Improved visual outcome in familial retinoblastoma with late preterm or

early term delivery after prenatal RB1 mutation identification.

Sameh E. Soliman, M.D.1,2,3, Helen Dimaras, Ph.D.2,3,4, Vikas Khetan, M.B. B.S.5, Elise Héon,

M.D., F.R.C.S.C.2,3,4, Helen S.L. Chan, M.B. B.S., F.R.C.S.C., Brenda L. Gallie, M.D.,

F.R.C.S.C.

1Ophthalmology Department, Faculty of Medicine, Alexandria University

2The Department of Ophthalmology & Vision Sciences, University of Toronto.

3The Hospital for Sick Children, Toronto, Canada.

4The Division of Visual Sciences, Toronto Western Research Institute, Toronto, Canada

5Sankara Nethralya Hospital, Chennai, India.

Pediatrics (H.S.L.C.), Molecular Genetics and Medical Biophysics (B.L.G.), University of

Toronto, Toronto, Ontario, Canada; the Division of Hematology/Oncology (H.D., H.S.L.C.) and

Departments of Ophthalmology & Vision Sciences (E.H., B.L.G., V.K.) and Pediatrics

(H.S.L.C.),; (H.D., B.L.G.);, Toronto, Canada.

Address reprint requests to Dr. Brenda Gallie at the Department of Ophthalmology and Vision

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

Canada, or at brenda@gallie.ca.

Running Head: early delivery of familial RB.

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

pre-term delivery, near-term delivery, amniocentesis

Submitted as Original Article to JAMA Ophthalmology.

Abstract

Purpose: To study the ocular and visual outcomes of infants with familial retinoblastoma when

they are prenatally molecularly diagnosed and safely delivered preterm to initiate treatment, and

compare to outcomes of those diagnosed postnatally.

Methods: A retrospective study of Sickkids medical records documented children born between

1 June 1996 and 1 June 2014, seen at Sickkids because of relation to a retinoblastoma proband.

Patients who inherited their family’s RB1 mutation were included in this study. Data collected

included: relation to proband; sex; gestational age at birth; pregnancy or delivery complications;

date of RB1 molecular testing, type of sample tested and result; timing and location of first and

all subsequent tumor appearance(s) in each eye; treatment history; International Intraocular

Retinoblastoma Classification of each eye; laterality; Tumour Node Metastasis staging; date of

last follow-up; and visual outcome.

Results: Of 21 infants shown to carry their parent’s RB1 mutation, 12 had been tested prenatally

and 9 after birth. Of the infants tested prenatally, 9 were induced at 36-38 weeks gestation and 3

were born spontaneously preterm. Immediate postnatal examination revealed vision-threatening

tumors present only in 25% (3/12) of infants prenatally diagnosed, compared to 67% (6/9) of

those diagnosed postnatally. All patients eventually developed tumors in both eyes. Good vision

was maintained in all prenatally diagnosed patients; treatments included focal therapy (all); later

systemic chemotherapy (5), enucleation and stereotactic radiation (1). Full-term infants received

focal therapy (all), systemic chemotherapy (4), stereotactic radiation (2), and enucleation of one

eye (4), with poorer visual outcome.

Conclusion: Expedient intervention and optimal outcomes were facilitated by prenatal molecular

detection coupled with safe late preterm and near term delivery.

Word Count ( /350)

Introduction

Retinoblastoma, the most common primary ocular malignancy in children, is initiated when

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

progresses when mutations in other specific genes occur.1,2 Both alleles may be lost only in the

somatic cell from which the tumor arises, however, in about 50% of children, a germline

mutation predisposes to the development of multiple retinal tumors during childhood, and other

cancers later in life. Ten percent of patients display a family history of disease, inheriting a

family-specific mutation from a parent.1,3

Familial retinoblastoma tends to be discovered earlier during the first six months if properly

screened and children with germline null alleles might have retinoblastoma tumor(s) already at

birth, which are often in the posterior pole of the eye where they threaten vision.4-8 Most of these

children will develop more tumors in the first year of life, which tend to be located more

peripherally. The preservation of vision with treatment of these small tumors is often difficult,

because focal treatment in proximity to the optic nerve and macula may damage vision. The

child is bilaterally affected in either simultaneous or sequential involvement. 4,7 Low penetrance

mutations (10% of cases)3 and mosiacism can cause low burden of disease with fewer tumors

and higher unilaterally affected children9. The timing of first tumors after birth has not been

studied.

