file · Web viewEarly delivery of children at risk for retinoblastoma 12

Impact of early delivery of children with familial

retinoblastoma after prenatal RB1 mutation

identification

Sameh E. Soliman, MD; Helen Dimaras, PhD; Vikas Khetan, MB, BS; Elise Héon, MD, FRCSC;

Helen S. L. Chan, MB, BS, FRCSC; Brenda L. Gallie, MD, FRCSC

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

Sciences, the Hospital for Sick Children, 525 University Avenue, 8th floor, Toronto, ON M5G 2L3,

Canada, or at [email protected]

Authors’ Affiliations:

Departments of Ophthalmology & Vision Sciences, (Soliman, Dimaras, Héon , Gallie) and

Division of Hematology/Oncology, Pediatrics (Chan), Hospital for Sick Children, Toronto,

Canada; Division of Visual Sciences, Toronto Western Research Institute, Toronto, Canada

(Héon , Gallie); Ophthalmology Department, Faculty of Medicine, Alexandria University, Egypt

(Soliman); Sankara Nethralya Hospital, Chennai, India (Khetan); Departments of Pediatrics

(Chan), Molecular Genetics (Gallie), Medical Biophysics (Gallie) and Ophthalmology & Vision

Sciences (Dimaras, Héon, Gallie), and the Division of Clinical Public Health (Dimaras) University

of Toronto, Toronto, Ontario, Canada.

Financial Support: None

Conflict of Interest: No conflicting relationship exists for any author

mailto:[email protected]

Early delivery of children at risk for retinoblastoma

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Running head: Early delivery of familial retinoblastoma

Word count: 3262 /3000 words

Numbers of figures and tables: 3 figures and 2 tables

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

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


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At A Glance:

All the children of a parent who had retinoblastoma as a child are at risk to also develop

retinoblastoma. However, if the unique RB1 mutation of that parent is molecularly

defined, their children can be determined prenatal to be at near 100% vs 0% risk to

develop retinoblastoma.

We compared children with familial retinoblastoma delivered spontaneously without

prenatal RB1 testing, to those with prenatal RB1 mutation identification and planned early

delivery. All children eventually developed tumors in both eyes.

Planned early term delivery resulted in more infants born with no tumors, whose tumors

could be detected early and treated when very small with less invasive therapies. This

resulted in better visual outcomes for the children identified prenatal to be at risk.

Early term delivery resulted in no perinatal complications.

Prenatal RB1 mutation detection is a good anticipatory planning tool for the family and

child.


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Abstract (342/350)

IMPORTANCE: Familial retinoblastoma can be predicted by prenatal RB1 mutation detection. Early

delivery following prenatal detection for treatment of smaller tumors may achieve better outcomes with

minimal therapy.

OBJECTIVE: To compare overall outcomes and intensity of treatment for children with familial

retinoblastoma diagnosed postnatally or by obstetrical ultrasound, and those diagnosed by prenatal RB1

mutation identification and delivered preterm.

DESIGN: A retrospective, observational study.

SETTING: This study was conducted at The Hospital for Sick Children (SickKids), a retinoblastoma referral

center in Toronto, Canada.

PARTICIPANTS: All children born between 1 June 1996 and 1 June 2014 with familial retinoblastoma

who were cared for at SickKids.

EXPOSURE(S): Cohort 1 consisted of infants where were spontaneously delivered and had postnatal

RB1 testing. Cohort 2 consisted of infants who were identified by amniocentesis to carry the affected

relative’s known RB1 mutant allele and had planned early term or late preterm delivery (36-37 weeks

gestation). All children received treatment for eye tumors.

MAIN OUTCOME MEASURES: Primary study outcome measurements were gestational age, age at first

tumor, eye classification, treatments given, visual outcome, number of anesthetics, pregnancy or

delivery complications and estimated treatment burden.

RESULTS: Of Cohort 1 (n=9) infants, 67% (6/9) already had vision-threatening tumors at birth. Of Cohort

2 (n=12) infants, 25% (3/12) had vision-threatening tumors at birth. Both Cohorts eventually developed


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tumors in both eyes. Useful vision (better than 0.1, legal blindness) was achieved for 78% of Cohort 1

compared to 100% of Cohort 2 (p<0.02). At first eye tumor diagnosis, 11% of Cohort 1 had both eyes

Group A (smallest and least vision-threatening tumors) compared to 67% of Cohort 2 (p<0.01). Eye

salvage (defined as avoidance of enucleation and external beam irradiation) was achieved in 33% of

Cohort 1 compared to 97% of Cohort 2 (p<0.002). There were no complications related to preterm

delivery.

