introduction - sharedocs.ca file · web viewretinoblastoma is the most common childhood intraocular...
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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
Chengyue Zhang, MD.
Hilary Racher, PhDHeather MacDonald,1 ,5 Brenda L. Gallie, MD.1, 5 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, Beijing, China.
5 Heather affiliation??
5 6 Brenda affiliationDepartments of Ophthalmology, Molecular Genetics, and Medical Biophysics,
University of Toronto, Toronto, Canada.
2Impact Genetics, Bowmanville, Ontario.Corresponding author:
Brenda L. Gallie:. Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada M5G
1X8Address: 525 University Ave, room 806, Toronto, Ontario, Canada. M5G 2L3. Telephone: +1 xxxxx,-
294-9729
email: [email protected]
2
Disclosures:
Both SS and HR contributed equally to this review and would be considered as first co-first
authersauthors.
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.
3
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. GA 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 theThis review is organized in the context of a based conversation
betweenconversation between a family with a retinoblastoma child and their treating attendingphysician
who is mostly the ophthalmologist orbut can be any member of the retinoblastoma multidisciplinary team
of physicians, nurses and genetic counselors. All the questions are true and high frequentlybased on real
life examples frequently askedcommon questions that all ocular oncology physiains face on regular
basisby thepatient’s parents. The main aim of Tthis scenario aimsis to tryThe goal of this article is to
simplify the information aroundconcepts of retinoblastoma genetics for ophthalmologists to help them
improve theirassist in the care of patients and their familiesy care. mmmmmmm
Key Words: retinoblastoma, RB1 gene, bilateral, unilateral, DNA sequencing, genetic counseling
prenatal screening
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4134/56000 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 [JEFFRY]
Retinoblastoma is the most common childhood intraocular malignancy in childhood that might affects
one or both eyes.{Dimaras, 2015 #10881} ItBecause of the strong links between clinical care and genetic
causation,{Knudson, 1971 #11106} retinoblastoma 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 Worldwide, aAbout 89,000 children are newly diagnosed with
retinoblastoma new cases perevery year worldwide(1/16,000 live births).{Seregard, 2004
#10380;Dimaras, 2015 #10881} but most have no access to knowledge of the keyimportant role of
genetics plays in Understanding retinoblastoma genetics is crucial in multiplemany aspects of
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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 genetic etiology of retinoblastoma in
the context of individual children and families, lead by the questions commonly asked by parents.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 child presented with left leukocorea (white pupil). The family
noticed the white pupil at a family photograph 5 days ago. They sought medical advise to their family
physician who suspected retinoblastoma and referred them urgently to the pediatric ophthalmologist. The
family history is irrelevant and the mother is 33 weeks pregnant. The child was extremely uncooperative
but the ophthalmologist was able to visualize a white retinal mass in the left eye. He couldn’t examine
except the inferior retina, an intact optic 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?
Retinoblastoma is a malignant tumorcancer that arises from a developing retinal cell in babies and young
children. The exact cell of origin is unknown but there are many theories suggesting either a
conephotoreceptor precursor cell or an inner nuclear layer {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
6
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 presentingHow can this cancer show up inat such a young
age?
Retinoblastoma arises fromThe cell of origin of retinoblastoma is most likely a developing cone
photoreceptor precursor cell that has lost both copies of the RB1 tumor suppressor gene, and remains in
the inner nuclear layer of the retina, unable to migrate to the outer retina and function normally.{Dimaras,
2015 #10881;Rootman, 2013 #11096;Xu, 2014 #9924} The susceptible developing cell that becomes
cancer is 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.
For your daughter, Despite the fact that 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
causedHow can a gene cause cancer in a baby?
“No one knows what really causes the damage to the RB1 gene. MaybeOn theory is that the mutation is
caused by a random cosmic ray (cite?)passes through Planet Earth and hits that large, important
gene.retinoblastoma genetics is challenging to understand, but once understood it largelycan greatly affect
the level of care presented to retinoblastoma the patients and their families. It helps alleviate the
psychological burden ofon the families regarding moving forward with their life choices regarding the
7
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.
“In Tumors are initiated by biallelic mutation of the retinoblastoma tumor suppressor gene (RB1) in a
precursor retinal cell. nearly 50% of patients Thethe first RB1RB1 mutationgene is presentdamaged in
most, or all, normal in constitutional cells in nearly 50% of patients, who are therebyresulting in
predispositioned to developing retinoblastoma. A retinal tumor develops when after the second RB1
allelegene is also damaged in a developing retinal cell in a somatic cell.{Dimaras, 2015 #10881} The RB1
gene, located on chromosome 13q14, encodes the RB protein (pRB), an important regulator of the cell
division cycle in most cell types, regulator and the first tumor suppressor gene discovered.{Friend, 1986
#10882} After a cell completes mitosis, Normally, dephosphorylated the pRB represses expression of the
E2F gene, thereby blockingprotein 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 phasecell division.{Nevins, 2001 #15292;Cobrinik, 2005 #15298;Sage, 2012 #7850}
In order for the cell to enterTo resume S phasecell division, cyclin-dependent kinases re-phosphorylate
pRB, which removes the ability of pRB to bind to thereleasing expression of E2F gene promoter.
