Prenatal versus Postnatal Expectant management versus

conventional screening in infants neonates withfor familial

retinoblastoma

Sameh E. Soliman, MD,1,2 Helen Dimaras, PhD,1,3,4,

11

Vikas Khetan, MD, BS,1,5 Jane A.

Gardiner, MD, FRCSC,1,6 Helen S. L. Chan, MB, BS, FRCPC,7,8 Elise Héon, MD, FRCSC,1,3,9

Brenda L. Gallie, MD, FRCSC1,3,9,10

Corresponding Author: Dr. Brenda Gallie at the Department of Ophthalmology and Vision

Sciences, the Hospital for Sick Children, 555 University Avenue, Toronto, ON M5G 1X8,

Canada, or at brenda@gallie.ca

Authors’ Affiliations:

1Departments of Ophthalmology & Vision Sciences, The Hospital for Sick Children, Toronto,

Canada

2Ophthalmology Department, Faculty of Medicine, Alexandria University, Egypt.

3Department of Ophthalmology & Vision Sciences, Faculty of Medicine, University of Toronto,

Toronto, Ontario, Canada.

4Division of Clinical Public Health, Dalla Lana School of Public Health, University of Toronto,

Toronto, Ontario, Canada.

5Sankara Nethralaya Hospital, Chennai, India.

6Department of Ophthalmology and Vision Science, University of British Columbia, Vancouver,

British Columbia, Canada

7Division of Hematology/Oncology, Pediatrics The Hospital for Sick Children, Toronto, Canada

8Department of Pediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada.

9Division of Visual Sciences, Toronto Western Research Institute, Toronto, Ontario, Canada.

10Departments of Molecular Genetics and Medical Biophysics, Faculty of Medicine, University of

Toronto, Toronto, Ontario, Canada.

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

11

Child Health Evaluative Sciences, SickKids Research Institute, Toronto, ON, Canada

Financial Support: None

Conflict of Interest: No financial conflicting relationship exists for any author. BLG is an unpaid

medical director for Impact Genetics Inc.

Running head: Expectant management inPrenatal screening for familial retinoblastoma

Word count: 2746 / 3000 words

Numbers of figures and tables: 4 figures and 2 tables; 1 supplementary table

Key Words: prenatal retinoblastoma, retinoblastoma gene mutation, RB1, molecular testing,

late pre-term delivery, near-term delivery, amniocentesis

Meeting Presentation: Association for Research in Vision and Ophthalmology, Seattle,

Washington, USA, 2016

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Précis: 35/35

Expectant management of familial retinoblastoma (Pprenatal RB1 mutation detection and planned early-

term delivery) was advantageous to infants with familial retinoblastoma, achieved was associated with

more often no tumors at birth, earlier tumor detectionsince more had no tumors at birth, needed less

invasive therapies and achieved better visual outcomes, compared than to those conventionally managed.

postnatal screening. in familial retinoblastoma,

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Abstract (34834513/350 words)

OBJECTIVE To compare overall outcomes of postnatal screening for children withinconventional

managementpostnatal screening of familial retinoblastoma conventionally screened postnatal, compared

toto prenatal those expectantly managedscreening with prenatal RB1 mutation identification and planned

early term delivery.

DESIGN A retrospective, observational study.

PARTICIPANTS Twenty children with with familial retinoblastoma, born between June 1996 and June

2014, examined within first week after birth and cared for at The Hospital for Sick Children (SickKids)

Toronto, Canada.

METHODS Conventional Cohort 1 included spontaneously delivered infants neonates screened

examined within one week of birth and confirmed to carry their family’s RB1 mutant allele who had

postnatal RB1 testingpost-natally. Expectant Cohort 2 consisted of infants identified by amniocentesis to

carry their relative’s knownfamily’s RB1 mutant allele, who wereand scheduled for early term delivery

(36-38 weeks gestation). All children received Ttreatment for eye tumorsretinoblastoma was performed at

The Hospital for Sick Children, (SickKids) Toronto, Canada.

MAIN OUTCOME MEASURES Main outcome measures were gestational age at birth, age at first

tumor in each eye, eye stage, treatments given, ocular salvage, treatment success (defined as avoidance of

enucleation and/or external beam irradiation), visual outcome, number of anesthetics, pregnancy or

delivery complications and estimated treatment burden.

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

RESULTS Vision-threatening tumors were present at birth in 50% (4/8) of Conventional Cohort 1

infants, and 25% (3/12 ) of Expectant Cohort 2 infants. Eventually, aAll infants eventually developed

tumors in both eyes. At first treatment, 12% (1/8) of Cohort 1 had eyes Group AA T1a/T1a or A0 T1a/T0

(smallest and least vision-threatening tumors) compared to 67% (8/12) of Cohort 2 (Fisher Exact test

(FET), p=0.02). Null RB1 germline alleles induced earlier tumors than low-penetrance alleles (FET,

p=0.03). Treatment success was achieved in 38% (3/8) Cohort 1 patients compared to 92% (11/12)

Cohort 2 patients children (FET, p<0.002). Acceptable vision (better than 0.2) was achieved for 50%

(8/16) of Cohort 1 eyes compared to 88% (21/24) of Cohort 2 eyes (FET, p=0.014). Useful vision (better

than 0.1, legal blindness) was achieved for 88% (8/9) of Cohort 1 children compared to 100% (12/12) of

Cohort 2 children. There were no complications related to early term delivery. The median follow-up was

5.6 years 5.6 yearsC.ohort and 5.8 years Cohort 2.

CONCLUSIONS AND RELEVANCE When a parent had retinoblastoma, expectant management with

prenatal molecular diagnosis with late preterm/nearearly -term delivery increased theincreases the

likelihood of infants born with no detectable tumors, and better vision outcomes with less invasive

therapy. Prenatal molecular diagnosis facilitates anticipatory planning for both child and family.