CONCLUSIONS AND RELEVANCE: Prenatal molecular diagnosis with late preterm/near-term delivery

resulted in more eyes with no detectable retinoblastoma tumors at birth, and better vision outcomes

with less invasive therapy. Prenatal molecular diagnosis facilitates anticipatory planning for both child

and family.


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INTRODUCTION

Retinoblastoma, the most common primary ocular malignancy in children, is commonly 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, or a germline mutation (about 50% of children)

predisposes to the 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 RB1 germline mutation may already have retinoblastoma tumor(s) at birth,

often in the posterior pole of the eye where they threaten vision.4-8 Because focal laser treatment

near the optic nerve and macula may compromise vision, treatment of these small tumors can be

difficult. Most of these children are bilaterally affected, with either simultaneous or sequential

detection of tumors.4,7 Later developing tumors tend to be located peripherally.7,9 Low penetrance

(10% of families)3 and mosaic10 mutations result in fewer tumors and more frequent unilateral

phenotype.10 The timing of first tumors after birth has not yet been studied.

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

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

years of life, including under anaesthesiaanesthesia.. Early diagnosis when tumors are small and

treatable with less invasive therapies is thought to optimize salvage of the eye and vision.6,7,11

Full term birth is defined as live birth after 37 weeks gestation.12 Preterm birth is defined as

live birth occurring before completion of 37 weeks. The American College of Obstetrics and

Gynecology has suggested the description of ‘early term’ be ascribed to infants born after

completion of 37 but before 39 weeks gestation.12,13 The main concern with preterm or early term


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delivery is the potential effect on neurological and cognitive development and later school

performance measured in children with a wide range of indications for early delivery.14-16 For

otherwise normal children with high risk of cancer and visual dysfunction from large macular

tumors, blindness17 may exceed such risks of early delivery.

We present the first report of outcomes of late preterm/early term late preterm or early term

delivery for children demonstrated prenatal to carry the RB1 mutant allele of a parent. We show

that such children had earlier detection and treatment of small tumors, lower treatment

morbidity, and better tumor control and visual outcome, than children born spontaneously

without precise genetic diagnosis.

Methods

Study Design

Research ethics board approval (REB approval number 1000028725) was obtained from The

Hospital for Sick Children (SickKids). Data collected for children 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; age and

location of first and all subsequent tumor(s)(s) in each eye; treatments used; number of

anaesthetics; International Intraocular Retinoblastoma Classification18 of each eye (IIRC); Tumor

Node Metastasis (TNM)11 staging for eyes and child;11 treatment duration; date of last follow-up;

and visual outcome at last follow-up. RB1 mutation testing was performed by Impact Genetics

(formerly Retinoblastoma Solutions), as previously described.19

Sameh Gaballah, 12/10/15,

Only include if required by journal; otherwise it is not important.


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The gestational age at birth for each child was calculated (39 weeks was considered full

term). Vision threatening tumors were defined as IIRC18 Group B or worse, which includes

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 counted. Treatment success was defined as avoidance of enucleation or

external beam irradiation or extraocular disease. Acceptable visual outcome was defined as

visual acuity better than 0.1 decimal (20/200). Legal blindness is defined as visual acuity worse

than 0.1.

Data analysis

Basic descriptive statistics were used for comparisons between patients diagnosed postnatal

(Cohort 1) and those provided prenatal testing and planned late preterm or preterm 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.

Results

Patient Demographics

Twenty-one children with familial retinoblastoma were reviewed (11 males, 10 females) and

eligible for this study (Supplementary Table 1, Figure 1). Diagnosis for Cohort 1 (9 children,

43%) was by observation of tumor or postnatal testing for the parental RB1 mutation. Six were

born full term and 3 were delivered late preterm because of pregnancy-induced hypertension


9

(child #7), fetal ultrasound evidence of retinoblastoma20 (child #9) or spontaneous delivery (child

#8). The 12 children (57%) in Cohort 2 were prenatally diagnosed to carry their family’s RB1

mutation and planned for late preterm or early term delivery: 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 late preterm or early term delivery (36-38 weeks gestation).

