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Genetics and Molecular Diagnostics in

Retinoblastoma - An Update

Authors:

Sameh E. Soliman, MD,1-2 Hilary Racher, PhD,3 Chengyue Zhang, MD,4

Chengyue Zhang, MD.

Hilary Racher, PhDHeather MacDonald,1 ,5 Brenda L. Gallie, 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:

Gallie Brenda, 01/05/17,

Organizing Text: Number the pages of the manuscript consecutively, beginning with the introduction as page 1. The text of an original article should not exceed 4,000 words with up to 8 images and tables and 50 references while that of a review article should not exceed 6,000 words with up to 8 images and tables and 100 references. The text of an annual review should not exceed 15,000 words with up to 200 references.


Title page: Include on the title page (a) complete manuscript title; (b) authors’ full names, highest academic degrees, and affiliations; (c) name and address for correspondence, including fax number, telephone number and email address; (d) address for reprints if different from that of corresponding author; and (e) sources of support that require acknowledgement.

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

Sameh Gaballah, 05/01/17,

Plz Brenda add your preferred phone for publications

mailto:[email protected]

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

4


Review articles should emphasize new developments and areas of controversy in clinical or laboratory ophthalmology. An unstructured abstract of no more than 250 words should be submitted on a separate page.

4134/56000 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

5


Organizing Text: Number the pages of the manuscript consecutively, beginning with the introduction as page 1. The text of an original article should not exceed 4,000 words with up to 8 images and tables and 50 references while that of a review article should not exceed 6,000 words with up to 8 images and tables and 100 references. FOR US 5,000 WORDS AND 80 REFS


unfinished


Delete redundant or not so important ones

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

Sameh Soliman, 01/05/17,

I have DD in mind I was thinking of having images of both her eyes but I found the imaging not done by cynthia or Leslie. I think very poor quality for publication. BG Not sure who you mean…….


We have not included floolowup for the eyes OR surveillance for second cancers.

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

8

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

9

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?’

10

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

11


Hilary, please write a sentence to describe the epigenetic changes in brief.

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

12

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.

13

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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Can we delete unilateral?

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]

15

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}

16

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


This whole nice little “editorial” does not really belong in this review of genetics…..?????

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


Hilary and heather, I know we have one case at least and there was one in the literature which I can not find!Can you look?

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


References Jeffrey.

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

Hilary Racher, 01/05/17,

Should I use the new Skalet table instead?


Can you write this down Hilary. I think this already available on Impacts website.

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, of the genetic test reducesing the need for other

28


CDN or US $?

possible scenarios to be discussed. with the family. This enhances the educational component of genetic

counseling and also provides further time for psychosocial support to be provided to the family.

Q19: Can genetic counseling suffice alone? If yes, what are the benefits of genetic testing?

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


Is it presumed or sure?

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


I find this a common question asked. We can answer Not yet, but there is research going on to target the epigenetic changes and MYCN. If approve this question, Hilary can add a line here ?She will add the epigenetic changes previously and can add what possible mechanisms here.

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


Jeffry - Build more details from the Dimaris Nature Primer paper into this paragraph


I would remove eye salvage because in developing countries the concept of eye salvage before mortality should be changed. I prefer to speak on mortality only as the paragraph started.


I still think that this is an over-statement. I don’t think this number is true? What is your reference here Jeffrey??

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


I would recommend a figure to show clinical features. BG: NO, this will be elsewhere in the revie issue. We chould stick to genetics.BG: not sure, this is one article in a review issue where we are assigned the genetics…..

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


Table attached

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


Sameh – define true H0 (*) vs most likely H0

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


Sameh – add section on retinoma

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


Can we delete unilateral?


Please Hilary, can we rephrase to a simpler sentence?

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


I preferred putting this here. Open for discussion.

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


Moved to RB1 mutation section


Hilary, Can you please write a small paragraph explaining this with citations?

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


-?A and B pockets-Also describe the role in genomic instability (Demaris. Rushlow)


I think this comes after Knudson hyposthesis and before the penetrance.

-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


Hilary, please organize this part

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


I am finding difficulty citing here Hilary. Can you please give me any hints about the papers?

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


Reference?

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


Is it presumed or sure?


I need a hint to references from here.

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


References Jeffrey.

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

73

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.

74

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

75

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

76


Sameh this is wrong!

introduction - sharedocs.ca file · web viewword count : (/5000) key words ... rb1 gene, bilateral,...

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