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Retinoblastoma, the most common primary ocular malignancy in children, is commonly usually

initiated when both alleles of the RB1 tumor suppressor gene are inactivated in a precursor retinal cell,

followed by progressive mutations in other specific genes.1,2 Both alleles may be lost only in the retinal

cell from which one tumor arises (non-heritable retinoblastoma), or, in 50% of children, a germline RB1

mutation (50% of children) predisposes to development of multiple retinal tumors during childhood and

other cancers later in life. Ten percent of patients inherit a family-specific mutation from a parent.1,3

Children with an RB1 germline mutation may have retinoblastoma tumor(s) at birth, often in the

posterior pole of the eye, where they threaten vision.4-7 Focal laser treatments near the optic nerve and

macula may compromise vision, making treatment of these small tumors difficult. Most of these children

are bilaterally affected, with either simultaneous or sequential detection of tumors.4,6 Later developing

tumors tend to be located peripherally where focal treatment does not affect vision.6,8 Low penetrance

(10% of families)3 and mosaic9 mutations result in fewer tumors and more frequent unilateral phenotype.9

The timing of first tumors after birth has not yet been studied.

The new TNMH (Tumor, nNode, mMetastasis and Heritability) classification for retinoblastoma

replaced the international intraocular retinoblastoma classification (IIRC)10 withand its modifications and

inconsistencies11,12 to accurately predict the prognosis. T1a represents the non-visually threatening tumors

while T1b represents the visually threatening tumors; they corresponds, corresponding to IIRC10 group A

and B eyes, respectively. T2 and uphigher represents the more advanced tumors, with tumor seeding,

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

higher risk features and up to extra-ocular spread. Retinoblastoma is the first tumor to be classified

according to its heritability reflecting the higher impact of the genetic status on management. (Table 1)

It is recommended that infants with a family history of retinoblastoma be examined for tumor

detection and management treatment as soon as possible after birth and repeatedly for the first few years

of life, often (conventional screening), including under general anesthesia.13Early diagnosis when tumors

are small (cT1) and treatable with less invasive therapies is thought to optimize salvage of the eye and

vision.5,6,14 We have managed familial retinoblastoma by screening the fetus for the RB1 mutation of the

proband parent. If the child carries the RB1 mutation and has a near 100% risk to develop bilateral

tumors, we suggest delivery at early full term (37 weeks of gestation){13,15 with full retinal examination on

day 1. Further management is conventional screening and treatment.

The aim of this study it was to performWe present a retrospective comparative review of outcomes of

children examined within one week of birth and shown to carry their family’s RB1 mutant allele post-

natally, compared to those found to carry their family’s RB1 mutant allele on prenatal testing and

delivered early deliveryexpectant prenatal genetic diagnosis vs versus conventional postnatal

management of children with familial retinoblastoma. We hypothesized that earlier diagnosed

retinoblastoma tumors would be smaller and easier to treat with better visual outcome.We show that

children with expectant management had earlier detection and treatment of small tumors, lower treatment

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

morbidity, and better tumor control and visual outcome, compared to children born spontaneously prior to

genetic diagnosis.

Methods

Study Design

Research Ethics Board approval was obtained from The Hospital for Sick Children (SickKids). The

Study was in accordance with study conformed to the tenets of the Declaration of Helsinki. Family

privacy was preserved by implementing a coding system to hide personal identifiers during data

collection and the code key was accessed only by SS and BLG. Data collected for children with familial

retinoblastoma (all H1 children will be included, Table 1) born between 1 June 1996 and 1 June 2014

included: relation to proband; laterality of retinoblastoma in proband; sex; gestational age at birth;

pregnancy, prenatal abdominal ultrasound (if done); delivery or perinatal complications; type of genetic

sample tested and result; penetrance of RB1 mutation; timing of first examination; age and location of

first tumor(s) in each eye; treatments used; TNMH{, 2009 #11254} staging for eyes and child;

International Intraocular Retinoblastoma Classification (IIRC)10 of each eye; Tumor Node Metastasis

(TNM)15 staging for eyes and child; active treatment duration; date of last follow-up; and visual outcome

at last follow-up. The gestational age at birth for each child was calculated (39 weeks was considered full

term). Eyes with vision threatening tumors were defined as cIIRC Group BT1b or worse, which includes

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

tumor size and proximity to optic nerve or macula. Treatments were summarized as focal therapies (laser

therapy, cryotherapy and periocular subtenon’s injection of chemotherapy) or systemic therapies

(systemic chemotherapy or stereotactic external beam irradiation). Active treatment duration (time from

diagnosis to last treatment) and number of examinations under anesthesia (EUAs) were countedmeasured.

Treatment success was defined as avoidance of enucleation or external beam irradiation. Acceptable

visual outcome in an eye was defined as visual acuity better than 0.2 decimal (20/100) in an eye. Useful

vision is was defined as overall visual acuity better than 0.1 in the better eye and legal blindness as

overall visual acuity worse or equal to 0.1 in the best eye.

Data analysis

Basic descriptive statistics were used for comparisons between patients seen screened postnatally (in

first week) and conventionally screened (Cohort 1) and those provided expectant management defined as

prenatal testing and planned late preterm or early term delivery (Cohort 2). These included Student T-

Test, Chi Square Test, Fisher Exact Test, Mann Whitney Test and Mood’s Median Test. Correlations and

Kaplan-Meyer Survival Graphs were plotted using Microsoft Excel 2007 and Prism 6 for Mac.

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Results

Patient Demographics

Twenty children with familial retinoblastoma were reviewed (10 males, 10 females) and 20 were

eligible for this study (Figure 1). Diagnosis for Conventional Cohort 1 (8 children, 40%) was by

observation of tumor or postnatal testing for the parental RB1 mutation. Six were born full term and 2

were delivered late preterm because of pregnancy-induced hypertension (child #7), or fetal ultrasound

evidence of retinoblastoma16 (child #8). The 12 children (60%) in Expectant Cohort 2 were prenatally

diagnosed to carry their family’s RB1 mutation: 3 were spontaneously premature (children #10, 13, 15;

28-37 weeks gestation) and 9 were referred to a high-risk pregnancy unit for elective late preterm/early

term delivery (36-38 weeks gestation).