Molecular diagnosis

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

and 2 were unilaterally (mother #8, father #19) affected. The familial RB1 mutations were

previously detected except for the unilaterally affected parent of #8. This unilaterally affected

parent had not been tested, because she believed that since she had unilateral retinoblastoma, her

children were not at risk for retinoblastoma. Cohort 1 children were (#1-9) 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 16 families. Five had low penetrance RB1 mutations (whole

gene deletion, #19; weak splice site mutations, #15, 18, 21; and a missense mutation,19,21 #5)

(Supplementary Table 1). No proband in this study was mosaic for the RB1 mutation. All study

subjects were eventually bilaterally affected. At birth, 9/16 (56%) infants with null RB1

mutations had tumors, affecting 14/31 (45%) eyes, but 0/5 infants with low penetrance mutations

had tumors at birth (Table 1a, b, P=0.02 for eyes, P=0.04 for children; Fisher’s exact test). (The

Group A eye of Child #8 was excluded from per eye calculations as the child was first examined

at 3 months of age with Group A/D tumors, so first detectable tumor in the Group A eye is

unknown. We presume the Group D eye had tumor at birth, Table 1).


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The age at first tumor per child (per eye??) was insignificantly younger for those with null

mutations (mean 59, median 20 days), than those with low penetrance mutations (mean 107,

median 119 days) (P=0.03*, Phi=0.38, Mood’s median test). Figure x shows the data (Ivana’s

plot)…..The gestational age at first tumor tended to be insignificantly younger was also

significantly younger for those with null mutations (mean 48, median 25 days) tended to be

younger but was not significantly different, than for those with low penetrance mutations (mean

83, median 81 days). (Figure 2) (P=0.03, Phi=0.32, Mood’s median test).

Classification of Eyes at Birth

Of Cohort 1 eyes, 53% (9/17) had tumor at birth, compared to 21% (5/24) of Cohort 2 eyes

(P=0.05*, Chi Square test), excluding the IIRC18 Group A eye of child #8, as above (Table 1a).

We assumed that child #8 had tumor at birth since he had Group D IIRC18 in the right eye at 3

months of age. Of Cohort 1 children, 66% (6/9) and 25% (3/12) of Cohort 2 had tumor in at least

one eye at birth (Table 1b, Figure 1). At birth, 39% of eyes (7/18) in Cohort 1 had a visually

threatening tumor (IIRC18 Group B or worse) compared to 17% of eyes (4/22) in Cohort 2.

Tumors emerged at a younger age in the macular and peri-macular region (IIRC18 Group B), as

previously described.22 The median age of diagnosis of 15 IIRC18 B eyes (all threatening optic

nerve and fovea, 6 also >3 mm size) was 9 days, tending younger than the 92 days for 24 IIRC18

A eyes (< 3mm and away from optic nerve and fovea).18 The gestational age showed the same

tendency (7 and 60 days respectively).

Bilateral IIRC18 Group A eyes were present at initial diagnosis in 2/9 (22%) children in Cohort 1

compared to 8/12 (67%) in Cohort 2 (P=0.0109, Fisher exact test) (Table 2a). At first diagnosis,

tumors were not threatening vision (IIRC18 Group A) in 8/17 (47%) Cohort 1 eyes, compared to

Gallie Brenda, 12/10/15,

REAL AGE OR GESTATIONAL

Gallie Brenda, 12/08/15,

REDO THE TEST!!!


Ivana plot


REDO THE MM MEDIAN TESTWord the sentence according to the stats. I reworded this and will put the Mood Median test on Ivana plot diagram.


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17/24 (71%) Cohort 2 eyes (Table 2b). One eye was an IIRC18 D eye and presented at age of 3

months (child #8).

Treatment Course

All infants were frequently examined from birth onwards (except child #8 who presented at age

3 months) as per the National Retinoblastoma Strategy Guidelines for Care.11 If there were no

tumors at birth, each child was examined awake every week for 1 month, every 2 weeks for 2

months. After 3 months of age, the children had an examination under general anesthesia (EUA)

every 2-4 weeks. If there was 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 (all),

chemotherapy using vincristine, carboplatin, etoposide and cyclosporine (Toronto

protocol)23{Chan, 2005 #21688} (4), stereotactic radiation (2), and enucleation of one eye (5)

(Supplementary Table 1, Figure 1). Cohort 2 patients were treated with focal therapy (all);

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

focal therapy alone (avoidance of systemic chemotherapy or EBRT) was possible in 4/9 (44%) of

Cohort 1 and 7/12 (58%) of Cohort 2. (Table 2b).

