Improved visual Outcome outcome in familial retinoblastoma with late

preterm or early term delivery after prenatal RB1 mutation identification.

Helen Dimaras, Ph.D., Sameh E. Soliman, M.D., ** Vikas Khetan, M.B. B.S.*, Elise Héon,

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

From the Department of Ophthalmology & Vision Sciences (H.D., E.H., B.L.G.), 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.), The Hospital for Sick Children, Toronto, Canada; the Division of Visual Sciences,

Toronto Western Research Institute, Toronto, Canada (H.D., B.L.G.); and the Campbell Family

Institute for Cancer Research, Princess Margaret Hospital (B.L.G.), Toronto, Canada. *Current

Affiliation: Sankara Nethralya Hospital, Chennai, India.

**Ophthalmology Department, Faculty of Medicine, Alexandria University

Address reprint requests to Dr. Brenda Gallie at the Campbell Family Institute for Cancer

Research, Ontario Cancer Institute/Princess Margaret Hospital, Room 8-415, 610

University Avenue, Toronto, ON M5G 2M9, 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.

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 vision4-7 + munier

ref), ). Most of these children will develop more tumors in the first year of life, which tend to be

located in more peripheral to the macular area. 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 can cause low burden of disease with

fewer tumors and higher unilaterally affected children8. 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) 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). 9,10 The main concern

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

development and later school performance ,11-13 but visual dysfunction from a larger macular

tumor can cause similar neurocognitive defects due to blindness14 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 40 weeks.

Methods

Study Design

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

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

children with familial retinoblastoma seen at SickKids medical records documented children

diagnosed with retinoblastomaand born between 1 June 1996 and 1 June 2014, seen at SickKids

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

laterality of RB retinoblastoma in proband; sex; gestational age at birth; birth dateage;

pregnancy, prenatal abdominal ultrasound if done; delivery or perinatal complications; date of

molecular testing, type of sample tested and result; type of penetrance of mutaion detected;

timing and location of first and all subsequent tumor (s)appearance(s) in each eye; treatment

history; number of anaesthetics; International Intraocular Retinoblastoma Classification of each

eye (IIRC); (Murpheree paper)Tumor Node Metastasis (TNM) staging; date of last treatment;

date of last follow-up; and visual outcome at last follow-up in Snellen and LogMAR values. If

any of the above data was not explicitly noted in the medical record, it was deduced from the

available clinical information.

RB1 mutation testing was performed by Retinoblastoma Solutions before 2013, and Impact

Genetics after 2013, as previously described.15

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

Eyes with vision threatening tumor was defined any tumor involving the macular area i.e. IIRC

group B (in posterior pole) or higher. Treatment burden was evaluated by an impact score

ofbased on i) duration of active treatment (time from diagnosis till last treatment), and ii) adding

systemic chemotherapy to the focal therapies. And , 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 by as best eye visual acuity < 20/200

in both eyes (<0 in 1-LogMAR scale). or only eye if one is enucleated). For proper statistical

analysis, (1-LogMAR visual acuity) value will be used to describe best corrected visual acuity

for each eye.

Statistics

Basic descriptive statistics were used for all comparisons between patients who underwent

prenatal testing and preterm delivery and those who were diagnosed post-natally.

Results

Patient Demographics

During the period studied, we reviewed Tthe files records of 21 familial retinoblastoma

children were reviewed (11 males, 10 females). Twelve children (57%) were prenatally

diagnosed to carry an RB1 mutation and planned to be deliveredfor late preterm or early term

delivery. Three of these were spontaneously premature (Patients 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). The molecular diagnosis was confirmed performed

postnatal in 9 children (43%). In these children, 6 were delivered at full term and 3 were

delivered late preterm due to pregnancy-induced hypertension (1), fetal ultrasound evidence of

retinoblastoma (1) or spontaneous (1). (Table 1)

Molecular diagnosis

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

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

mutations were previously detected and known except for the unilaterally affected parent of child

8, whose mutation was discovered identified after finding her son’s mutation as she was tested

previously and considered to be a non germline mutation. Childrens 1-9 were tested post-natally

in blood; one child inherited a low penetrance mutation; while,. Childrens 10-21 were tested

prenatally on amniocentesis at 16-33 weeks gestation; four children had low penetrance

mutations (Table 1).