Molecular diagnosis

All study subjects were offspring of retinoblastoma probands. Nineteen probands were bilaterally and

1 was unilaterally affected (father #19). The familial RB1 mutations were previously detected. Cohort 1

children (#1-8) were tested postnatal for their family’s RB1 mutation by blood; Cohort 2 children (#10-

21) were tested prenatal by amniocentesis at 16-33 weeks gestation.

Null RB1 mutations were present in 15 families. Five families had low penetrance RB1 mutations

(whole gene deletion, #18; weak splice site mutations, #14, 17, 20; and a missense mutation,17,18 #5)

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

(Supplementary 1). No proband in this study was mosaic for the RB1 mutation. Eventually, aAll study

subjects were eventually bilaterally affected. At birth, 8/15 (53%) infants with null RB1 mutations had

tumors, affecting 13/23 (53%) eyes, but 0/5 infants with low penetrance mutations had tumors at birth

(Table 12, p=0.02 for eyes, P=0.1 for children; Fisher’s exact test).

At first tumor per child, children with null mutations tended to be younger (mean 59, median 20 days)

than those with low penetrance mutations (mean 107, median 114 days). At first tumor per eye, children

with null mutations (mean 83, median 39 days) were significantly younger than those with low

penetrance mutations (mean 134, median 119 days) (p=0.03, Median Mood test) (Figure 2).

Classification of Eyes at Birth

Of Cohort 1 eyes, 50% (8/16 ) had tumor at birth, compared to 21% (5/24) of Cohort 2 eyes (Table

2a3a, Figure 1). At birth, tumor was present in at least one eye in 5/8 of Cohort 1 children, 63% (5/8) and

25% (3/12) of Cohort 2 had children Tumor in at least one eye at birth (Table 2b3b, Figure 1). At birth,

38% 6/16 of eyes (6/16) in Cohort 1 had a visually threatening tumor (cIIRC10 Group BT1b or worse)

compared to 17% (4/22) of Cohort 2 of eyes (Table 2a).

Tumors detected at birth tended to be in the peri-macular region (IIRC10 Group BcT1b), and tumors

detected later were smaller and not threatening vision, as previously described (Figures 1, 3).19,20 The

median age of diagnosis of When first tumor was diagnosed, median age was 9 days of for 15 IIRC10

Group BcT1b eyes (threatening optic nerve and fovea, and 6 with at least 1 tumor >3 mm), was 9 days,

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

tending younger than the 92 days for 24 IIRC10 Group AcT1a eyes (tumors < 3 mm and away from optic

nerve and fovea). The correctedAgeAfter correcteding for gestational age age when first tumor was

diagnosed per eye, at first tumor diagnosis for these eyes showed the same tendency was observed

(medians 7 and 60 days respectively).

At initial tumor diagnosis, 2/8 (25%) children in Cohort 1 had IIRC10 Group A/A or A/0cT1a/cT1a or

cT1a/cT0 eyes, compared to 8/12 (67%) in Cohort 2 (P=0.02, Fisher exact test) (Figure 1, Table 2b3b). At

first diagnosis, tumors were not threatening vision (IIRC10 Group AcT1a) in 7/16 (50%) Cohort 1 eyes,

compared to 17/24 (71%) Cohort 2 eyes , (Table 2a3a). Vision-threatening tumor (Group BcT1b or

worse) were was present at first diagnosis in 38%6/16 of Cohort 1 compared to 17%4/24 of Cohort 2

(Figure 1, Table 2a3a).

The Cohort 1 showed larger and more posterior tumors than Cohort 2 (Figure 3). Before 37 weeks

gestation, 7.5% (3/40) of eyes showed tumors, and in all tumors were within the posterior pole. At 42

weeks gestation (2 weeks after full term birth), 37.5% (15/40) of eyes showed tumors, and in 80% (12/15

eyes the tumors) were within the posterior pole.

Treatment Course

All infants were frequently examined from birth onwards as per the National Retinoblastoma Strategy

Guidelines for Care.14 If there were no tumors at birth, each child was examined awake every week for 1

month, every 2 weeks for 2 months, and after 3 months of age, had EUAs every 2-4 weeks. If there was

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

tumor at birth, the children had EUAs every 2-4 weeks until control of tumors was achieved. Cohort 1

patients were treated with focal therapy (8)all), chemotherapy using vincristine, carboplatin, etoposide

and cyclosporine (Toronto protocol)21 {Chan, 2005 #21688}(5), stereotactic radiation (2), and enucleation

of one eye (4) (Supplementary Table 1, Figure 1). Cohort 2 patients were treated with focal therapy

(12all); chemotherapy (5), enucleation of one eye and stereotactic radiation (1) (Figure 1). Treatment by

focal therapy alone (avoidance of systemic chemotherapy or external beam irradiation, EBRT) was

possible in 2/8 (25%) of Cohort 1 patients and 7/12 (58%) of Cohort 2 patients (Table 2b).

The median active treatment duration was 523 days (range 0-2101 days) in Cohort 1, compared to 447

days (range 0-971 days) in Cohort 2 (p=0.77, Mood median test). The median number of EUAs was

equal: 29 (range 18-81) Cohort 1 and 29 (range 20-41) Cohort 2 (p=1, Mood median test).

Outcomes

There were no adverse events associated with spontaneous preterm, induced late preterm or early term

birth. There were no pregnancy, delivery or perinatal complications reported for any infant. Median

Follow upfollow-up (mean, median) wasas 8, 5.6 years; (Cohort 1=, 8.4, 5.6 years; and Cohort 2,= 7.6,

5.8 years) (Supplementary Table 1). At the last follow up, median age of Cohort 1 was mean 10 years

(median 10, range 3-19) and Cohort 2 was mean 9 years (median 9, range 3-16).

Neither enucleation nor external beam irradiation were required (defined as treatmentTreatment

success was achieved ) in 33% (3/8) of Cohort 1 and 92% (11/12 ) of Cohort 2 patients (P=0.02, Fisher

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

exact test) (Table 2b3b). Kaplan Meier 5 year ocular survival for Cohort 1 was 62% compared to 92% for

Cohort 2 (P=0.03, Log-rank (Mantel-Cox test) (Figure 4). One child (#6) (11%) in Cohort 1 showed high-

risk histopathologic features22-24 in the enucleated eye and received adjuvant treatment. The one eye

enucleated in Cohort 2 showed no high-risk features. All children were are presently alive without

metastases inat the close of the study.

