic clinical features: translucent white mass,{Gallie, 1982 #5686}
Pathology of retinoma reveals fleurettes structures that are not proliferative. Genetic analysis of retinoma
and adjacent normal retina and retinoblastoma shows loss of both RB1 alleles, and early genomic copy
23
number changes that are amplified further in the adjacent retinoblastoma. {Gallie, 1982 #5686}
{Theodossiadis, 2005 #5649}
Clinical Features
Leukocorea (white pupil) is main clinical presentation usually detected by parents either directly or in
photographs (photo-leukocorea). Strabismus due early macular involvement is the second most
common.50 In developing countries, buphthalmos and proptosis due to advanced and extraocular disease
respectively represents a higher percentage.43 Less common presentations include; heterochromia irides,
neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.50 Retinoblastoma
(unilateral or bilateral) might be associated with a brain tumor in the pineal, suprasellar or parasellar
regions (Trilateral retinoblastoma)51,52.{Popovic, 2007 #11607;Antoneli, 2007 #14202;de Jong, 2015
#14413} It might present in a syndromic form (13q deletion syndrome) associated with some facial
features as high and broad forehead, thick and everted ear lobes, short nose, prominent philtrum and thick
everted lower lip, bulbous tip of the noseassociated with various degrees of hypotonea and mental
retardation53-55 (Baud et al 1999 PMID: ; Bojinova et al 2001 PMID: ; Skrypnyk and Bartsch 2004 PMID:)
The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and
ocular toxicariasis.50
Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a
brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma
(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median age
of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014
PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,
from very few to nearly half of all patients and is dependent on early detection and small tumor size (de
Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy
and autologous stem-cell rescue.
24
Grouping/Retinoblastoma Cancer Staging
Treatment and prognosis depend on the stage of disease at initial presentation. The main Factors
involvedpredictive of outcomes include in grouping are size, and site of thelocation of tumor origin,
amountextent of subretinal fluid, size and sitepresence of tumor seeds and the presence of high risk
features on pathology.56 Multiple grouping staging systems have predicted likelihood to salvage an eye
without using radiation therapy; for the intraocular retinoblastoma existed with thethe International
Intraocular Retinoblastoma Classification (IIRC)47 being has been the recently the most reliable, but
published evidence is in the last decadebecause significantly different versions have emerged.1 Recently,
it has been replaced by tThe 2017 TNMH classification is based on international consensus and evidence
from an international survey of 1728 eyes, with algorithms evaluating initial features and outcomes by 5
different eye staging systems.56 The main factors involved in grouping are size and site of the tumor,
amount of subretinal fluid, size and site of tumor seeds and the presence of high risk features. (Table X)
Retinoblastoma is the first cancer to be stagedin which staging recognizes the impact of by genetics in
addition to the clinical features due to the high impact of genetic status on managementon outcomes: . If
there ispresence of a positive family history, bilateral or trilateral disease or documentedhigh sensitivity
positive RB1mutation testing, the disease is staged as is H1; without these features or testing of blood,
HX; and H0 for those relatives who are shown to not carry the. Otherwise it is considered as H0. A true
H0 is with documented negative specific RB1 mutation status.56 We propose H0* for patients with 2 RB1
mutant alleles in blood that are not detectable in blood, reducing risk of a heritable RB1 mutation to <1%.