Tumors at Birth

The aim of elective preterm delivery is to diagnose tumors early in their development, as they

emerge and are easy to control. In the prenatally diagnosed cohort, 75% (9/12) were free of

visible tumor at birth (Figure 1); 33% (3/9) of patients postnatally detected were tumor free at

birth (p=0.09). After excluding the low penetrance mutation cases (which by definition results in

a lesser burden of disease), 58% (3/8) of prenatally detected full penetrance mutations and 25%

(2/8) of patients with were tumor-free at birth (p=0.31) (Table 2). Only one patient was not seen

at birth (Case 8) and was seen at 3 months with bilateral RB but as one eye had a group D IIRC

eye it was assumed that he had tumor at birth.

Looking at the numbers on a per eye basis, 79% (19/24) of eyes in prenatally diagnosed

babies were free of tumor at birth, compared to 33% (6/18) of postnatal diagnosed babies

(p=0.026*) (Table 2b).

All patients eventually developed tumors in both eyes regardless of the mutation whether full

or low penetrance. Tumors emerged first in the macular and peri-macular region. By the

International Intraocular Retinoblastoma Classification,16 58% (7/12) of prenatally diagnosed

infants had bilateral Group A eyes at the time of initial tumor development in each eye (the

classification that denotes the lowest level of tumor involvement, free foveal center and hence

least visual threat), compared with only 22% (2/9) of postnatally diagnosed infants (Table 2a). on

a per eye basis 39% (7/18) of postnatally diagnosed cohort eyes were visually threatening in

comparison with 17% (4/24) in the prenatal diagnosis cohort eyes (p=0.15)

The mean age at first tumor showed statistical significant difference between full penetrance

and low penetrance mutation eyes (90 days versus 134 days respectively, P=0.04*), after

correcting the age for the gestational age (as some of them were preterm), the mean age at first

tumor became 79 days versus 110 days respectively, p=0.24. (Case eight were excluded from

this calculations as the child presented at 3 months of age)

Treatment Course

All infants were frequently examined from birth onwards (except case 8 that presented at 3

months) as per the National Retinoblastoma Strategy Guidelines for Care.17 For patients who

were prenatally diagnosed and delivered late preterm or early term, treatments included focal

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

(1) (Figure 1, 2). Postnatally diagnosed infants received focal therapy (all), chemotherapy (4),

stereotactic radiation (2), and enucleation of one eye (4) (Figure 1, 3). Successful treatment by

focal therapy alone (avoidance of systemic chemotherapy) was possible in 58% of prenatally

diagnosed babies (7/12) compared with only 22% (2/9) of postnatally diagnosed infants (Table

2a, Figure 2).

The treatment burden showed no statistical significant difference between those prenatally or

postnatally diagnosed (percentage of patients requiring systemic therapy and active treatment

duration). The mean active treatment duration was 579 days (0-2101 days) in the postnatal

detection group compared to 473 days (0-971 days) in the prenatal detection group.

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 group were considered blind (visual acuity less than 20/200 both eyes) in

comparison none of the patients in the prenatal detection group (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 .17 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.17 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 studies18-20 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.21

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. 22 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.5,22-25 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 resistance26,27 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, 26,27 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,28-30 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 retinoblastoma31 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).7,32 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%).33,34 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 groups 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 groups 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),11-13 but visual

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

blindness14 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 outcome21 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 pregnancy35. 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 mutation36, and in the other, 3 of 5 tested fetuses

of a proband were terminated once molecular testing confirmed the mutation in the offspring.37

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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