Children were legally Legal blindness afflicted in 12% (1/8 ) of Cohort 1 children and 0% ofno Cohort

2 children (Table 1a3a). Visual outcomes considered acceptable (were better than 0.2) for 50% (8/16) of

eyes in Cohort 1 and 88% (21/24) of eyes in Cohort 2 (P=0.01, Fisher exact test) (Table 1a3a). Final

visual acuity better than 0.5 (20/40) was achieved in 50% (9/18) of eyes in Cohort 1 compared to 71%

(17/24 ) of Cohort 2 eyes. (p=0.3, Mood median test)

Combined treatment success and good vision per eye was documented 50% (8/16) of Cohort 1 and

88% (21/24) of Cohort 2 (P=0.02*, Fisher exact test) (Table 2a, Figure 1). A trend to negative correlation

was found between gestational age and final visual outcome (r=-0.03) with better visual outcome

observed for earlier births.

Discussion

This is the first report on outcomes of expectant management by elective early term or late preterm

delivery of infants confirmed by prenatal RB1 mutation testing to be at risk for familial retinoblastoma

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

shows benefit for the child. The fewer, and smaller, tumors present at birth wereThis approach allowed

treatment of Small tumors treated as they emerged, resulting in better ocular and visual outcomes and

with less intensive medical interventions, in very young children. Our data suggests that infants at high

risk of developing retinoblastoma (prenatally confirmed RB1+/-), the risk of vision and eye loss despite

intensive therapies after spontaneous delivery outweighings the theoretical risks associated with induced

late pretermof early delivery (Figure 1, Table 2). Consistent with previous reports,23 63% of children with

a germline gene mutation already had tumors at full term birth, compared to 25% at planned late preterm

or early term delivery (Table 2b).

It is practical now standard of care13 to identify 97% of the germline mutations in bilaterally affected

probands and ~15% of unilateral probands who carry a germline gene mutation.3,9,25 When the proband's

unique mutation is known, molecular testing of family members can determines which relatives also carry

thate mutation and are at risk to develop retinoblastoma and other cancers. We report 12 infants identified

in utero by molecular testing to carry the family’s mutant RB1 allele of a parent. The tested Infants who

shown to did not inherit carry their family’s mutation or H0 (not shown) required no surveillance and

were not included in this study. Amniocentesis (to collect sample for genetic testing) was performed in

the second half of pregnancy, where risks of miscarriage are low (0.1-1.4%).26,27

The Our data confirms that the earliest tumors commonly involve the perimacular region, threatening

loss of central vision, while later developing tumors that develop later are usuallytended to be peripheral

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

(Figure 3), with less visual impact.19,20,28 The smaller mMacular and perimacular tumors are at diagnosis

and treatment the better the visual outcome, since are difficult to manage by laser therapy or radioactive

plaque , since these threaten the optic nerve and central vision. Systemic chemotherapy effectively

shrinks tumors such that focal therapy can be applied with minimal visual damage. Child #8 (Cohort 1)

had a tumor at 36 weeks gestation large enough for detectiondetected by obstetrical ultrasound, which

developed drug-resistance following reduced-dose chemotherapy as a newborn,16 ultimately requiring

enucleation at the age of 14 years. Systemic chemotherapy in neonates is difficult challenging due tosince

severe adverse effects may occur due to the unknowns of immature liver and kidney function to in

metabolizmetabolism ofinge the drugs , increasing th.ewith potential of severe adverse effects. The

Conventional recommendation is to eitherdose reductione chemotherapy dosages by 50%, particularly for

infants in the first three months of life,29 or administer a single agent carboplatin chemotherapy.30 open the

door to However, reduced doses carry risk of selection of ing for multidrug resistantce in the tumor cells,

making later recurrences difficult to treat.31-33 Periocular topotecan for treatment of small-volume

retinoblastoma34 may increase the effectiveness of focal therapy without facilitating resistance. Intra-

arterial chemotherapy is not feasible in such tiny babies and thebabies; the youngest age reported is 2

months.35

Imhof et al6 screened 135 children at risk of familial retinoblastoma starting 1-2 weeks after birth

without molecular diagnosis and identified 17 retinoblastoma cases. At first diagnosisscreening,

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

70%12/17 infants had retinoblastoma. At first diagnosis, 14/32 eyes with had vision threatened

threatening tumorin 41% of eyes; 41%12/32 had eye salvagetreatment failure (required radiation and/or

enucleation); one child developed metastasis; 20 eyes had achieved vision >20/100 and 74% 2/17

children achieved vision ><20/2100 (legal blindness). In comparison to our, Cohort 2, showed 17%4/24

eyes visionshowed vision threatening tumors, 8%3/24 eyes had eye salvagetreatment failures, and

88%21/24 eyes had vision >20/100 without any child metastasis or legal blindness.

Early screening of at-risk infants with positive family history as soon as possible after birth is the

internationally accepted convention for retinoblastoma.6,36 Rothschild et al36 retrospectively reviewed 16

children that undergone intensive screening (defined as first week then every month up to 18 months) and

developed familial retinoblastoma; 15/16 had systemic chemotherapy; 2/16 had irradiation; 12/13

children achieved vision >20/200 without data on vision per eye. In our cohort 2, less children required

systemic chemotherapy (5/12) and irradiation (1/12) with similar visual outcome per child.

Two additional reports indicated showed prenatal molecular testing for retinoblastoma: in one, the

fetus sibling of a proband was found not to carry the sibling’s mutation;35 and in the other, 2 of 5 tested

fetuses of a mosaic proband were born without the parental mutation.36

Early screening of at-risk infants with positive family history as soon as possible after birth is the

internationally accepted convention for retinoblastoma.6,33 33

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

In our series, amniocentesis (to collect sample for genetic testing) was performed in the second half of

pregnancy, where risks of miscarriage are low (0.1-1.4%).34,35 We show that planned late preterm/early

term delivery (36-38 weeks gestation) for those confirmed to carry their family’s RB1 mutation, resulted

in smaller tumors with less macular involvement and better visual outcome. We observed no difference in

treatment burden between Cohorts, and treatment courses were similar. However, earlier delivery and

treatment correlated with better overall patient outcomes.