-Pedigree defining H0 (*define a true H0 vs most likely H0), H1, HX
Treatments
Multiple treatments are now available and the choice depends on the laterality of disease and the
grouping of the tumor. Chemotherapy (systemic or intraarterial chemotherapy) to reduce the size of the
tumor followed by consolidation focal therapies (Laser therapy or cryotherapy) is the main stay of
treatment.1 Enucleation for eyes with advanced tumors or in unilateral disease where the other eye is
25
normal is more appropriate and definitive. Other therapies include; intravitreal chemotherapy for vitreous
disease, plaque radiotherapy or periocular chemotherapy. External beam radiation therapy has extremely
limited indications nowadays due to its extensive cancer risks and complications.1
Metastasis and Second Cancers
Germline retinoblastoma carry the risk of development of second primary cancers, most commonly
osteosarcoma and fibrosarcoma. Sometimes it might be confused with metastatic retinoblastoma. Fine
needle aspiration cytopathology has minimal role in differentiation as both metastasis and second cancers
appear as blue round cell tumors. Genetic analysis might help to differentiate57…. (Hilary to write details
and choose appropriate site) –Cite Racher paper
Add differential diagnosis? NO, ELSEWHERE IN JOURNAL ISSUE; BUT ONE SENTENCE
ONLY….MERGE THE ABOVE HEADINGS INTO TWO PARAS…AT MOST.
Add retinoblastoma/retinoma? ONLY THE GENETICS OF IT
Inheritance pattern [Hilary]
Knudson two-hit hypothesis: In most cases, retinoblastoma develops when both copies of the RB1
gene are inactivated. This concept was first formulated in 1971, when Knudson used retinoblastoma as
the prototypic cancer to derive the two-hit hypothesis (Knudson, 1971).31 In heritable retinoblastoma, the
first mutational event is inherited via the germinal cells, while the second event occurs in the somatic
cells. In nonheritable retinoblastoma, both mutation events occur in the somatic cells. Heritable
retinoblastoma encompasses 45% of all reported cases (MacCarthy et al 2009; Moreno et al 2014; Wong
et al {risk of subse malig neoplasms in long term hereditary rb surviv…}2014).32-34 The clinical
presentation of heritable retinoblastoma consists of 80% bilateral and 15-18% unilateral (cite).1 In
26
nonheritable retinoblastoma the majority (98%) of cases have somatic biallelic RB1 loss in the tumor,
while the remaining 2% have no mutation in either copy of RB1 but instead have somatic amplification of
the MYCN oncogene 35
Heritable Retinoblastoma and Penetrance
In heritable retinoblastoma, the offspring of each patient has a 50% risk of inheriting the RB1
pathogenic change. Whether the individual for whom inherited the RB1 mutation develops
retinoblastoma depends on the RB1 DNA alteration. Typically, nonsense and frameshift germline
mutations, which lead to absence of RB1 expression or truncated dysfunctional RB1 protein, show nearly
complete (90%) penetrance. Often the second mutational event in the retinal cell is loss of the second
RB1 allele (LOH, loss of heterozygosity). In these families the presentation is typically unilateral,
multifocal or bilateral retinoblastoma. In a smaller subset of hereditary retinoblastoma, reduced
expressivity and reduced penetrance is observed (citations). In these families, when retinoblastoma
develops, it is often late onset and less severe, presenting as unilateral, unifocal (reduced expressivity)
and in some carrier family member retinoblastoma never develops (reduced penetrance). The types of
RB1 mutations reported that result in reduced expressivity/penetrance are diverse. Many consist of
mutations which reduced the expression of the RB1 protein. Examples include, (1) mutations in exons 1
and 2 25,36 (2) mutations in exons 26 and 2726,37{Mitter, 2009 #18935;Mitter, 2009 #7347} (3) intronic
mutations38,39 (Schubert et al 1997 PMID: 9341870; Lefevre et al 2002 PMID: 12011162 ; ) and (4)
missense mutations (cite).40,41 In addition, large deletions that encompass the RB1 gene and the MED1
gene cause reduced expressivity/penetrance (Dehainault et al 2014 PMID: 24858910; Bunin et al 1989
PMID: 2915374 ; ).42,43 Dehainault et al showed that RB1 -/- cells cannot survive in the absence of MED4.
Patients with 13q14 deletion syndrome more often have unilateral tumors only, in comparison to patients
with gross deletions with one breakpoint in the RB1 gene whom typically present with bilateral 44-46Rb
(Mitter et al 2011 PMID: ; Matsunaga et al 1980 PMID: ; Baud et al 1999; Albrecht et al 2002 PMID: ).