Infants in families with a history of retinoblastoma are recommended to be screened as soon

as possible after birth, and then repeatedly for the first few years of life, including under

anaesthesia, aiming at early diagnosis when tumors are small and easy to treat with ocular and

visual salvage. 6,7

Preterm birth is defined as a live birth occurring before completion of 37 weeks gestation.

Subcategories of preterm birth include: extremely preterm (<28 weeks gestation), very preterm

(28 to <32 weeks) and moderate-to-late preterm (32 to <37 weeks). Full term birth is generally

defined as a live birth occurring at 40 weeks gestation. Infants born after completion of 37 and

before 39 weeks gestation are technically considered early term. (8-9). 10,11 The main concern

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

development and later school performance ,12-14 but visual dysfunction from a larger macular

tumor can cause similar neurocognitive defects due to blindness15 despite never studied in a

comparative manner.

We now present the first report of prenatal genetic screening and late preterm or early term

delivery for treatment of retinoblastoma for children demonstrated to carry the RB1 mutant allele

of a parent. We show that for children at 50% risk to inherit a germline RB1 mutant allele,

prenatal molecular diagnosis and preterm delivery allowed detection and treatment of small,

early tumors, resulting in lower treatment morbidity, better tumor control and visual outcome,

compared to those born full term at 39-40 weeks.

Methods

Study Design

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

The Hospital for Sick Children (SickKids). A retrospective review of medical records of all

children with familial retinoblastoma seen at SickKids and born between 1 June 1996 and 1 June

2014, with relation to a retinoblastoma proband. Data collected included: relation to proband;

laterality of retinoblastoma in proband; sex; gestational age at birth; age; pregnancy, prenatal

abdominal ultrasound if done; delivery or perinatal complications; type of genetic sample tested

and result; penetrance of RB1 mutaion; timing and location of first and all subsequent tumor (s)

in each eye; treatments used; number of anaesthetics; International Intraocular Retinoblastoma

Classification16 of each eye (IIRC); Tumor Node Metastasis (TNM) staging for eyes and child17;

date of last treatment; date of last follow-up; and visual outcome at last follow-up in Snellen and

LogMAR values. RB1 mutation testing was performed by Retinoblastoma Solutions before 2013,

and Impact Genetics after 2013, as previously described.18

The corrected age for gestation for each child was calculated (taking 39 weeks as full term).

Eyes with vision threatening tumor were defined any tumor threatening the optic nerve or

macular area, in posterior pole. Treatment burden was evaluated by an impact score (Figure 1)

based on i) duration of active treatment (time from diagnosis to last treatment), ii) use of

systemic chemotherapy or radiation, and iii) number of examinations under anesthesia (EUAs).

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

visual outcome was defined as visual acuity > 20/200 (>0 in 1-LogMAR scale) (cut edge of legal

blindness). A blind child is defined as best eye visual acuity < 20/200 (<0 in 1-LogMAR scale).

Statistics

Basic descriptive statistics (student t-test, chi square test and Fisher exact test) were used for

all comparisons between patients who underwent prenatal testing and preterm delivery and those

who were diagnosed post-natally.

Results

Patient Demographics

The records of 21 familial retinoblastoma children were reviewed (11 males, 10 females)

(Supplementary Table 1). Diagnosis was by observation of prenatal retinoblastoma tumor (child

#9) or postnatal tumor (child #8) or postnatal testing for the parental RB1 mutation for Cohort 1:

6 were delivered full term and 3 late preterm because of pregnancy-induced hypertension (#7),

fetal ultrasound evidence of retinoblastoma19 (#9) or spontaneous (#8). Twelve children (57%)

(Cohort 2) were prenatally diagnosed to carry an RB1 mutation and planned for late preterm or

early term delivery: 3 were spontaneously premature (#10, 13, 15; 28-37 weeks gestation) and 9

were referred to a high-risk pregnancy unit for elective 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 affected (mother #8, father #19). The familial RB1 mutations

were previously detected except for the unilaterally affected parent of #8, whose mutation was

identified after finding her son’s mutation as she was tested previously and considered to be a

non germline mutation. Cohort 1 children (#1-9) were tested postnatal on blood; Cohort 2

children (#10-21) were tested prenatal on amniocentesis at 16-33 weeks gestation.