{Knudsen, 2008 #15310} pRB functions to regulate proliferation in most cell types.{Cobrinik, 2005
#15298} OftenI, n many cell types, loss of the RB1 gene is compensated by increased expression of
itsother related proteins., However, in certain susceptible cells,, such as the retinal cone cell precursors,
compensatory mechanisms are not sufficient, leading to uncontrolled cell division is uncontrolled, and
tumorigenesiscancer is initiated.{Xu, 2014 #9924}
Often the second mutational event in the retinal cell is loss of the second RB1 allele (LOH, loss of
heterozygosity).
Q4: What causes retinoblastoma to be unilateral versus bilateral?
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
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retinoblastoma as the prototypic cancer to derive the two-hit hypothesis.{Knudson, 1971 #11106} In
heritable retinoblastoma (sometimes called germline retinoblastoma), the first mutational eventRB1 allele
(M1) is mutant in all cells, including is inherited via the germinalgermline reproductive cells, while the
second eventallele (M2) occursis mutated in the somaticretina initiating cancer cells. Often the M2 event
in the retinal cell is loss of the normal RB1 allele and duplication of the mutant M1 allele (LOH, loss of
heterozygosity). 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
#9935;Wong, 2014 #15170} The clinical presentation of heritable retinoblastoma consists ofwith either
80% bilateral (80%),and 15-18% unilateral (15%) or trilateral (5%) tumors.{Dimaras, 2015 #10881}
Germline retinoblastoma carries an increased the risk of development of second primary cancers higher
than normal, most commonly osteosarcoma, and fibrosarcoma and melanoma. due to loss of RB1 gene.
This is why these children should be keptese personspatients can benefit from regular under surveillance
for such cancers for their lifetime. for the rest of their lives.
Of non-heritable retinoblastoma, 98% have both RB1 M1 and M2 ariseevents with in a retinal cell.
TIn non-heritable retinoblastoma (non-germline retinoblastoma) the majority (98%) of cases have somatic
biallelic RB1 loss in the tumor, while theIn the remaining 2% , have no the retinoblastoma is induced by
mutation in either copy of RB1 but instead have somatic amplification of the MYCN oncogene, in the
presence of normal RB1 genes.{Rushlow, 2013 #11102}” Germline retinoblastoma carries the risk of
development of second primary cancers, most commonly osteosarcoma and fibrosarcoma due to loss of
RB1 gene. This is why these children should be kept under surveillance for the rest of their lives.
Q5: Mother: What caused these mutations? Did I cause them?
No one is to blame for the mutations causing retinoblastoma. There are Many environmental causes in the
environment that can causeforces induce thisDNA mutationsdamage, including cosmic rays, X-rays, DNA
viruses, UV irradiation and smoking. and irradiation????. This is sporadic and cannot be anticipated
or prevented. TThe DNA damage may be here are many ways in which the function of the pRB is
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impaired including point mutations, small and large deletions, promotor methylation shutting down RB1
expression and rarely, chromothripsis.{Lohmann, 1999 #9272;McEvoy, 2014 #8499} The majority of
RB1 mutations arearise de novo, unique to a specific patient or family. y, However, there are some known
recurrent mutations are found in found across many unrelated individuals, such as those that affect . One
subset of recurrent mutations involve 11 sites CpG DNA sequence sites, which are hyper-mutable and
which make up ~22% of all RB1 mutations.{Rushlow, 2009 #10337;Richter, 2003 #11998}
When there is no family history of retinoblastoma, The origin of a de novo RB1 germline mutation
may can arise either pre- or post-conception. PMost often, pre-conception mutagenesis of RB1 usually
occurs during spermatogenesis, perhaps because cell division (and opportunity for mutation) is very
active during spermatogenesis, but not during oogenesis.{Zhu, 1989 #6514;Dryja, 1997 #15586;Munier,
1998 #10955} AFurthermore, advanced paternal age has been shown to increases risk for retinoblastoma,.
{Toriello, 2008 #15506} This might be due to the larger number of cell divisions during spermatogenesis
than oogenesissuggesting that or the increased rate for base substitution errors may increase in aging men
compared to women. In cases of pre-conception mutagenesis, The probandaffected child carries the de
novo RB1 mutation in every cell within their body, and typically presentings with 4-5 tumors and
bilateral retinoblastoma. In contrast, if post-conception RB1 mutagenesis occurs post-conception, during
embryogenesis,. Depending on the embryological stage of development, only a portion (1-50%) of cells
will carrying the RB1 mutation (ie. mosaicismand the person will be mosaic for the RB1 mutation)a few
or numerous tissues may be mosaic for the RB1 mutation. If the mutation arises al event occurs during
retinal development, the presentation is oftenchild will have unilateral retinoblastoma.{Dimaras, 2015
#10881}
Q6: Father: So, only RB1 mutation is sufficient forcauses retinoblastoma to
develop?
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?’