One way in which the severity of risk can be evaluated is through the disease-eye-ratio (DER) (Lohmann
27
et al 1994). 47 The DER is calculated by taking the number of eyes affected divided by the total number of
eyes of carriers within the family.
In some instances of hereditable reduced expressivity/penetrance retinoblastoma, the parental origin
impacts whether or not an individual develops retinoblastoma and subsequently whether their carrier
offspring are at risk to develop retinoblastoma, a phenomenon termed the parent-of-origin effect (Klutz et
al 2002 PMID: 12016586; Schuler et al 2004 PMID: 15763650; Eloy et al 2016 PMID: 26925970).48-50 A
recent study by Eloy et al50 helped shed light on a potential molecular mechanism to explain the parent-
of-origin effect. Using the c.1981C>T (p.Arg661Trp) reduced penetrance/expressivity missense
mutation, the researchers discovered that differential methylation of the intron 2 CpG85 skews RB1
expression in favour of the maternal allele. In other words, when the p.Arg661Trp allele is maternally
inherited there is sufficient tumor suppressor activity to prevent pRB RB development; 90.3% of carriers
of maternally inherited p.Arg661Trp remain unaffected. However, when the mutation is paternally
transmitted, very little RB1 is expressed, leading to haploinsufficiency and pRB RB development in
67.5% of cases. A similar inheritance pattern was also reported for the intron 6 c.607+1G>T substitution
(Klutz et al 2002 PMID: 12016586).48
Trilateral: In approximately 5% of heritable cases, in addition to retinal tumors in one or both eyes, a
brain tumor (pineal, suprasellar or parasellar) will develop, a condition termed trilateral retinoblastoma
(de Jong et al 2015 PMID: 26374932). The onset of the brain tumor is relatively early, with the median age
of onset 17 months after retinoblastoma is diagnosed and before the age of 5 years (de Jong et al 2014
PMID: 26374932). The survival outcome for trilateral Rb patients has improved over the last 2 decades,
from very few to nearly half of all patients and is dependent on early detection and small tumor size (de
Jong et al 2014 PMID: 26374932). Improved survival is largely due to the use of high-dose chemotherapy
and autologous stem-cell rescue.
28
13q deletion syndrome
In patients with large interstitial 13q14 deletions that include the RB1 gene, variable clinical features
are present in addition to retinoblastoma, termed 13q14 deletion syndrome. Common facial features
includes high and broad forehead, thick and everted ear lobes, short nose, prominent philtrum and thick
everted lower lip, bulbous tip of the nose and mental retardation (Baud et al 1999 PMID: ; Bojinova et al
2001 PMID: ; Skrypnyk and Bartsch 2004 PMID: ). Patients with 13q14 deletion syndrome more often
have unilateral tumors only, in comparison to patients with gross deletions with one breakpoint in the RB1
gene whom typically present with bilateral Rb (Mitter et al 2011 PMID: ; Matsunaga et al 1980 PMID: ;
Baud et al 1999; Albrecht et al 2002 PMID: ).
?mechanism ?non-allelic homologous recombination.