The median active treatment duration was 458 days (0-2101 days) in Cohort 1, compared to 447

days (0-971 days) in Cohort 2. The median number of EUAs in Cohort 1 was 25 (range 18-81)

and for Cohort 2 was 29 (range 20-41). .

Outcomes

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

early term birth. There were no pregnancy, delivery or perinatal complications reported for any

of the infants. Follow up (mean, median) was 8, 5.6 years; Cohort 1, 8.4, 5.6 years; and Cohort 2,


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7.6, 5.8 years (Supplementary Table 1). At the last follow up, the mean age of Cohort 1 was 10

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

Neither enucleation nor external beam irradiation were required (defined as treatment success) in

44% of Cohort 1 and 92% of Cohort 2 (P=0.05*, Fisher exact test) (Table 2). Kaplan Meier

ocular survival for Cohort 1 was 62% compared to 92% for Cohort 2 (P=0.02, Log-rank (Mantel-

Cox) test) (Figure 23). One child (#6) (11%) in Cohort 1 showed high riskhigh-risk histo-

pathologic features in the enucleated eye and still under active treatment. All children are still

alive.

Children were legally blind (visual acuity less than 0.1 (20/200) using both eyes) in 22% of

Cohort 1 and 0% of Cohort 2 (P=0.02, Fisher exact test) (Table 2a). Visual outcomes were better

than 0.1 for 50% of eyes in Cohort 1 and 92% of eyes in Cohort 2 (P=0.02, Fisher exact test)

(Table 2b). Seventy one percent of eyes (17/24) of Cohort 2 had final visual acuity better than

0.5 (20/40) compared to 50% (9/18) of eyes in Cohort 1.

Treatment success (avoidance of enucleation and/or stereotactic radiation) and good vision per

eye was documented 50% (9/18) of Cohort 1 and 88% (21/24) of Cohort 2 (P=0.014*, Fisher

exact test) (Table 2b, Figure 1). A negative correlation trend was found between gestational age

and final visual outcome (r=-0.03) with better visual outcome observed for earlier deliveries

(Figure 34).

Discussion

This is the first report on the outcome of early delivery of children for retinoblastoma (true?). We

show that prenatal molecular diagnosis of familial retinoblastoma and elective late preterm/early

term delivery allowed treatment of tumors as they emerged, resulting in better ocular and visual


13

outcomes and less intensive medical interventions in very young children. This data illustrates

that for infants with high risk of developing retinoblastoma (RB1+/- or family history) the risk of

vision and eye loss despite intensive therapies with spontaneous delivery, outweighs the risks

associated with induced late preterm delivery (Figure 1). Consistent with previous reports,5 67%

of children with a germline gene mutation already had tumors at full term birth, compared to

25% when the germline mutation was detected prenatally with planned late preterm or early term

delivery (Table 2a).

It is practical to identify 96% of the germline mutations in bilaterally affected probands and to

identify the >15% of unilateral probands who carry a germline gene mutation.3,10,24 When the

proband's unique mutation is identified, molecular testing of family members can determine who

else carries the mutation and is at risk to develop retinoblastoma. We report 12 infants identified

in utero by molecular testing to carry the mutant RB1 allele of a parent. The 50% of tested

infants who do not inherit their family’s mutation require no surveillance.

Without molecular information, repeated retinal examination is recommended for all first degree

relatives until age 7 years, the first 3 years under general anesthesia.11 Such repeated clinical

screening may impose psychological and financial burden on the children and families. Early

molecular RB1 identification of the childrenchildren, who are not at risk and require no clinical

intervention, costs significantly less than clinical screening for tumors.19,25

The earliest tumors commonly involve the macular or paramacular region, threatening loss of

central vision, while tumors that develop later are usually peripheral, where they have less visual

impact.5,26-29 In our study, the risk of a vision-threatening tumor dropped from 39% to 17% by

prenatal mutation detection and planned early delivery (Table 2/Figure #). Macular and

paramacular tumors are difficult to manage by laser therapy or application of a radioactive

Helen Dimaras, 12/10/15,

Reference the data table or Figure that shows this.Done


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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. In

our study, child #9 (Cohort 1) had a tumor at 36 weeks gestation large enough for detection by

obstetrical ultrasound, which showed drug-resistant tumor following reduced-dose chemotherapy

as a newborn,20 ultimately requiring enucleation. Systemic chemotherapy in neonates is difficult

due to the unknowns of immature liver and kidney function to metabolize the drugs, increasing

the potential of severe adverse effects. The conventional recommendation is to either reduce

chemotherapy dosages by 50%, particularly for infants in the first three months of life,30 or

administer a single agent carboplatin chemotherapy.26 However, reduced doses carry risk of

selecting for multidrug resistance 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.