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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. There are two answers to this question: (i) RB1 mutation only causes a benign
precursor to retinoblastoma, retinoma, and other genes are modified to cause progression to cancer;
{Dimaras, 2008 #13250} and (ii) 2% of retinoblastoma have normal RB1 and are caused by a different
gene.
In addition to loss of RB1, specific alterations in copy number of other genes are common in RB1-/-
retinoblastoma. There are gains (4-10 copies) in oncogenes MDM4, KIF14 (1q32), MYCN (2p24), DEK
and E2F3 (6p22), and loss of the tumor suppressor gene CDH11 (16q22-24).{Corson, 2007
#9909;Theriault, 2014 #8591} Other less common genomic alterations in retinoblastoma tumors include
differential expression of specific microRNAs{Huang, 2007 #8613} and recurrent single nucleotide
variants/insertion-deletions in the genes BCOR and CREBBP.{Kooi, 2016 #14338} In comparison to the
genomic landscape of other cancers, retinoblastoma is one of the least mutated.{Kooi, 2016 #14338}”
There is a newly recognized form of retinoblastoma with normal RB1 genes.
In a small subset (Two percent 2%) of unilateral unilateral patients have RB1+/+ MYCNA tumors, with,
no RB1 mutation is identified. Instead, striking the MYCN oncogene is amplifiedcation (28-121 instead
of the normal 2 DNA copies)copies) of the MYCN oncogene is detected.{Rushlow, 2013 #11102}
PatientsThese children are with RB1+/+ MYCN are clinically distinct from RB-/- patients, showingdiagnosed
at median age 4.5 months compared to 24 months for non-heritable unilateral RB-/- patients, and the
tumors are much younger age at diagnosis, distinct histologically, features and larger, more invasive
tumorswith advanced features at diagnosis.
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}{Theriault, 2014 #19306} These regions contain
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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}
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?
Retinoma is a premalignant precursor to retinoblastoma with characteristic clinical features:
translucent white mass, reactive retinal pigment epithelial growthproliferation and calcific foci.{Gallie,
1982 #10343} Pathology of retinoma reveals fleurettess structures{Tso, 1970 #3456} that are not
proliferative.{Dimaras, 2008 #13250}. Genetic analysisComparison of retinoma and adjacent normal
retina, retinoma and retinoblastoma shows in retinoma loss of both RB1 alleles, and early genomic copy
number changes, that are amplified further in the adjacent retinoblastoma.{Dimaras, 2008 #13250} Many
retinoblastoma have underlying elements of retinoma. ItRetinoma can transform to retinoblastoma even
after many years of stability.{Theodossiadis, 2005 #5578}
Could we have discovered retinoblastoma earlier?
The only way to find retinoblastoma tumor early is to lookexamine the eye with specific expertise,
which we cannot do for every child. RetinoblastomaIf we know to look because a relative had
retinoblastoma, the smallest visible tumors starts as are a roundedround, white white retinal masslesions
that obscure the underlying choroidal pattern.
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} Next, tTumor seeds
floatspread out free of the main tumor a result of poor cohesive forces between tumor cells into the
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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. Advanced tumors may induce iris neovascularization. Rapid
necrosis of tumor can cause an aseptic orbital inflammatory reaction resembling orbital cellulitis,
sometimes showing central retinal artery occlusion.{Balmer, 2007 #8320;Balmer, 2006 #8323;Murphree,
2005 #11984} Untreated, retinoblastoma spreads into the optic nerve and brain, or hematogenous spread
occurs through choroid, particularly to grow in bone marrow. Direct tumor growth through the sclera can
present as orbital extension and proptosis.
The earliest signs of retinoblastoma detectable by parents are leukocorea (white pupil), either
directly or in photographs (photo-leukocorea) and strabismus when the macula is involvement by tumor.
{Balmer, 2007 #8320} In developing countries, buphthalmos and proptosis due to advanced and
extraocular disease respectively is common.{Canturk, 2010 #13461} Less common presentations include;
heterochromia irides, neovascular glaucoma, vitreous hemorrhage, hypopyon or aseptic orbital cellulitis.
{Balmer, 2007 #8320} Retinoblastoma (unilateral or bilateral) might be associated with a brain tumor in
the pineal, suprasellar or parasellar regions (Trilateral retinoblastoma){Popovic, 2007 #9156;Antoneli,
2007 #10877} with the median age of diagnsosis 17 months after retinoblastoma and before the age of 5
years. Retinoblastoma might present as 13q deletion syndrome, with facial features and various degrees
of hypotony and mental retardation.{Baud, 1999 #8118;Bojinova, 2001 #13205;Skrypnyk, 2004 #5276}
The main differential diagnosis includes Coats’ disease, persistent hyperplastic primary vitreous and
ocular toxicariasis.{Balmer, 2007 #8320}
Q8: Doeos all affected individuals with RB1 mutations develop retinoblastoma?
Depending on the exact RB1 mutation, most, but not all, carriers of an RB1 mutation will develop
retinoblastoma and other cancers throughout life.
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In heritable retinoblastoma, eachEach offspring of a patientperson carrying an RB1 mutant gene has a
50% risk ofto inheriting the RB1 pathogenic changemutant gene [Figure # Pedigree – full penetrance].