A concern with late preterm or early term delivery is its reported effect on neurological and cognitive

development and later school performance.37-39 Additionally, The studies reporting on preterm and early

term babies tend to include many children with complex reasons for early delivery. However, the visual

dysfunction from a large macular tumor common in retinoblastoma patients is equally concerning, as it

can cause similar neurocognitive defects due to blindness,39 although no comparative studies address this.

Additionally, the studies reporting on preterm and early term babies tend to include many children with

complex reasons for early delivery. In contrast, retinoblastoma children are otherwise healthy normal

babies, except for the cancer growing in their eye(s). Early term delivery requires an interactive team of

neonatologist, ophthalmologist and oncologist to reach the best timing for better outcome.40

Neurocognitive development in retinoblastoma patients can be affected by multiple factors including

blindness, multiple EUAs, and external beam irradiation.41,42 A comparative study is recommended to

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

evaluate neurocognitive deficits after early delivery versus after advanced retinoblastoma with its

treatment.

Two additional reports showed prenatal molecular testing for retinoblastoma: in one, the fetus sibling

of a proband was found not to carry the sibling’s mutation;43 and in the other, 2 of 5 tested fetuses of a

mosaic proband were born without the parental mutation.44

Counseling on reproductive risks is important for families affected by retinoblastoma including

unilateral probands.43 Current optimized therapies result in very low mortality, and most retinoblastoma

patients survive and will consider having children. Prenatal diagnosis also enables pre-implantation

screening to ensure an unaffected child and informs parents who may wish to terminate an affected

pregnancy.44 It is our experience that retinoblastoma survivors and their relatives with full understanding

of the underlying risks are often interested in early diagnosis to optimize options for therapy in affected

babies rather than termination of pregnancy. Since germline RB1 mutations predispose to future, second

cancers, perhaps it may be worth investigating the role of cord blood banking for infants that are

prenatally molecularly diagnosed with RB1 mutant allelesretinoblastoma, as a potential stem cell source

in later anti-cancer therapy.

The current study The is limited by ourthe small sample size, in a single institute, being an

observational retrospective data analysis with lack of randomization in both groups. Despite the small

number, this is considered the biggest cohort describing prenatal screening to date as only case reports

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

were published,45-48 multiple outcomes were Strength is the statistically significant outcome parameters

mainly tumors at birth, treatment success and visual outcome despite the small sample size.,,

andfromoriginating in. Needs more…weaknesses

We conclude that the infants with familial retinoblastoma, who are likely to develop vision-threatening

macular tumors, have an improved chance of good visual outcome with decreased treatment associated

morbidity, with expectant managementprenatal molecular screeningdiagnosis and induced late

preterm/early term delivery rather than conventional postnatal screening of at risk children with positive

family history.

Acknowledgements

We acknowledge the important contributions of Ivana Ristevski to the figures.

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Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

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10. Murphree AL. Intraocular retinoblastoma: the case for a new group classification.

Ophthalmology clinics of North America. 2005;18:41-53.

11. Shields CL, Mashayekhi A, Au AK, et al. The International Classification of Retinoblastoma

predicts chemoreduction success. Ophthalmology. 2006;113(12):2276-2280.

12. Novetsky DE, Abramson DH, Kim JW, Dunkel IJ. Published international classification of

retinoblastoma (ICRB) definitions contain inconsistencies--an analysis of impact. Ophthalmic

Genet. 2009;30(1):40-44.

13. Canadian Retinoblastoma S. National Retinoblastoma Strategy Canadian Guidelines for Care:

Strategie therapeutique du retinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44

Suppl 2:S1-88.

21

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

14. National Retinoblastoma Strategy Canadian Guidelines for Care / Stratégie thérapeutique du

rétinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44(Supp 2):S1-88.

15. Born Too Soon: The Global Action Report on Preterm Birth. Geneva: World Health

Organization;2012.

16. Sahgal A, Millar BA, Michaels H, et al. Focal stereotactic external beam radiotherapy as a vision-

sparing method for the treatment of peripapillary and perimacular retinoblastoma: preliminary

results. Clinical Oncology (Royal College Of Radiologists). 2006;18(8):628-634.

17. Cowell JK, Bia B. A novel missense mutation in patients from a retinoblastoma pedigree showing

only mild expression of the tumor phenotype. Oncogene. 1998;16(24):3211-3213.

18. Richter S, Vandezande K, Chen N, et al. Sensitive and efficient detection of RB1 gene mutations

enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72(2):253-269.

19. Balmer A, Munier F, Gailloud C, Uffer S, van Melle G. [New retinal tumors in hereditary

retinoblastoma]. Klinische Monatsblatter fur Augenheilkunde. 1995;206(5):328-331.

20. King BA, Parra C, Li Y, et al. Spatiotemporal Patterns of Tumor Occurrence in Children with

Intraocular Retinoblastoma. PLoS One. 2015;10(7):e0132932.

21. Chan HS, Gallie BL, Munier FL, Beck Popovic M. Chemotherapy for retinoblastoma.

Ophthalmology clinics of North America. 2005;18(1):55-63, viii.

22. Aerts I, Sastre-Garau X, Savignoni A, et al. Results of a multicenter prospective study on the

postoperative treatment of unilateral retinoblastoma after primary enucleation. Journal Of

Clinical Oncology. 2013;31(11):1458-1463.

23. Chantada G, Luna-Fineman S, Sitorus RS, et al. SIOP-PODC recommendations for graduated-

intensity treatment of retinoblastoma in developing countries. Pediatr Blood Cancer.

2013;60(5):719-727.

24. Sastre X, Chantada GL, Doz F, et al. Proceedings of the consensus meetings from the

International Retinoblastoma Staging Working Group on the pathology guidelines for the

examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma.

Archives of pathology & laboratory medicine. 2009;133(8):1199-1202.