Null RB1 mutations were present in 16 families; 5 had low penetrance RB1 mutations (whole

gene deletion #19; weak splice site mutations #15, 18, 21; and C712R18). No proband in this

study was mosaic for the RB1 mutation. All study subjects were eventually bilaterally affected.

No tumors were detected at birth (IIRC16 Group 0) in 7/15 (47%) infants and 17/30 (57%) eyes

with null RB1 mutations, and in 5/5 (100%) infants and 10/10 eyes with low penetrance

mutations (p=0.04* for patients, p=0.02* for eyes; Fisher exact test) (Table 1).

The age at first tumor in either eye was significantly younger for those with null mutations

(mean 89, median 74 days), than those with low penetrance mutations (mean 134, median 119

days) (P=0.04*, Mann Whitney test). However, the gestational age at first tumor for those with

null mutations (mean 77, median 43 days) was not significantly different than for those with low

penetrance mutations (mean 110, median 81 days) (P=0.24*, Mann Whitney test). (Child #8 was

excluded from these calculations as the child was first examined at 3 months of age with Group

A/D tumors, so age at first diagnosis is unknown.)

Stage of Tumors at Birth

Thirty-three percent (3/9) of Cohort 1 and 75% (9/12) of Cohort 2 were free of visible tumor

at birth (Table 1a, Figure 1) (p=0.09). We assumed that child #8 had tumor at birth since he had a

group D IIRC16 eye at 3 months of age. Of eyes, 79% (19/24) of Cohort 1 eyes were tumor-free

at birth, compared to 33% (6/18) of Cohort 2 eyes (p=0.026*, Chi Square test), excluding the

IIRC16 Group A eye of child #8 (Table 1b).

All patients eventually developed tumors in both eyes regardless of whether their RB1

mutation was full or low penetrance. Tumors emerged first in the macular and peri-macular

region (IIRC16 Group B), as previously described20. The median gestational age of diagnosis of

24 IIRC16 A eyes (< 3mm and away from optic nerve and fovea) was 103 days, and of 16 IIRC16

B eyes (all threatening optic nerve and fovea, 6 also >3 mm) was 38 days, significantly younger

reflecting the early development of visually threatening tumors (Table 3?). Bilateral IIRC16

Group A eyes were present at initial diagnosis (optimal situation for achieving good vision with

minimally invasive therapy) in 2/9 (22%) children in Cohort 1 and 7/12 (58%) in Cohort 2

(Table 2a). IIRC16 Group A was the initial diagnosis of 9/18 (50%) eyes in Cohort 1, and 11/22

(68%) eyes in Cohort 2 (p=0.15, Table 2b).

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.21 Cohort 1

patients were treated with focal therapy (all), chemotherapy (4), stereotactic radiation (2), and

enucleation of one eye (4) (Supplementary Table 1, Figures 1, 3). Cohort 2 patients were treated

with focal therapy (all); later systemic chemotherapy (5), enucleation of one eye and stereotactic

radiation (1) (Figure 1, 2). Successful treatment by focal therapy alone (avoidance of systemic

chemotherapy or EBRT) was possible in 2/9 (22%) of Cohort 1 and 7/12 (58%) of Cohort 2

(Table 3, , Figure 2).

The treatment burden showed no statistical significant difference between Cohort 1 and 2

(percentage of patients requiring systemic therapy and active treatment duration). The mean

active treatment duration was 579 days (0-2101 days) in Cohort 1 compared to 473 days (0-971

days) in Cohort 2.

Outcome

There were no adverse effects associated with induced or natural preterm or early term birth,

and no pregnancy, delivery or perinatal complications reported for any of the infants. Overall

mean follow up was 8 years (median 5.6 years) (Table 1).