Typically, Nonsense and frame-shift germline mutations, which lead to absent ce of RB1 expression or
truncated dysfunctional pRB protein, result in 90% bilateral retinoblastoma 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,For partially functional RB1 mutant
alleles, reduced penetrance and expressivity and reduced penetrance is observed, with later onset and
fewer tumors{Soliman, 2016 #15159}, and . In these families, when retinoblastoma develops, it is often
late onset and less severe, presenting as unilateral, unifocal (reduced expressivity) and in some carriers
family member retinoblastoma never develop retinoblastoma.s (reduced penetrance). The types of
reported RB1 mutations that result in reduced expressivity or penetrance are diverse. Many consist
ofSome reduced penetrance mutations that reduced RB1 protein expression:. Examples include, (1i)
mutations in exons 1 and 2,{Sanchez-Sanchez, 2007 #6108} (ii2) mutations near the 3’ end of the gene in
exons 246 andto 27,{Bremner, 1997 #12040;Mitter, 2009 #7216} (iii3) splice and intronic
mutations{Zhang, 2003 #8986;Schubert, 1997 #4830;Lefevre, 2002 #4903} and (iv4) missense
mutations.{Scheffer, 2000 #15178;Cowell, 1998 #10958} In additionStrangely, large deletions
encompassing RB1 gene and MED1 gene also cause reduced expressivity/penetrance, because RB1-/- cells
cannot survive in the absence of MED4..{Dehainault, 2014 #12140;Bunin, 1989 #4280} 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 grosslarge
deletions with one breakpoint in the RB1 gene whom typically present with bilateral disease.{Mitter, 2011
#7339;Matsunaga, 1980 #357;Albrecht, 2005 #10898} The severity of risk can be evaluated throughA
measure of expressivity of a mutant retinoblastoma allele is the disease-eye-ratio (DER) (calculated by
taking the number of eyes affected with tumors divided by the total number of eyes ofin carriers within
the familyof the mutation).{Lohmann, 1994 #10954}
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In some instances of hereditable reduced expressivity/penetrance retinoblastomaThere are two
specific RB1 mutations showing a parent-of-origin effect: intron 6 c.607+1G>T substitution{Klutz, 2002
#8593;Schuler, 2005 #5551} [Figure # Pedigree – reduced penetrance family with parent of origin….]
and c.1981C>T (p.Arg661Trp).Eloy et al{Eloy, 2016 #12079} proposed a potential molecular mechanism
to explain the parent-of-origin effect. Using the c.1981C>T (p.Arg661Trp) reduced
penetrance/expressivity missense mutation,mutation; Both may be explained by the researchers
discovered that differential methylation of the intron 2 CpG85, which skews RB1 expression in favor of
the maternal allele.{Buiting, 2010 #7661;Kanber, 2009 #16381} In other words, whenWhen the
p.Arg661Trp allele is maternally inherited there is sufficient tumor suppressor activity to prevent
pRBretinoblastoma development and 90.3% of carriers remain unaffected. However, when the
p.Arg661Trp allele is paternally transmitted, very little RB1 is expressed, leading to haploinsufficiency
and pRB development retinoblastoma in 687.5% of casescarriers. A similar inheritance pattern was also
reported for intron 6 c.607+1G>T substitution.{Klutz, 2002 #19004} [Figure # Pedigree – reduced
penetrance family]
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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??????
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 nose associated 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 governs the choice?
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;Mallipatna, 2017 #14252} The 2017 TNMH classification is based on international consensus
and evidence from an international survey of 1728 eyes, and separates more clearly s, with algorithms
evaluating initial clinical and pathological features andrelevant to outcomes, in retrospective comparison
to by 5 differentprevious eye staging systems.{Mallipatna, 2017 #14252} (Table X)
Retinoblastoma is the first cancer in which staging recognizes the impact of genetic status on
outcomes: presence of a positive family history, bilateral or trilateral disease or high sensitivity positive
RB1mutation testing, is stage H1; without these features orbfore 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 2M1 and M2 RB1 mutant alleles inof the bloodtumor that are not detectable
in blood, but with remaining low risk (<1%) of mosaicism. reducing risk of a heritable RB1 mutation to
<1%.
Multiple treatments are now available and the Choice of treatment depends on the laterality of
disease, and tumor stage and genetic status.he grouping Focal therapy only can control cT1a eyes, but
visually threatening or large cT1b tumors and cT2 eyes need chemotherapyof the tumor. Chemotherapy
(systemic or intra-arterial chemotherapy) to reduce the size of the tumor followed by consolidation focal
therapies (lLaser therapy or cryotherapy) isas the main stay ofinitial treatment. Enucleation forof eyes
with advanced tumors or in unilateral disease where the other eye is normal is more appropriate anda
definitive cure.{Dimaras, 2015 #10881}. OtherAncillary therapies for specific indications include plaque
17
radiotherapy and periocular chemotherapy. includeIntravitreal chemotherapy for vitreous disease has
recently dramatically improved safe eye salvage.{Munier, 2012 #8588;Munier, 2012 #8587} For persons
carrying RB1 mutations, ; intravitreal chemotherapy for vitreous disease, plaque radiotherapy or
periocular chemotherapy. eExternal beam radiation therapy has extremely limitedis rarely indicated ions
nowadays due to its extensive cancer risks and complicationsthe high risk of inducing later second
cancers.{Dimaras, 2015 #10881}
The main concept of treatment is that life salvageSaving life is is the main priority duringof
retinoblastoma treatment, planning followed by vision salvage; and the least important is eye salvage.