25. Lohmann D, Gallie BL. Retinoblastoma. In: Pagon RA AM, Ardinger HH, Wallace SE, Amemiya A,

Bean LGH, Bird TD, Fong C-T, Mefford HC, Smith RJH, and Stephens K., ed. GeneReviews™

[Internet]. Seattle (WA): University of Washington, Seattle; 1993-2015. Available at

http://www.ncbi.nlm.nih.gov/books/NBK1452/2015.

26. Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage

following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis.

Ultrasound in obstetrics & gynecology : the official journal of the International Society of

Ultrasound in Obstetrics and Gynecology. 2015;45(1):16-26.

22

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

27. Tabor A, Vestergaard CH, Lidegaard O. Fetal loss rate after chorionic villus sampling and

amniocentesis: an 11-year national registry study. Ultrasound in obstetrics & gynecology : the

official journal of the International Society of Ultrasound in Obstetrics and Gynecology.

2009;34(1):19-24.

28. Abramson DH, Greenfield DS, Ellsworth RM. Bilateral retinoblastoma. Correlations between age

at diagnosis and time course for new intraocular tumors. Ophthalmic Paediatrics And Genetics.

1992;13(1):1-7.

29. Chan HS, Grogan TM, DeBoer G, Haddad G, Gallie BL, Ling V. Diagnosis and reversal of multidrug

resistance in paediatric cancers. European Journal Of Cancer. 1996;32A(6):1051-1061.

30. Gombos DS. Retinoblastoma in the perinatal and neonatal child. Semin Fetal Neonatal Med.

2012;17(4):239-242.

31. Sreenivasan S, Ravichandran S, Vetrivel U, Krishnakumar S. Modulation of multidrug resistance 1

expression and function in retinoblastoma cells by curcumin. Journal of pharmacology &

pharmacotherapeutics. 2013;4(2):103-109.

32. Barot M, Gokulgandhi MR, Pal D, Mitra AK. In vitro moxifloxacin drug interaction with

chemotherapeutics: implications for retinoblastoma management. Exp Eye Res. 2014;118:61-71.

33. Yague E, Arance A, Kubitza L, et al. Ability to acquire drug resistance arises early during the

tumorigenesis process. Cancer Res. 2007;67(3):1130-1137.

34. Mallipatna AC, Dimaras H, Chan HS, Heon E, Gallie BL. Periocular topotecan for intraocular

retinoblastoma. Arch Ophthalmol. 2011;129(6):738-745.

35. Magan T, Khoo CT, Jabbour PM, Shields CL. Intraarterial Chemotherapy for Retinoblastoma in a

2-Month-Old Infant. Retinal cases & brief reports. 2016.

36. Rothschild PR, Levy D, Savignoni A, et al. Familial retinoblastoma: fundus screening schedule

impact and guideline proposal. A retrospective study. Eye (Lond). 2011;25(12):1555-1561.

37. Cheong JL, Doyle LW. Increasing rates of prematurity and epidemiology of late preterm birth.

Journal of paediatrics and child health. 2012;48(9):784-788.

38. Woythaler MA, McCormick MC, Smith VC. Late preterm infants have worse 24-month

neurodevelopmental outcomes than term infants. Pediatrics. 2011;127(3):e622-629.

39. Poulsen G, Wolke D, Kurinczuk JJ, et al. Gestational age and cognitive ability in early childhood: a

population-based cohort study. Paediatr Perinat Epidemiol. 2013;27(4):371-379.

40. Dimaras H, Kimani K, Dimba EA, et al. Retinoblastoma. Lancet. 2012;379(9824):1436-1446.

41. Bedny M, Saxe R. Insights into the origins of knowledge from the cognitive neuroscience of

blindness. Cogn Neuropsychol. 2012;29(1-2):56-84.

42. Brinkman TM, Merchant TE, Li Z, et al. Cognitive function and social attainment in adult survivors

of retinoblastoma: a report from the St. Jude Lifetime Cohort Study. Cancer. 2015;121(1):123-

131.

23

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

43. Soliman SE EM, Dimaras H. Knowledge of Genetics in Familial Retinoblastoma. Ophthalmic

Genet. 2016;In Press.

44. Dommering CJ, Garvelink MM, Moll AC, et al. Reproductive behavior of individuals with

increased risk of having a child with retinoblastoma. Clin Genet. 2012;81(3):216-223.

45. Lau CS, Choy KW, Fan DS, et al. Prenatal screening for retinoblastoma in Hong Kong. Hong Kong

Med J. 2008;14(5):391-394.

46. Castera L, Gauthier-Villars M, Dehainault C, et al. Mosaicism in clinical practice exemplified by

prenatal diagnosis in retinoblastoma. Prenatal Diagnosis. 2011;31(11):1106-1108.

47. Shah PK, Sripriya S, Narendran V, Pandian AJ. Prenatal genetic diagnosis of retinoblastoma and

report of RB1 gene mutation from India. Ophthalmic Genet. 2016:1-4.

48. Neriyanuri S, Raman R, Rishi P, Govindasamy K, Ramprasad VL, Sharma T. Prenatal genetic

diagnosis of retinoblastoma--clinical correlates on follow-up. Indian Journal Of Ophthalmology.

2015;63(9):741-742.

24

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Table 12. Tumors present at birth and type of RB1 mutation. *p value by Fisher's exact test

Eyes with tumors at birthChildren with tumors at

birthYES NO total % YES YES NO total % YES

Null RB1 mutation 13 10 23 57% 8 7 15 53%Low penetrance

RB1 mutation0 10 10 0% 0 5 5 0%

Total 33 20p=0.02* p=0.1

p value by Fisher's exact test. *Statistically significant

25

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Table 2a3a: Outcome parameters per eyeConventional Expectant

Cohort 1 (n=16)

Cohort 2 (n=24)

# % # % P valueTumor(s) at birth 8 50% 5 21% 0.09Visual prognosis at first tumor

Group A, O 10 62% 20 83%0.26

Group B, C 6 38% 4 17%

Ocular salvage 12 75%23

96% 0.13

Treatment Success§ 10 63%22

92% 0.04*

Visual Outcome

Acceptable vision∑ 8 50%21

88%0.01*

Poor Vision 8 50% 3 13%Combined Treatment Success & Acceptable vision

8 50%21

88% 0.01*

26

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Table 2b3b: Outcome parameters per childConventional ExpectantCohort 1 (n=8) Cohort 2 (n=12)