Mosaicism
{FIGURE ON MOSAICISM}
RB1 gene [Hilary]
Function: The RB1 gene, located on 13q14, encodes the pRB RB protein (pRB), which is an
important cell cycle regulator and the first tumor suppressor gene ever discovered (Friend et al 1986
PMID: ).41 After a cell completes mitosis, the pRB RB protein is dephosphorylated, permitting it to bind
to the promoter region of the E2F transcription factor gene, thereby repressing transcription and inhibiting
the progression of the cell cycle from G1 to S phase (Nevins et al 2001 PMID: ; Cobrinik 2005 PMID: ;
Sage et al 2012 PMID: ).42-44 In order for the cell to enter S phase, cyclin-dependent kinases
phosphorylate RB, which removes the ability of pRB RB to bind to the E2F gene promoter (Knudsen and
Knudsen 2008 PMID: ).45 pRB RB functions to regulate proliferation in most cell types (Cobrinik 2005
PMID:).43 Often, loss of RB1 is compensated by increased expression of its related proteins, however, in
certain susceptible cells, such as the retinal cone cell precursors, compensatory mechanisms are not
29
sufficient and tumorigenesis is initiated (Xu et al 2014 – Nature – Rb suppresses human cone-precur
PMID).46
-?A and B pockets
-Also describe the role in genomic instability (Demaris. Rushlow)
RB1 Mutations
Different ways in which RB1 can be disrupted: There are many ways in which the function of the
pRB RB protein is impaired including point mutations, small and large deletions, promotor methylation
and chromothripsis (Lohmann 1999 PMID: ; McEvoy et al 2014 PMID: ).47,48 The majority of RB1
mutations are de novo, unique to a specific patient or family, however, there are some known recurrent
mutations found across many unrelated individuals. One subset of recurrent mutations involved CpGOne
subset of recurrent mutations involve 11 CpG sites, which make up ~22% of all RB1 mutations (Rushlow
et al 2009 PMID: 19280657).49 The high recurrence of nonsense mutations at these sites is due to the
hypermutabilty and subsequent deamination of 5-methylcytosine (Richter et al 2003).50
The origin of a de novo RB1 mutation can arise either pre- or post-conception. Most often, pre-
conception mutagenesis occur during spermatogenesis (Munier et al 1998 PMID: 9837842; Dryja et al 1997
PMID: 9272170)51,52.51,52 Furthermore, advanced paternal age has been shown to increase risk for
retinoblastoma.53 This is thought to be due to the large number of cell divisions during spermatogenesis
and the increased rate for base substitution errors in aging men compared to women. In cases of pre-
conception mutagenesis, the proband carries the de novo RB1 mutation in every cell within their body and
typically presents with bilateral retinoblastoma. In contrast, post-conception RB1 mutagenesis occurs
during embryogenesis. Depending on the embryological stage of development, a few or numerous tissues
may be mosaic for the RB1 mutation. If the mutational event occurs during retinal development, the
presentation is often unilateral retinoblastoma.1
Coding sequencing mutations
30
Promoter methylation
Hot-spot mutations – CpG transition
Non-coding/regulatory changes
?in genetic counselling?? Origin of new mutations
Xu et al. new mutations are on fathers chromosome
Older fathers, but not older mothers for RB50
Greta Bunin
MYCN
PROGRESSIVE OTHER GENOMIC CHANGES IN ADDITION TO RB1
Other genomic changes in addition to alterations in RB1 [Hilary]
DEK, KIF14, E2F3, CDH11
In a small subset (2%) of unilateral patients, no RB1 mutantion is identified. Instead, striking
amplification (28-121 copies) of the MYCN oncogene is detected (Rushlow et al 2013 PMID: 23498719).35
Patients with RB1+/+ MYCNA are clinically distinct from RB-/- patients, showing much younger age at
diagnosis, distinct histological features and larger, more invasive tumors.