That’s why we prefer enucleation in advanced unilateral intraocular retinoblastoma with low visual
potential. The child’s job at this point is to play and enjoydevelop in a healthy life; away of all the many
procedures and their complications that may span over a couple of years for aat best a 50% chance to save
a blind eye andwith 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
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.
18
Q11: Is retinoblastoma lethal?
If untreated, retinoblastoma is lethal. If treated before metastasis occurs, there is a nearly acure is nearly
100% chance of life salvage. 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 options
becomes more challenging butand there is around 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 leiomyosarcoma, osteosarcoma, and fibrosarcomaand melanoma.{MacCarthy,
2013 #11093} When the enormous impact of external beam irradiation was recognized after 30 years of
being used on every child, it was recognizeddiscovered that more children with bilateral retinoblastoma
(H1) were dying of their second (third, etc) cancer than of retinoblastoma.{Eng, 1993 #10933}
SometimesOccasionally metastatic retinoblastoma may be confused with a second cancer; it might be
confused with metastatic retinoblastoma. Fine needle aspiration blue round cell tumors on cytopathology
has minimal role in differentiation as both the metastasis from and second cancers that appear asmay not
differentiate from retinoblastoma, but molecular demonstration of the same RB1 mutations as the
intraocular retinoblastoma will confirm metastases blue round cell tumors.. Mmolecular analysis might
help to distingguishfferentiate.{Racher, 2016 #13990}
19
Q12: How can we test for retinoblastoma mutations?
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 in DNA extracted from blood is high (example - 97% detection rate in a comprehensive RB1
laboratory). For this reason, the most optimal strategy for testing bilateral patients involves first testing
genomic DNA extracted from peripheral blood lymphocytes (PBL). In rare3% instances of bilateral
retinoblastoma patients, the predisposing RB1 mutation cannot be detected.hasmay be mosaic at a low
level occurred sometime during embryonical development. In these instances, Iidentification of M1 and
M2 RB1 mutations in 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 can leadassist in to the identification of a germline mutation,
including low level mosaic mutationsshould be investigated.{Astudillo, 2014 #10893;Rushlow, 2009
#10337;Canadian Retinoblastoma, 2009 #14251}
In contrast, given thatSimilarly, to detect the approximately 15% of unilateral patients carrying a
germline mutation, the the most optimal strategy is to first test testtumor DNA , thenand then
checkinvestigate for thosethese mutations in bloodextracted 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 onlyIf the tumorblood is not found to carry theone of
the tumor RB1 mutationss, this result dramatically reduces the risk of germline status is reduced to <1%
(Table) for parents, 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 impactsQuality of genetic results depends on the quality of DNA. For best
results, Fresh or frozen tumor samples are fineideal, but should be collected, as opposed to formalin fixed
paraffin embedded tumors generally produce, in which DNA is often highly degraded DNA., making it
20
often too fragmented for use in some molecular diagnostic methods. With regards to For blood 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.{provide high quality DNA.{Banfi, 2007 #15789}Banfi, 2007
#19549}
The Technologies and techniques: Given that there are many ways in which the RB1 gene can be
mutated in many ways, best identified by a series of, 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
usingby 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.
Deciding on whichThe most appropriate technology to use depends on the clinical question being asked..
NGS may be the most effective screening strategy to investigate for an unknown de novo mutation in an
affected proband, and may have a much lower limit of detection (analytic sensitivity) for identify low
level mosaic mutations in comparison to Sanger sequencing.{Chen, 2014 #14457} WhenTo screening
family members for a known sequencing-detectable RB1 mutation, targeted Sanger sequencing is more 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
#14457}
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
21
comparative genomic hybridization (aCGH) areare often used to to interrogate foridentify RB1 large
deletions (ex. 13q14 deletion syndrome) and duplications, and. 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
whichsuggest that NGS data can be interrogated for copy number variants,.{Devarajan, 2015 #9654;Li,
2016 #14646} While the data is promising; the current limitation of targeted NGS is thatbut sensitivity is
not yet optimized. 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. Allele-specific PCR (AS-PCR) has excellent sensitivity when the RB1 mutation is known,
{Rushlow, 2009 #10337} and with 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 mutations mosaicism levels as low as 1%.
mosaicism.
Microsatellite analysis: The second mutational event in the majority70% of retinoblastoma tumors is
consists of loss of heterozygosity (LOH),. LOH isa common event in many cancers and is strongly
associated with loss of the wild-typenormal allele in tumor from 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. 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 usefulimportant in identity
testing and in determining the presence of maternal cell contamination in prenatal diagnostic testsing.