Treatment success

92% of prenatally diagnosed mutation patients didn’t require either enucleation or external

beam irradiation in comparison to 44% in the postnatally diagnosed mutation patients (P=0.046*,

Fisher exact test). Kaplan Meier ocular survival graph for prenatal versus postnatal diagnosis is

shown in figure it is estimated at 67% in postnatal cohort compared to 92% in the prenatal cohort

at 4 years. None of the cases developed extra ocular or metastatic disease and all of them are

alive at the end of the study.

Visual outcome

As per eye, 92% of eyes of the prenatal detection and planned earlier delivery were of good

visual outcome compared to 56% of eyes in the postnatal detection; a difference found to be

statistically significant (P=0.010*, Fisher exact test). As per child, 22% of children in the

postnatal detection Cohort were considered blind (visual acuity less than 20/200 both eyes) in

comparison none of the patients in the prenatal detection Cohort (p=0.174, Fisher exact test)

Treatment success (avoidance of enucleation and/or stereotactic radiation) accompanied with

resultant good vision was documented for 88% (21/24) eyes in the prenatal diagnosis cohort,

compared to 50% (9/18) of eyes from the postnatal diagnosis cohort with significant difference

(p=0.014*, Fisher exact test) (Table 2b, Figure 1). A negative correlation was found between

gestational age and final visual outcome (r=-0.24) with better visual outcome in earlier

deliveries. (Figure )

Discussion

In the first study of its kind, we report that prenatal molecular diagnosis of familial

retinoblastoma was possible, and that elective late-preterm or early term delivery allowed

monitoring and treatment of tumors as they emerged, resulting in better ocular and visual

outcomes. Our cases illustrate well that the risk of visual loss from delayed therapy for macular

retinoblastoma tumors outweighs the risks associated with induced late preterm delivery (Figure

1).

With the most current technologies, it is practical to identify 96% of the germline mutations

in bilaterally affected probands, and to identify the 10% of unilateral probands who carry a

germline gene mutation by studying the tumor .21 When the family's unique mutation is identified

in the proband, molecular testing of family members can determine who else carries the mutation

and is at risk of developing the disease. We reported on in utero testing that identified 12

mutation carriers, but did not include the number of infants tested who did not inherit their

family’s mutation. These infants can go to full term and do not need examination to detect

retinoblastoma tumors, since they are at no greater risk of developing the disease than the general

population. Without molecular information, repeated retinal examination is essential for all first

degree relatives until the age of 7 years, the first 3 years under general anesthesia.21 Such

repeated screening can be a great burden on patients and their families, finances, time and

resource utilization than the one-time-only molecular genetic testing that would conclusively

determine their risk.4 Also multiple studies22-24 showed the deleterious effects of multiple

anesthesias in early infancy on the neurocognitive development of the child.

Optimal treatment for retinoblastoma today includes combined therapeutic modalities to

optimize vision and minimize morbidity of treatment, while achieving tumor control; the relative

ease or difficulty of this task is affected by the age and the degree of tumor burden at diagnosis.25

Retinoblastoma treatment in the first 3 months of life represents a challenge as most of the

therapeutic modalities as systemic chemotherapy, chemosurgery or radiation therapy can’t be

used. The only treatment options at this age are focal therapy (laser and cryotherapy) and

periocular chemotherapy.26 Consistent with previous reports,5 we showed that 62% of children

with a germline gene mutation already have tumors at birth. This percentage reduces to 31%

when the germline mutation is prenatally detected and earlier delivery (late preterm or early

term) was planned.

The earliest tumors commonly involve the macular or paramacular region, dangerously

risking loss of central vision, while tumors that develop later on are usually peripheral, where

they have less visual impact.5,26-29 In our cohort, the risk of having a vision threatening tumor

dropped from 39% to 17% by prenatal mutation detection and planned earlier delivery.