In addition to loss of RB1 or MYCN amplification, specific somatic copy number alterations
commonly occur in the progression of the retinoblastoma. Commonly seen are gains in 1q32, 2p24, 6p22
and losses at 13q and 16q22-24 (Corson and Gallie 2007 PMID: 17437278).54 These regions contain
important oncogenes (MDM4, KIF14, MYCN, DEK and E2F3) and tumor suppressor genes (CDH11),
thought to act as drivers promoting the growth of the cancer (Theriault et al 2014 PMID: 24433356).55
31
Other less common alterations that have been identified in retinoblastoma tumors include differential
expression of some microRNAs56 (Huang et al 2007 PMID: 18026111) and recurrent single nucleotide
variants/insertion-deletions in the genes BCOR and CREBBP (Kooi et al 2016 PMID: 27126562).57 In
comparison to the genomic landscape of other cancers, retinoblastoma is one of the least mutated57 (Kooi
et al 2016 PMID: 27126562)
Molecular diagnosis [Hilary]
Strategic testing - Tumor testing first for unilateral/PBL for bilateral
Technologies and techniques
NGS [flow chart of molecular techniques]
Cytogenetic strategies (FISH/microarray)
RNA for discovery and VUS functional studies
Protein studies
The most optimal strategy for retinoblastoma molecular genetic testing is guided by the patient’s
tumor presentation. If the patient is bilaterally affected, the probability of finding a germline mutation in
the RB1 gene is high (example - 97% detection rate in comprehensive laboratory). For this reason, the
most optimal strategy for testing bilateral patients involves first testing genomic DNA extracted from
peripheral blood lymphocytes (PBL). In rare instances of bilateral retinoblastoma, the predisposing RB1
mutation has occurred sometime during embryonal development. In these cases, the RB1 mutation may
only be present in some cells and may not be detected in DNA from PBL. Therefore, in the event that no
mutation is identified in the blood of a bilaterally affected patient, DNA from tumor should be
investigated.58
In contrast, given that approximately 15% of unilateral patients carry a germline mutation, the most
optimal strategy is to first test DNA extracted from a tumor sample. Upon identification of the tumor
32
mutations, targeted molecular analysis can be performed on DNA from PBL to determine if the mutation
is present is the patient’s germline. When only the tumor is found to carry the RB1 mutations, this result
dramatically reduces the risk of recurrence in siblings and cousins. In addition, this targeted approach can
allow for a more sensitive assessment of the PBL DNA, which can be useful in the detection of low level
mosaic mutations, more common in unilateral cases (cite).58
Sample preparation impacts the quality of DNA. For best results, fresh or frozen tumor samples
should be collected, as opposed to formalin fixed paraffin embedded tumors, in which DNA is often
highly degraded, making it often too fragmented for use in some molecular diagnostic methods. With
regards to genomic DNA from PBL, it is best to collect whole blood in EDTA or ACD, as these
anticoagulants have minimal impact on downstream molecular methods (Banfi et al 2007
PMID:17484616).59
Technologies and techniques: Given that there are many ways in which the RB1 gene can be mutated,
several molecular techniques are required to assess for the whole spectrum of oncogenic events.
DNA sequencing: Single nucleotide variants (SNVs) and small insertions/deletions can be identified
using DNA sequencing strategies including Sanger dideoxy-sequencing or massively parallel next-
generation sequencing (NGS) methods (Singh et al 2016 PMID: 27582626; Li et al 2016 PMID: 27155049;
Chen et al 2014 PMID: 24282159).60-62 While both strategies function to produce DNA sequences, NGS
has the added advantage of producing millions of DNA sequences in a single run, in contrast to one
sequence per reaction with Sanger. Deciding on which technology to use depends on the clinical question
being asked. When screening family members for a known sequencing-detectable RB1 mutation, targeted
Sanger sequencing is a more cost and time effective strategy. In contrast, NGS may be the most effective
screening strategy to investigate for an unknown de novo mutation in an affected proband. Another added
advantage to NGS is the ability to perform deep sequencing, which allows for a much lower limit of
detection (analytic sensitivity) for identify low level mosaic mutations in comparison to Sanger
sequencing (Chen et al 2014 PMID: 24282159)62 .
33
Copy number analysis: Large RB1 deletions or duplications that span whole exons or multiple exons
typically cannot be easily detected by DNA sequencing. Instead, techniques including multiplex ligation-
dependent probe amplification (MLPA), quantitative multiplex PCR (QM-PCR) or array comparative
genomic hybridization (aCGH) are often used to interrogate for large deletions (ex. 13q14 deletion
syndrome) and duplications. In addition, these techniques can also be used to identify other genomic
copy number alterations seen in retinoblastoma tumors, such as MYCN amplification. Recently, new
developments in bioinformatics analysis has created ways in which NGS data can be interrogated for
copy number variants59 (Devarajan et al 2015; Li et al 2016 PMID: 27155049).61,63 While the data is
promising, the current limitation of targeted NGS is that capture efficiency is uneven, which reduces the
sensitivity of detecting CNVs in comparison to conventional methods.