Imhof et al7 screened 135 children at risk of familial retinoblastoma starting 1-2 weeks after birth

without molecular diagnosis and identified 17 retinoblastoma cases (13% of screened children at

risk). Of these, 70% had retinoblastoma in at least one eye at first examination and 41% of eyes

had vision threatening macular tumors; 41% of patients (7/17) had eye salvage failure (defined

by radiation or enucleation) and one case metastasized. Of eyes, 74% (27/34) had good visual

acuity (defined by vision >20/100). These Some of these results are similar to our Cohort 1, who

werewas also diagnosed postnatally but with additional molecular confirmation of disease risk.

In comparison, Cohort 2 showed fewer less vision threatening tumors (17%), fewer treatment

failures (8%) and better visual outcome (88%).

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

internationally accepted convention for retinoblastoma.7,35 In our series, amniocentesis (to collect


“Fewer” is for things you can count; “less” is for things you can’t count.


Which results (data points) are you saying are similar? Since the paper also had a case that metastasized, which we did not have.


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sample for genetic testing) was performed in the second half of pregnancy, where risks of

miscarriage are low (0.1-1.4%).36,37 We show that for infants those confirmed to carry their

family’s RB1 mutation, planned late preterm/ or early term delivery (36-38 weeks gestation)

resulted in smaller tumors with less macular involvement and better visual outcome. We did not

observe a difference in treatment burden between our two Cohorts, likely because treatment

course did not differ; however, early delivery and thus earlier treatment appeared to change

patient outcomes.

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

cognitive development and later school performance.14-16 One could argue that 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,17 though this has not

studied in a comparative manner. Moreover, the results from studies reporting on preterm and

early term babies may also be difficult to generalize, as they tend to include many children with

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

normal babies, save for the tumor growing in their eye. Early term delivery requires an

interactive team of neonatologist, ophthalmologist and oncologist to reach the best timing for

better outcome.38 We show that safe late preterm/early term preterm delivery resulted in lower

tumor burden at birth (Cohort 2) that was significantly easier to treat than in Cohort 1 (Figure 23,

Table 2). Safe late preterm and early term delivery resulted in more infants born tumor-free,

facilitating frequent surveillance to detect tumors as they emerged, enabling focal therapy of

small tumors with minimal damage to vision (Figures 1, 3 2).

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

unilateral probands. In developed countries, where current therapies result in extremely low


Fetuses? Since ‘infant’ suggests already born.


Is the convention not to refer to pregnancies as first, second or third trimester?


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mortality, most retinoblastoma patients will survive to have children. Prenatal diagnosis also

enables pre-implantation genetics (to ensure an unaffected child) and informs parents who wish

to terminate an affected pregnancy.39 There have been two prior reports indicating pre-natal

molecular testing for retinoblastoma; in one, the fetus sibling of a proband was found not to carry

the sibling’s mutation,40 and in the other, 2 of 5 tested fetuses of a mosaic proband were born

without the parental mutation.41

This is the first report that elective safe late preterm/early term late-preterm delivery of

prenatally diagnosed infants with familial retinoblastoma results in improved outcomes. 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. We also surmise that since germline

mutations predispose to future, second cancers in affected individuals, perhaps it is worth

investigating the role of cord blood banking infants that are prenatally molecularly diagnosed

with retinoblastoma, as a potential stem cell source in later anti-cancer therapy. We conclude that

the infants with familial retinoblastoma likely to develop vision-threatening macular

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

associated morbidity with prenatal molecular diagnosis and safe, late-preterm delivery.

Acknowledgements

We would like to acknowledge Ivana….?? Who constructed some graphs in the statistical analysis.

Author contributions:


17

SS and BG had full access to all the data in the study and take responsibility for the integrity of

the data and the accuracy of the data analysis.