22
Methylation analysis: In addition to genetic changes, Epigenetic changes have been recognized as
another mechanism ofcan also initiate retinoblastoma development.{Ohtani-Fujita, 1993 #2258}
Hypermethylation of the RB1 promoter CpG island results in transcription inhibition of the RB1 gene
transcription in and has been identified in 10-12% of retinoblastoma tumors, commonly involving both
alleles.{Richter, 2003 #11998;Zeschnigk, 1999 #15496} This epigenetic eventgene silencing event
primarily occurs somaticallyin somatic cells, howeverbut, rare instance of heritable RB1 promoter
mutations in the RB1 promoter and translocations disrupting RB1 regulatory sites or translocations
involving the X chromosome, have been reported to alsomayhave been shown to cause constitutional
RB1 promoter hypermethylation.{Quinonez-Silva, 2016 #12111} Occasionally hypermethylation of the
RB1 promotor is found in blood, strongly suggestion the presence of a translocation, for example to the X
Chromosome. (Jones et al 1997 PMID: 9199583)
RNA analysis: In rare instanceRarely, 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 tothat disrupt RB1
transcription by interfering with correct splicing in patients with retinoblastomretinoblastoma can a.
{Zhang, 2008 #7502;Dehainault, 2007 #5872} In order to investigate for deep intronic changes,are bestbe
detected by analysis of the RB1 transcript by reverse-transcriptase PCR (RT-PCR) is performed.{Zhang,
2008 #7502;Dehainault, 2007 #5872} RNA studies are also useful in clarifying the pathogenicity of
intronic sequenceing alterations. detected by conventional DNA sequencing. {Zhang, 2008
#7502;Dehainault, 2007 #5872} Alternatively, As NGS sequencing costs continue to decrease,; whole
genome sequencinge (WGS) may become the method of choice to become the method of choice to
uncover deep intronic changes.
Protein studies
23
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 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 todiscover assess forcan be used to investigate for balanced translocations, which increases the in
carriers, which increases the informing about risk for k of recurrenceretinoblastoma in subsequent
offspringgenerations.{.{Baud, 1999 #8118}
Q13: Are these tests available worldwide?
No, they are mainly present in developed countries.{, 2015 #10824} In China, many families with
retinoblastoma children do not understand the benefits of genetic testing and genetic counseling in
treatment and follow-up.{Dimaras, 2015 #10881} MeanwhileIn addition, the health insurance canno’t
cover the cost for ittesting. SoGiven all the obstacles, mentioned above result in thethere is limited
application of genetic testing and genetic counseling nationwide, which also lead to the redundant
economic burden on the affected families. TRecently, the Chinese government started a new policy that
allowsed every family the ability to have one more child nowadays. Therefore, genetic testing and
genetic counseling should be put into good use, especially for the families carrying thea 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 defectiveinsufficient 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}
24
Q14: What is done after finding the RB1 mutation?
Targeted familial testingFamily 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} is used to determine if a predisposing RB1 mutation has occurred de novo, through investigation
of parental DNA from PBL is investigated. Even If neither parent is identified to be a carrierthe mutation
is found in neither parent, a small risk offor low level mosaicism still exists, leaving a low level risk for ,
recurrencesiblings risk in siblings is still increased due to the risk of germline mosaicism. Offspring of
any family member carrying the RB1 mutation can be tested during pregnancy or immediately after birth
(see below). DNA from PBL for all siblings of affected patients should be tested for the proband’s
mutation. As well, DNA from PBL for children of all affected patients’s should also be tested for the
predisposing mutation. Table Y shows the risk of having retinoblastoma in different family relatives.
If the proband is mosaic for the RB1 ’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, since mosaicism cannot be inherited.
to carry the predisposing mutation. However, the children of a mosaic proband shouldneeds to be
testinged as early as possible, be tested, as their risk ofto inheriting the predisposing RB1 mutation canis
up to be as high as 50%; if they do carry the mutation, they are at population risk for bilateral
retinoblastoma. depending on the mutation burden in the probands germline.
When a RB1 mutation has been identified in a family, tThe Kknown 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 upcomingfuture child? I am 33
weeks pregnantren?
GPrenatal genetic testing is usuallycan be performed early in the course of the pregnancy and is
available in many countries around the worldwide. Two early procedures are available: i1) chorionic
villus sampling (CVS) and 2) amniocentesis. CVS is a test typically performed between 11-14 weeks
25
gestation, which involves during which as obtaining a sample of the placenta by is obtained either by
trans-vaginally or trans-abdominally approach; . and ii) amniocentesisAmniocentesis is a test performed
after 16 weeks of gestation, which involving wherebyobtains as sample of the amniotic fluid trans-
abdominallyis gathered with a transabdominal approach. CVS has aThe procedure-associated risk of
miscarriage of CVS is ~1%, while of a. Amniocentesis is has a procedure-associated risk of miscarriage
between 0.1-0.5%. Though uncommon, there is a risk for Maternal cell contamination of the sample is
that occurs more frequently with CVS,.{Akolekar, 2015 #9479} and is checkedassessed for inby the
clinical molecular lab.