Low level mosaic detection: Somatic mosaicism can arise in either the presenting patient or their
parent. Detecting a mosaic mutation can be difficult depending on the individual’s level of mosaicism.
As described in the DNA sequencing section, NGS is one tool that can be used detect low level
mosaicism. In addition, allele-specific PCR (AS-PCR) is an another strategy that can be used in
situations where the RB1 mutation is known (Rushlow et al 2009 PMID: 19280657).17 This strategy
involves the generation of a unique set of primers specific to the mutation of interest and can detect
mosaicism levels as low as 1%.
Microsatellite analysis: The second mutational event in the majority of retinoblastoma tumors
consists of loss of heterozygosity (LOH). LOH is common event in many cancers and is strongly
associated with loss of the wild-type allele in individuals with an inherited cancer predisposition
syndrome (Canvenee et al 1983 PMID: 6633649).64 Polymorphic microsatellite markers distributed
throughout chromosome 13 can be used to detect a change from a heterozygous state in blood compared
to the homozygous state in a tumor with LOH. Microsatellite marker analysis is also useful in identity
testing and in determining the presence of maternal cell contamination in prenatal diagnostic testing.
34
Methylation analysis: In addition to genetic changes, epigenetic changes have been recognized as
another mechanism of retinoblastoma development (Ohtani-Fujita et al 1993 PMID: 8455933). 65
Hypermethylation of the RB1 promoter CpG island results in transcription inhibition of the RB1 gene and
has been identified 10-12% of retinoblastoma tumors (Richter et al 2003).18,66(Zeshnigk et al 1999 PMID:
10528863) This epigenetic event primarily occurs somatically, however, rare instance of heritable
mutations in the RB1 promoter and translocations disrupting RB1 regulator sites have been reported to
also cause RB1 promoter hypermethylation (Quinonez-Silva et al 2016 PMID: 26753011). 67
RNA analysis: In rare instance, no RB1 mutation is identified in the coding, promoter or flanking
intronic sequence in blood from a bilateral patient. Conventional molecular methods do not interrogate
all RB1 intronic nucleotides due to the large amount of sequence and repetitive nature of intronic DNA.
However, deep intronic sequencing alterations have been identified to disrupt RB1 transcription in
patients with retinoblastoma (Zhang et al PMID: 18181215; Dehainault et al., 2007 PMID:17299438). 68,69
Inorder to investigate for deep intronic changes, analysis of the RB1 transcript by reverse-transcriptase
PCR (RT-PCR) is performed. RNA studies are also useful in clarifying the pathogenicity of intronic
sequencing alterations detected by conventional DNA sequencing (Zhang et al PMID: 18181215;
Dehainault et al., 2007 PMID: 17299438). Alternatively, as sequencing costs continue to decrease; whole
genome sequence (WGS) may become the method of choice to uncover deep intronic changes.
Protein studies
Cytogenetic strategies: Karyotype, fluorescent in situ hybridization (FISH) or array comparative
genomic hybridization (aCGH) of peripheral blood lymphocytes can be used to identify large deletions
and rearrangements in patient’s suspected of 13q14 deletion syndrome (Caselli et al 2007 PMID:
17502991; Mitter et al 2011 PMID: 21505449). 41,70 In parents of 13q14 deletion patients, karyotype analysis can
be used to assess for balanced translocations, which increases the risk of recurrence in subsequent
offspring (Baud et al 1999 PMID: 10450867).51
35
Genetic Counseling (Heather/Hilary)
Importance of high detection rate
Targeted familial testing/prenatal testing, preconception testing
Targeted familial testing1,58: To determine if a predisposing RB1 mutation has occurred de novo,
parental DNA from PBL is investigated. Even if neither parent is identified to be a carrier, recurrence risk
in siblings is still increased due to the risk of germline mosaicism. DNA from PBL for all siblings of
affected patients should be tested for the proband’s mutation. As well, DNA from PBL for children of all
affected patient’s should also be tested for the predisposing mutation.