# % # % P valueTumor(s) at birth 5 63% 3 25% 0.17IIRC AA or AO at first tumor diagnosis

1 13% 8 67% 0.02*

TreatmentFocal therapy only 2 25% 7 58%

0.2Systemic chemotherapy 6 75% 5 42%

Treatment Success§ 3 38% 11 92% 0.02*

Ocular salvage 4 50% 11 92% 0.1

Visual Outcome 0%Useful vision¥ 7 88% 12 100%

1Legal Blind¶ 1 13% 0 0%

* significant result; § Avoided enucleation or external beam radiation; ¥ vision > 0.1 in better seeing

eye;¶ vision ≤ 0.1 in better seeing eye; ∑ vision > 0.2 in an eye.

27

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

Figure Legends

Figure 1. Tumor timing, therapy and outcomes. Patients in Cohorts 1 and 2 plotted with gestational age at birth (pink symbols), international

intraocular retinoblastoma classification (IIRC) at birth and at diagnosis, treatment at time of first tumor occurrence (blue diamonds), labeled with

number of EUAs, final visual outcome and follow-up time.

Figure 2. RB1 germline mutation influences timing of tumor development. First tumor per eye for children with null mutations was

significantly earlier (mean 83, median 39 days) than for those with low penetrance mutations (mean 134, median 119 days) (p=0.03, Median

Mood test).

Figure 3: Topographic representation of the first tumor(s) in each eye. The case number is written on each tumor. The size of the tumor is

proportional to the size at diagnosis. Tumors are color coded according to corrected age in weeks at initial diagnosis.

Figure 4. Kaplan Meier ocular survival (avoidance of radiation or enucleation) for Cohort 1 was 62% compared to 92% for Cohort 2 (P=0.03,

Log-rank Mantel-Cox test).

1. Corson TW, Gallie BL. One hit, two hits, three hits, more? Genomic changes in the development of retinoblastoma. Genes Chromosomes

Cancer. 2007;46(7):617-634.

2. Dimaras H, Gallie BL. Retinoblastoma: The Prototypic Hereditary Tumor. In: Heike Allgayer, Helga Rehder, Fulda S, eds. Hereditary

Tumors - From Genes to Clinical Consequences. Weinheim, Germany: WILEY-VCH Verlag GmbH & Co.KGaA; 2008:147-162.

3. Lohmann DR, Gallie BL. Retinoblastoma. In: Pagon RA, Adam MP, Ardinger HH, et al., eds. GeneReviews(R). Seattle (WA)2000.

28

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

4. Butros LJ, Abramson DH, Dunkel IJ. Delayed diagnosis of retinoblastoma: analysis of degree, cause, and potential consequences.

Pediatrics. 2002;109(3):E45.

5. Noorani HZ, Khan HN, Gallie BL, Detsky AS. Cost comparison of molecular versus conventional screening of relatives at risk for

retinoblastoma. Am J Hum Genet. 1996;59(2):301-307.

6. Imhof SM, Moll AC, Schouten-van Meeteren AY. Stage of presentation and visual outcome of patients screened for familial

retinoblastoma: nationwide registration in the Netherlands. Br J Ophthalmol. 2006;90(7):875-878.

7. Abouzeid H, Schorderet DF, Balmer A, Munier FL. Germline mutations in retinoma patients: relevance to low-penetrance and low-

expressivity molecular basis. Mol Vis. 2009;15:771-777.

8. Abramson DH, Du TT, Beaverson KL. (Neonatal) retinoblastoma in the first month of life. Arch Ophthalmol. 2002;120(6):738-742.

9. Rushlow D, Piovesan B, Zhang K, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009;30(5):842-

851.

10. Murphree AL. Intraocular retinoblastoma: the case for a new group classification. Ophthalmology clinics of North America. 2005;18:41-

53.

11. Shields CL, Mashayekhi A, Au AK, et al. The International Classification of Retinoblastoma predicts chemoreduction success.

Ophthalmology. 2006;113(12):2276-2280.

12. Novetsky DE, Abramson DH, Kim JW, Dunkel IJ. Published international classification of retinoblastoma (ICRB) definitions contain

inconsistencies--an analysis of impact. Ophthalmic Genet. 2009;30(1):40-44.

13. Canadian Retinoblastoma S. National Retinoblastoma Strategy Canadian Guidelines for Care: Strategie therapeutique du retinoblastome

guide clinique canadien. Can J Ophthalmol. 2009;44 Suppl 2:S1-88.

14. National Retinoblastoma Strategy Canadian Guidelines for Care / Stratégie thérapeutique du rétinoblastome guide clinique canadien.

Can J Ophthalmol. 2009;44(Supp 2):S1-88.

15. Born Too Soon: The Global Action Report on Preterm Birth. Geneva: World Health Organization;2012.

16. Sahgal A, Millar BA, Michaels H, et al. Focal stereotactic external beam radiotherapy as a vision-sparing method for the treatment of

peripapillary and perimacular retinoblastoma: preliminary results. Clinical Oncology (Royal College Of Radiologists). 2006;18(8):628-634.

17. Cowell JK, Bia B. A novel missense mutation in patients from a retinoblastoma pedigree showing only mild expression of the tumor

phenotype. Oncogene. 1998;16(24):3211-3213.

29

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

18. Richter S, Vandezande K, Chen N, et al. Sensitive and efficient detection of RB1 gene mutations enhances care for families with

retinoblastoma. Am J Hum Genet. 2003;72(2):253-269.

19. Balmer A, Munier F, Gailloud C, Uffer S, van Melle G. [New retinal tumors in hereditary retinoblastoma]. Klinische Monatsblatter fur

Augenheilkunde. 1995;206(5):328-331.

20. King BA, Parra C, Li Y, et al. Spatiotemporal Patterns of Tumor Occurrence in Children with Intraocular Retinoblastoma. PLoS One.

2015;10(7):e0132932.