Genetic testing results can be used by the family and health care team to manage the pregnancy. If the
fetus does not carry the 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
thefetus carries the familial mutation, is identifiedthe parents have several choices. , Some couples may
consider decideing to stop the pregnancy, while; others couples willmay know they wish to decide to
continue the pregnancy regardless of test results. If the parents are concerned byofby the risk of
miscarriage associated with early invasive prenatal testing. Where available, couples can also they can
consider the option of late amniocentesis , performed 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 RB1 mutation) or “H1”, confirmed
to carry the mutation. with the pregnancy and apply appropriate interventions,If the fetus has the familialy
RB1 mutation, such as early deliveryearly pre-term delivery achieves showed lesssmaller tumors and
treatment burden with higher treatment success, eye preservation and visual outcome .{Soliman, 2016
#15159} than delivery at full term, 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
26
available, couples can also consider the option of late amniocentesis, performed between 30-34 weeks
gestation. When amniocentesis is performed late into the pregnancy, the key complication becomes early
delivery rather than miscarriage.{Akolekar, 2015 #9479} The risk for procedure-associated significant
preterm delivery is low (<3%). Results of genetic testing will be available with enough time to plan for
early delivery when a mutation has been inherited.
In many countries around the world, the option for prenatal genetic testing is not available, and. Even
where available, some couplesparents may electchoose to not do prenatal no invasive testing during the
course of the pregnancy. For these conceptions, if the pregnancy isIf the risk for retinoblastoma in the
fetus is at 50% risk for inheriting a RB1 mutation, it is crucialit is important that the pregnancy does does
not go post-datesnot go past 40 weeks. 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?
ThisRB1 mutation detection can be performed either Prenatal, as discussed earlierpreviously, 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 intoto be 100% risk. Both
screening methods are effective in improving visual outcome and eye salvage thancompared to non-
screened children., However, prenatal screening allows for planning for earlier delivery in positive
children (late preterm/early term); this was shown to have less number of tumors at birth (20% versus 50
%) with only 15 % visual threatening tumors in prenatatlprenatal screening. Prenatal screening with early
delivery showed less tumor and treatment burden with higher treatment success, eye preservation and
visual outcome.{Soliman, 2016 #15159}
27
Preconception testing Q17: Can we plan our next pregnancy to avoid
havingpassing on this RB1 mutation?
In In sSome countries around the world, preimplantation genetic diagnosis (PGD) with there is an in vitro
fertilization is an option available to couples called preimplantation genetic diagnosis (PGD).{Dhanjal,
2007 #9216;Dommering, 2004 #10248;Xu, 2004 #9246;Girardet, 2003 #9219} For PGD, a couple
undergoes in vitro fertilization. Conceptions are tested for the familial mutation at an early stage of
development (typically 8 -cells) for the presence of the familial mutation. OnlyThose those conceptions
that do not carry the RB1 mutation will beare used for fertilizationimplanted. The procedure is costly,
ranging from $10,000-$15,000 per cycle; . Inin 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 tThe full medical
implications of PGD are not yet understood; there is emerging evidence that there may beof a low risk for
epigenetic changes in the conception as a result of the procedure. For couples that undergo PGD, itIt is
recommended that typical prenatal testing be pursued during the course of the pregnancy to confirm the
results.{Dhanjal, 2007 #9216;Dommering, 2004 #10248;Girardet, 2003 #9219;Xu, 2004 #9246}
Molecular Screening for Retinoblastoma
WQ168: what is genetic counseling?
Genetic counseling is both a psychosocial and educational process for patients and their families to with
the aim of helpinghelp them 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} When genetic testing is not available or unaffordable, genetic counseling is still very important.
. 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 supports informed and
precise genetic counseling and results inthe specificity, reducesing the need for other possible scenarios to
28
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?
In countries Where genetic testing is not available or unaffordable, genetic counseling is the option. It
was found that Genetic testing to support precision medicine for only those who need it (carry the RB1
mutant allele) is more cost effective than examining all the at-risk family members.{Noorani, 1996
#13637;Richter, 2003 #11998}
Q17: what are the risks for the relatives in the family?
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 tumourss. 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
29
families. Genetic testing companieslaboratories 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 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 Bblood 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?
Today there is no prevention of retinoblastoma, only treatment when the tumors are very small, even
invisible. But ……..bg to write
30
Q18: What are the long term risks for germ line RB1 mutation?
Are these tests available worldwide?
High sensitivity RB1 mutation testing is established in core labs mainly in high-income countries.{, 2015
#10824} In low- and middle-income countries, genetic counseling as a specialty does not exist, so many
families with retinoblastoma children do not understand the benefits of genetic testing and counseling in
retinoblastoma treatment and follow-up.{Dimaras, 2015 #10881} In many places, existing health
insurance does not cover costs of genetic testing. Given all these obstacles, there is limited application of
genetic testing and genetic counseling worldwide, overall health care costs are increased to deal with late
diagnoses, increasing economic burden on affected families. The Chinese government’s new policy to
allow families to have one more child will make the need to genetic testing and counseling even more
iimportant. A novel international collaboration between the companies Impact Genetics in Canada and
Geneseeq in China is optimizing expertise to achieve high quality RB1 testing for families.