If the proband’s mutation was identified to be mosaic (ie postzygotic in origin) in DNA from PBL,
parents and siblings of the proband are not at risk to carry the predisposing mutation. However, the
children of mosaic affecteds should be tested as their risk of inheriting the predisposing RB1 mutation can
be as high as 50% depending on the mutation burden in the probands germline.
When a RB1 mutation has been identified in a family, The Known RB1 mutation of the proband can
be tested in his offspring. couples may consider a number of options with respect to planning a pregnancy.
Genetic testing performed early in the course of the pregnancy is available in many countries around the
world. Two early procedures are available: 1) chorionic villus sampling (CVS) and 2) amniocentesis.
CVS is a test typically performed between 11-14wks gestation during which as sample of the placenta is
obtained either by transvaginal or transabdominal approach. Amniocentesis is a test performed after 16
weeks of gestation whereby as sample of the amniotic fluid is gathered with a transabdominal approach.
CVS has a procedure-associated risk of miscarriage of ~1%. Amniocentesis has a procedure-associated
risk of miscarriage between 0.1-0.5%. Though uncommon, there is a risk for maternal cell contamination
which occurs more frequently with CVS.71
36
Results of genetic testing can be used by the family and health care team to manage the pregnancy. If
a mutation is not identified, the pregnancy can proceed with no further intervention as there is no
increased risk for retinoblastoma beyond the general population risk. If the mutation is identified, some
couples may consider deciding to stop the pregnancy; other couples will decide to continue with the
pregnancy and appropriate intervention, such as early delivery, will be put into place to improve
outcomes.72
Some couples know that they wish to continue their pregnancy regardless of the genetic testing results
and are concerned by the risk of miscarriage associated with early invasive prenatal testing. Where
available, couples can also consider the option of late amniocentesis, performed between 30-34wks
gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early
delivery rather than miscarriage.71 The risk for procedure-associated significant preterm delivery is low
(<3%). Results of genetic testing will be available with enough time to plan for early delivery when a
mutation has been inherited.
In many countries around the world, the option for prenatal genetic testing is not available. Even
where available, some couples may elect to do no invasive testing during the course of the pregnancy.
For these conceptions, if the pregnancy is at 50% risk for inheriting a RB1 mutation, it is crucial that the
pregnancy does not go post-dates. Induction of labour should be seriously considered if natural delivery
has not occurred by the due date.58,72
In some countries around the world, there is an in vitro fertilization option available to couples called
preimplantation genetic diagnosis (PGD).73-76 For PGD, a couple undergoes in vitro fertilization.
Conceptions are tested at an early stage of development (typically 8-cell) for the presence of the familial
mutation. Only those conceptions that do not carry the mutation will be used for fertilization. The
procedure is costly, ranging from $10,000-$15,000 per cycle. In some countries, there may be full or
partial coverage of the costs associated with procedure. In addition to cost, couples must consider the
medical and time impact of undergoing in vitro fertilization. Couples also need to be aware that the full
37
medical implications of PGD are not yet understood; there is emerging evidence that there may be a low
risk for epigenetic changes in the conception as a result of the procedure. For couples that undergo PGD,
it is recommended that typical prenatal testing be pursued during the course of the pregnancy to confirm
the results73-76
72Surveillance for mets and second cancer
Benefits of genetic counsellingcounseling (Table of risk% [skalet etc] [impact new data?] ie: siblings,
offspring, cousins, faroff relatives, stats below population risk]
Genetic counselling is both a psychosocial and educational process for patients and their families with
the aim of helping families better adapt to the genetic risk, the genetic condition, and the process of
informed decision making.77-79 (Uhlmann et al. (2009), Shugar (2016)). Genetic testing is an integral
component of genetic counselling that results in more informed and precise genetic counselling. Concrete
knowledge of the genetic test outcomes results in specificity, reducing the need for other possible
scenarios to be discussed with the family. This enhances the educational component of genetic
counselling and also provides further time for psychosocial support to be provided to the family.