Study concept and design: Soliman, Gallie,

Acquisition, analysis, or interpretation of data: Soliman, Dimaras, Khetan, Gallie

Drafting of the manuscript: Soliman, Dimaras, Khetan, Gallie

Critical revision of the manuscript for important intellectual content: Dimaras, Gallie, Chan,

Héon

Statistical analysis: Soliman, Dimaras, Gallie

Study supervision: Chan, Héon, Gallie


Only one or two authors to be written as per the JAMA guidelines


18

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of retinoblastoma. Genes Chromosomes Cancer. 2007;46(7):617-634.2. Dimaras H, Gallie BL. Retinoblastoma: The Prototypic Hereditary Tumor. In: Heike Allgayer,

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

27. Abramson DH, Niksarli K, Ellsworth RM, Servodidio CA. Changing trends in the management of retinoblastoma: 1951-1965 vs 1966-1980. J Pediatr Ophthalmol Strabismus. 1994;31(1):32-37.

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. Abramson DA, Gallie BL. Retinoblastoma. Current Opinion in Ophthalmology. 1992;3:302-311.30. 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):1051-1061.31. 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. 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.

36. 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.

37. 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.

38. Dimaras H, Kimani K, Dimba EA, et al. Retinoblastoma. Lancet. 2012;379(9824):1436-1446.39. 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.

http://www.ncbi.nlm.nih.gov/books/NBK1116/2013

https://clinicaltrials.gov/ct2/show/NCT00110110?term=retinoblastoma&intr=cyclosporin&rank=1



20

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

41. 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.


21


22

Author contributions:

SS and BG had full access to all the data in the study and take responsibility for the integrity of

the data and the accuracy of the data analysis.

Study concept and design: Soliman, Gallie,

Acquisition, analysis, or interpretation of data: Soliman, Dimaras, Khetan, Gallie

Drafting of the manuscript: Soliman, Dimaras, Khetan, Gallie

Critical revision of the manuscript for important intellectual content: Dimaras, Gallie, Chan,

Héon

Statistical analysis: Soliman, Dimaras, Gallie

Study supervision: Chan, Héon, Gallie


Only one or two authors to be written as per the JAMA guidelines


23

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.

4. Butros LJ, Abramson DH, Dunkel IJ. Delayed diagnosis of retinoblastoma: analysis of degree, cause, and potential consequences. Pediatrics. 2002;109(3):E45.

5. Abramson DH, Mendelsohn ME, Servodidio CA, Tretter T, Gombos DS. Familial retinoblastoma: where and when? Acta Ophthalmol Scand. 1998;76(3):334-338.

6. 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.

7. 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.

8. 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.

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

10. Rushlow D, Piovesan B, Zhang K, et al. Detection of mosaic RB1 mutations in families with retinoblastoma. Hum Mutat. 2009;30(5):842-851.

11. National Retinoblastoma Strategy Canadian Guidelines for Care / Stratégie thérapeutique du rétinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44(Supp 2):S1-88.

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

13. ACOG Committee Opinion No 579: Definition of term pregnancy. Obstet Gynecol. 2013;122(5):1139-1140.

14. 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.

15. Woythaler MA, McCormick MC, Smith VC. Late preterm infants have worse 24-month neurodevelopmental outcomes than term infants. Pediatrics. 2011;127(3):e622-629.

16. 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.

17. Bedny M, Saxe R. Insights into the origins of knowledge from the cognitive neuroscience of blindness. Cogn Neuropsychol. 2012;29(1-2):56-84.

18. Murphree AL. Intraocular retinoblastoma: the case for a new group classification. Ophthalmology clinics of North America. 2005;18:41-53.

19. 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.

20. 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.

21. 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.

22. 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.


24

23. Chan HSL. Combination Chemotherapy and Cyclosporine Followed by Focal Therapy for Bilateral Retinoblastoma NCT00110110. 2005; https://clinicaltrials.gov/ct2/show/NCT00110110?term=retinoblastoma&intr=cyclosporin&rank=1.

24. Lohmann D, Gallie BL. Retinoblastoma. In: Pagon RA AM, Bird TD, Dolan CR, Fong C-T, and Stephens K. , ed. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2013. Available at http://www.ncbi.nlm.nih.gov/books/NBK1116/2013.

25. Houdayer C, Gauthier-Villars M, Lauge A, et al. Comprehensive screening for constitutional RB1 mutations by DHPLC and QMPSF. Hum Mutat. 2004;23(2):193-202.