In Egypt{Soliman, 2016 #14713} genetic testing for retinoblastoma is not available and genetic
counseling is the only way to address the issues. Ophthalmologists with insufficient training in
retinoblastoma genetics are burdened with the task. Genetic counseling was found to increase knowledge
gaps remained in translation of this knowledge into appropriate screening action.{Soliman, 2016 #14713}
31
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 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…..
32
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
33
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 number changes that are amplified further in the adjacent
retinoblastoma. {Gallie, 1982 #5686}{Theodossiadis, 2005 #5649}
Clinical Features
34
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
35
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.
Grouping/Retinoblastoma Cancer Staging
36
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
37
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
38
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
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
39
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]
40
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 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
41
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
42
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 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.
43
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
44
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.
13q deletion syndrome
45
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}
46
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
sufficient and tumorigenesis is initiated (Xu et al 2014 – Nature – Rb
suppresses human cone-precur PMID).46
-?A and B pockets
47
-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
48
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
Promoter methylation
Hot-spot mutations – CpG transition
49
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
50
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
51
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
52
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
53
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 (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
54
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.
55
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 .
56
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.
57
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%.
58
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.
59
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
60
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
61
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
Genetic Counseling (Heather/Hilary)
Importance of high detection rate
Targeted familial testing/prenatal testing, preconception testing
62
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.
63
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
64
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.
65
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
66
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 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
67
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.
68
Patients with bilateral retinoblastoma at presentation are presumed to
have heritable retinoblastoma and a RB1 mutation. Genetic testing
provides more accurate information about the type of heritable
retinoblastoma and allows for straightforward testing to determine if
additional family members are at risk. Through genetic testing, a
patient may be found to have a large deletion extending beyond the
RB1 gene as part of the 13q deletion spectrum. Individuals with 13q
deletion syndrome are at risk for additional health concerns requiring
appropriate medical management and intervention. Results may
reveal a mosaic mutation which indicates that the mutation is
definitively de novo; only the individual’s own children are at risk and
no further surveillance or genetic testing is needed for other family
members. The results may find a low-penetrance mutation which
indicates the patient is at reduced risk to develop future tumours. As
genetic testing for retinoblastoma becomes more common place and
data accumulate, surveillance of the proband may one day be
matched more precisely to the level of risk for new tumours for
individuals with low penetrance mutations.
69
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
70
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
71
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
72
Benefits of genetic testing for the proband and family members
[Heather]
Prenatal vs Postnatal [Sameh]
Cost-effectiveness [Brenda/Crystal] {FIGURE/FLOW CHART}
Difficulties and opportunities across different jurisdictions/countries
[Jeffry/Sameh]
Compare/contrast Canada vs China vs Jordon
Societal/cultural challenges to GC
Conclusions
Retinoblastoma genetics is challenging to understand, but once understood It largely affect the level
of a core element in care presented toof retinoblastoma patients and their families. ItAction based on
knowledge of genetics in retinoblastoma improves outcomes for the eye , life and points to life and
treatment strategies to reduce early mortality and alleviate suffering. The whole family benefits
economically and in health, by precision in diagnosis of risk, based on testing, to establish who carries the
RB1 cancer-predisposing gene (H1), who does NOT carry the gene (H0), who has <1% risk to carry the
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gene (H0*) and who is not tested so at unknown risk (HX). Knowledge of the test resultshelps alleviates
the psychological burden ofon the families regarding moving forward with their life choices regarding the
affected child and future siblings and exposure to environmental carcinogens for the whole family. It also
helps the family to understand the risks of different family members giving them the chance of the level
of disclosure they wish.
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REFERENCES
Uhlmann, WR; Schuette, JL; Yashar, B. (2009) A Guide to Genetic Counseling. 2nd Ed. Wiley-
Blackwell.
Shugar, A. (2016) Teaching Genetic Counseling Skills: Incorporating a Genetic Counseling
Adaptation Continuum Model to Address Psychosocial complexity. J Genet Counsel. Epub ahead of print.
PMID: 27891554 DOI: 10.1007/s10897-016-0042-y
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Table X:
Subretinal Fluid (RD)
No≤ 5 mm
>5 mm - ≤ 1 quadrant
> 1quadran
t
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/CcT2b/
C cT2b/C cT2b/Ddiffuse vitreous/subretinal seeding cT2b/D
High
risk
feat
ures
Phthisis or pre-phthisis bulbi cT3a/ETumor invasion of the pars plana, ciliary body, lens, zonules, iris or anterior chamber cT3b/ERaised intraocular pressure with neovascularization and/or buphthalmos cT3c/EHyphema and/or massive vitreous hemorrhage cT3d/EAseptic orbital cellulitis cT3e/EDiffuse infiltrating retinoblastoma ??/E
Extraocular retinoblastoma cT4/??
clinical T (cT) versus International Intraocular retinoblastoma Classification (IIRC) (cT/IIRC); ?? Not
applicable ; RD Retinal detachment
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