Patients with bilateral retinoblastoma at presentation are presumed to have heritable retinoblastoma
and a RB1 mutation. Genetic testing provides more accurate information about the type of heritable
retinoblastoma and allows for straightforward testing to determine if additional family members are at
risk. Through genetic testing, a patient may be found to have a large deletion extending beyond the RB1
gene as part of the 13q deletion spectrum. Individuals with 13q deletion syndrome are at risk for
additional health concerns requiring appropriate medical management and intervention. Results may
reveal a mosaic mutation which indicates that the mutation is definitively de novo; only the individual’s
own children are at risk and no further surveillance or genetic testing is needed for other family members.
The results may find a low-penetrance mutation which indicates the patient is at reduced risk to develop
future tumours. As genetic testing for retinoblastoma becomes more common place and data accumulate,
38
surveillance of the proband may one day be matched more precisely to the level of risk for new tumours
for individuals with low penetrance mutations.
Patients with unilateral retinoblastoma greatly benefit from genetic testing and counselling.
Approximately 15% of patients with unilateral retinoblastoma will be found to have heritable
retinoblastoma. Correctly identifying these patients can be lifesaving, for both the patients and their
families. Genetic testing companies focused on enhanced detection of RB1 mutations are able to identify
nearly 97% of all retinoblastoma mutations. Genetic testing of the patient’s blood is sensitive enough
when thorough methods are used that not finding a mutation results in a residual risk of heritable
retinoblastoma low enough to remove the need for examinations under anesthesia. This reduces the health
risk for the patient and the cost to the health care system. Testing is even more accurate when a tumour
sample is collected and tested when available. When mutations are identified in the tumour and are
negative in blood, the results can eliminate the need for screening of family members and provide
accurate testing for the patient’s future children. Whether or not a tumour sample is available, finding a
RB1 mutation in a patient’s blood confirms that this patient has heritable retinoblastoma. This patient now
benefits from increased surveillance designed to detect tumours at the earliest stages and awareness of an
increased lifelong risk for second cancers. Members of the patient’s family can have appropriate genetic
testing to accurately determine who is at risk. As with patients with bilateral retinoblastoma, knowing the
specific type of mutation provides the most detailed provision of medical management and counselling.
63
Cost-effectiveness [Brenda/Crystal] {FIGURE/FLOW CHART}
Difficulties and opportunities across different jurisdictions/countries [Jeffry/Sameh]
Compare/contrast Canada vs China vs Jordon
Societal/cultural challenges to GC
39
In China, many families with retinoblastoma children do not understand the benefits of genetic testing
and genetic counseling in treatment and follow-up. Meanwhile, the health insurance can’t cover the cost
for it. So all the obstacles mentioned above result in the limited application of genetic testing and genetic
counseling nationwide, which also lead to the redundant economic burden on the affected families. The
Chinese government started new policy that allowed every family to have one more child nowadays.
Therefore, genetic testing and genetic counseling should be put into good use especially for the families
carrying the germline RB1 mutation.
8080References
Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-
Blackwell.
Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling
Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of print.
PMID: 27891554 DOI: 10.1007/s10897-016-0042-y
Benefits of genetic testing for the proband and family members [Heather]
Prenatal vs Postnatal [Sameh]
Cost-effectiveness [Brenda/Crystal] {FIGURE/FLOW CHART}
Difficulties and opportunities across different jurisdictions/countries [Jeffry/Sameh]
Compare/contrast Canada vs China vs Jordon
Societal/cultural challenges to GC
40
Conclusions
Retinoblastoma genetics is challenging to understand, but once understood It largely affect the level
of care presented to retinoblastoma patients and their families. It helps alleviate the psychological burden
of the families regarding moving forward with their life choices regarding the affected child and future
siblings. It also helps the family to understand the risks of different family members giving them the
chance of the level of disclosure they wish.
41
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
42
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
43
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