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

27. Abramson DH, Niksarli K, Ellsworth RM, Servodidio CA. Changing trends in the management of retinoblastoma: 1951-1965 vs 1966-1980. J Pediatr Ophthalmol Strabismus. 1994;31(1):32-37.

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. Abramson DA, Gallie BL. Retinoblastoma. Current Opinion in Ophthalmology. 1992;3:302-311.30. 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):1051-1061.31. 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. 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.

36. 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.

37. 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.

38. Dimaras H, Kimani K, Dimba EA, et al. Retinoblastoma. Lancet. 2012;379(9824):1436-1446.39. 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):216-223.40. Lau CS, Choy KW, Fan DS, et al. Prenatal screening for retinoblastoma in Hong Kong. Hong Kong

Med J. 2008;14(5):391-394.41. 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.

http://www.ncbi.nlm.nih.gov/books/NBK1116/2013



Early delivery of children at risk for retinoblastoma 25

Table 1: Occurrence of tumors at birth. (*, significantSignificant difference.)

Table 1a Table 1bEyes with tumors at birth Children with tumors at birth

(excluding IIRC A eye of child #8 first examined at age 3 months)

(child #8 included)

YES NO Total % YES YES NO Total % YESNull RB1 mutation 14 17 31 45% 9 7 16 56%Low penetrance RB1 mutation 0 10 10 0% 0 5 5 0%

Total 41 21Fisher's exact test p=0.02* p=0.04*

Cohort 1 9 8 17 53% 6 3 9 67%Cohort 2 5 19 24 21% 3 9 12 25%Total 41 21Fisher's exact test p=0.05* p=0.09

Elise Heon, 12/08/15,

What is the number of cases you are referring to. in subsections it would be useful to see n=...


Table 2: Outcome parameters and their level of significance

Table 2a: Outcome parameters per child

Cohort 1(n=9) Cohort 2 (n=12)

No % No % P value

Tumor(s) at birth 6 67% 3 25% 0.087IIRC AA at first tumor 2 22% 8 67% 0.009*Treatment

Focal therapy only 3 33% 7 58%0.39Systemic chemotherapy 6 67% 5 42%

Treatment success 3 33% 11 92% 0.002*Ocular salvage 4 44% 11 92% 0.046*Visual Outcome

Acceptable vision (better than 0.1) 7 78% 12 100% 0.017*Legal Blind 2 22% 0 0% Table 2b: Outcome parameters per eye

Cohort 1(n=18) Cohort 2(n=24)

No % No % P value

Tumor(s) at birth 10 56% 5 21% 0.027*Good visual prognosis (A) at first tumor 8 44% 17 71% 0.1Treatment success 11 61% 22 92% 0.025*Ocular salvage 13 72% 23 96% 0.07Visual Outcome

Acceptable vision (better than 0.1) 9 50% 21 88% 0.014*Poor Vision 9 50% 3 13%able 2: Outcome parameters and their level of significa



28


29

Figure 1: Schematic representation of each child in Cohort 1 (postnatal RB1 detection) and Cohort 2

(prenatal RB1 detection) from delivery until time of first tumor, IIRC at first tumor per eye, treatment

burden (focal, systemic chemotherapy, or radiation treatment). Number of EUAs, visual acuity at last

follow up and follow up duration.


30


31

I suggest you put all VA in black , if there are no reason for bolding, do not bold some only or bold all, I

would ljustify the VA to the left


32

Figure 2: IVANA PLOT of Age versus type of RB1 mutation.


33

Figure 23: Kaplan Meiyer curves of eye salvage without radiationtreatment success percentage showing a

significant treatment success in Cohort 2 versus Cohort 1.

.

Perc

enta

ge

Time in months


Is ‘percentage’ the right term for the y-axis?Also, the ‘time in months’ is counting from what ‘time 0’? birth? Start of treatment?


34

Good point from Helen, explain “0” and percentage of what? Children without irradiation?


35

Figure 34: Correlation between visual acuity per eye at last follow up (decimal) and gestational age at

delivery in weeks showing a negative correlation.

26 28 30 32 34 36 38 400

0.5

1

1.5

2

f(x) = − 0.0284788135593221 x + 1.66274293785311R² = 0.0313248065403926

Gestational age (weeks)

Visu

al A

cuity

(dec

imal

)

Elise Heon, 12/08/15,

Better eye or both eyes


36

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