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

radioactive plaque, since these will damage the optic nerve or central vision. Systemic

chemotherapy effectively shrinks tumors such that focal therapy can be applied with minimal

visual damage. In our setting, the Toronto Protocol using high dose, short duration cyclosporine

to counteract multidrug resistance30,31 has allowed many retinoblastoma tumors to be treated with

combination chemotherapy and focal therapy without resorting to radiation. In our experience,

infants as young as 30 days tolerate the Toronto Protocol with cyclosporine A, 30,31 however

systemic chemotherapy in neonates has other associated morbidities. We recognize the

conventional recommendation to reduce chemotherapy dosages by 50%, particularly for infants

in the first three months of life, to offset the immaturity of their liver and kidneys,32-34 but note

that this also breeds the optimal conditions for cancer cells to develop multidrug resistance,

making later recurrence difficult to treat. The development of periocular topotecan for treatment

of small-volume retinoblastoma35 also assisted in the number of patients that were able to be

treated by focal therapy alone, avoiding systemic modalities on the young infants, and a greater

rate of eye salvage with good visual outcome (Table 2).

Imhof et al7 in the Netherlands screened 135 children at risk of familial retinoblastoma 1-2

weeks after birth without molecular diagnosis and discovered 17 cases of familial RB (13% of

screened children at risk) and 70% of them had RB in at least one eye at first examination and

41% of eyes had vision threatening tumor to the macula. 41% (7/17) of patients had failure of

treatment (EBRT or enucleation) and one case of metastasis. 73.5% of eyes (27/34) had good

visual acuity (defined by vision >20/100) that will reduce to 56% (19/27) if we consider eyes

with EBRT as failure. These results correspond to our postnatal screening cohort showing similar

results. On the contrary, the prenatal diagnosis and planned earlier delivery cohort showed less

vision threatening tumors (17%), less treatment failure (8%) and better visual outcome (88%).

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

the internationally accepted model (whether intensive screening is utilized or not).7,36 Here we

propose the prenatal screening of the known mutation in the probands by amniocentesis in the

second half of pregnancy where the risks of miscarriage are minimal (0.1-1.4%).37,38 For those

who are confirmed to have the mutation; planned late preterm or early term delivery at 36-38

weeks of gestation and as a result a smaller tumor with less macular involvement leading to

better visual outcome is anticipated. there was no difference between the two Cohorts in the

treatment burden and the systemic chemotherapy usage as we didn't change the treatment course

by early delivery but changed the treatment outcome by catching the tumors at earlier stage also

multiple focal treatments in both Cohorts were for small new tumors that occurred due to the

nature of the germline tumor and not related to early delivery or prenatal detection.

The main concern with late preterm or early term delivery is its reported effect on

neurological and cognitive development and later school performance (30-32),12-14 but visual

dysfunction from a larger macular tumor can cause similar neurocognitive defects due to

blindness15 despite never studied in a comparative manner. So, earlier delivery must be discussed

thoroughly through the team of neonatologist ophthalmologist and oncologist to reach the best

timing for better outcome25 so rather than focusing on the combination of treatments to tackle

burdensome disease, we showed safe preterm delivery resulted in a decreased tumor burden at

birth that was significantly easier to treat (Figure 2, Table 2). Safe preterm delivery resulted in

more infants born tumor-free, facilitating frequent surveillance to detect tumors as they emerged,

and focal therapy of smaller, easier to control masses, causing minimal damage to vision (Figure

1,2).

Counseling on reproductive risks is imperative for families affected by retinoblastoma even

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

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

published literature has been cited as useful in preimplantation genetics (to ensure an unaffected

child) or to inform parents who wish to terminate an affected pregnancy39. 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 mutation40, and in the other, 3 of 5 tested fetuses

of a proband were terminated once molecular testing confirmed the mutation in the offspring.41

We are first to report that elective safe late-preterm delivery of prenatally diagnosed infants with

retinoblastoma results in improved outcomes. It is our experience that for retinoblastoma

survivors and their relatives who understand fully the underlying risks, they are more interested

in early diagnosis to optimize options for therapy in affected babies rather than to consider

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. A long-term

study could show the impact of such an approach to patient outcomes in their adulthood. We

conclude that since infants with familial retinoblastoma are likely to develop vision-threatening

macular tumors, prenatal molecular diagnosis and safe, late-preterm delivery will increase the

chance of good visual outcome with decreased treatment associated morbidity.

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