21. Chan HS, Gallie BL, Munier FL, Beck Popovic M. Chemotherapy for retinoblastoma. Ophthalmology clinics of North America.

2005;18(1):55-63, viii.

22. Aerts I, Sastre-Garau X, Savignoni A, et al. Results of a multicenter prospective study on the postoperative treatment of unilateral

retinoblastoma after primary enucleation. Journal Of Clinical Oncology. 2013;31(11):1458-1463.

23. Chantada G, Luna-Fineman S, Sitorus RS, et al. SIOP-PODC recommendations for graduated-intensity treatment of retinoblastoma in

developing countries. Pediatr Blood Cancer. 2013;60(5):719-727.

24. Sastre X, Chantada GL, Doz F, et al. Proceedings of the consensus meetings from the International Retinoblastoma Staging Working

Group on the pathology guidelines for the examination of enucleated eyes and evaluation of prognostic risk factors in retinoblastoma.

Archives of pathology & laboratory medicine. 2009;133(8):1199-1202.

25. Lohmann D, Gallie BL. Retinoblastoma. In: Pagon RA AM, Ardinger HH, Wallace SE, Amemiya A, Bean LGH, Bird TD, Fong C-T, Mefford HC,

Smith RJH, and Stephens K., ed. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2015. Available at

http://www.ncbi.nlm.nih.gov/books/NBK1452/2015.

26. Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus

sampling: a systematic review and meta-analysis. Ultrasound in obstetrics & gynecology : the official journal of the International Society

of Ultrasound in Obstetrics and Gynecology. 2015;45(1):16-26.

27. Tabor A, Vestergaard CH, Lidegaard O. Fetal loss rate after chorionic villus sampling and amniocentesis: an 11-year national registry

study. Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology.

2009;34(1):19-24.

28. Abramson DH, Greenfield DS, Ellsworth RM. Bilateral retinoblastoma. Correlations between age at diagnosis and time course for new

intraocular tumors. Ophthalmic Paediatrics And Genetics. 1992;13(1):1-7.

30

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

29. Chan HS, Grogan TM, DeBoer G, Haddad G, Gallie BL, Ling V. Diagnosis and reversal of multidrug resistance in paediatric cancers.

European Journal Of Cancer. 1996;32A(6):1051-1061.

30. Gombos DS. Retinoblastoma in the perinatal and neonatal child. Semin Fetal Neonatal Med. 2012;17(4):239-242.

31. Sreenivasan S, Ravichandran S, Vetrivel U, Krishnakumar S. Modulation of multidrug resistance 1 expression and function in

retinoblastoma cells by curcumin. Journal of pharmacology & pharmacotherapeutics. 2013;4(2):103-109.

32. Barot M, Gokulgandhi MR, Pal D, Mitra AK. In vitro moxifloxacin drug interaction with chemotherapeutics: implications for

retinoblastoma management. Exp Eye Res. 2014;118:61-71.

33. Yague E, Arance A, Kubitza L, et al. Ability to acquire drug resistance arises early during the tumorigenesis process. Cancer Res.

2007;67(3):1130-1137.

34. Mallipatna AC, Dimaras H, Chan HS, Heon E, Gallie BL. Periocular topotecan for intraocular retinoblastoma. Arch Ophthalmol.

2011;129(6):738-745.

35. Magan T, Khoo CT, Jabbour PM, Shields CL. Intraarterial Chemotherapy for Retinoblastoma in a 2-Month-Old Infant. Retinal cases & brief

reports. 2016.

36. Rothschild PR, Levy D, Savignoni A, et al. Familial retinoblastoma: fundus screening schedule impact and guideline proposal. A

retrospective study. Eye (Lond). 2011;25(12):1555-1561.

37. Cheong JL, Doyle LW. Increasing rates of prematurity and epidemiology of late preterm birth. Journal of paediatrics and child health.

2012;48(9):784-788.

38. Woythaler MA, McCormick MC, Smith VC. Late preterm infants have worse 24-month neurodevelopmental outcomes than term infants.

Pediatrics. 2011;127(3):e622-629.

39. Poulsen G, Wolke D, Kurinczuk JJ, et al. Gestational age and cognitive ability in early childhood: a population-based cohort study.

Paediatr Perinat Epidemiol. 2013;27(4):371-379.

40. Dimaras H, Kimani K, Dimba EA, et al. Retinoblastoma. Lancet. 2012;379(9824):1436-1446.

41. Bedny M, Saxe R. Insights into the origins of knowledge from the cognitive neuroscience of blindness. Cogn Neuropsychol. 2012;29(1-

2):56-84.

42. Brinkman TM, Merchant TE, Li Z, et al. Cognitive function and social attainment in adult survivors of retinoblastoma: a report from the St.

Jude Lifetime Cohort Study. Cancer. 2015;121(1):123-131.

31

Soliman et al. Expectant Management ofPrenatal screening for Familial Retinoblastoma

43. Soliman SE EM, Dimaras H. Knowledge of Genetics in Familial Retinoblastoma. Ophthalmic Genet. 2016;In Press.

44. Dommering CJ, Garvelink MM, Moll AC, et al. Reproductive behavior of individuals with increased risk of having a child with

retinoblastoma. Clin Genet. 2012;81(3):216-223.

45. Lau CS, Choy KW, Fan DS, et al. Prenatal screening for retinoblastoma in Hong Kong. Hong Kong Med J. 2008;14(5):391-394.

46. Castera L, Gauthier-Villars M, Dehainault C, et al. Mosaicism in clinical practice exemplified by prenatal diagnosis in retinoblastoma.

Prenatal Diagnosis. 2011;31(11):1106-1108.

47. Shah PK, Sripriya S, Narendran V, Pandian AJ. Prenatal genetic diagnosis of retinoblastoma and report of RB1 gene mutation from India.

Ophthalmic Genet. 2016:1-4.

48. Neriyanuri S, Raman R, Rishi P, Govindasamy K, Ramprasad VL, Sharma T. Prenatal genetic diagnosis of retinoblastoma--clinical

correlates on follow-up. Indian Journal Of Ophthalmology. 2015;63(9):741-742.

32