file · Web viewPrenatal versus Postnatal screening in neonates with familial retinoblastoma

Prenatal versus Postnatal Expectant management versus

conventional screening in infants neonates with familial

retinoblastoma

Sameh E. Soliman, MD,1,2 Helen Dimaras, PhD,1,3,4 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 [email protected]

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 Nethralya 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.

Sameh Gaballah, 07/01/16,

Reviewer 2:In the title, is 'infants' the right word for unborn babies? How about 'Prenatal vs post-natal screening for retinoblastoma in patients with a family history of this disease'


Sameh:I suggest to change the title to this Also, we can use newborns instead of neonates.

mailto:[email protected]

Soliman et al. Expectant Management of Familial Retinoblastoma

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 in 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

2


Précis: 35/35

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

term delivery) achieved was associated with more often no tumors at birth, earlier tumor detection, less

invasive therapies and better visual outcome, compared to conventional postnatal screening. in familial

retinoblastoma,

3


Reviewer 2:'achieved more often' seems like poor grammar. How about, 'was associated with'. Is 'no tumors at birth' an achievement?


Reviewer 2: In the Precis, 'Expectant management' suggests inactivity. Why not start the précis with the word 'Prenatal'?


Editor’s comment:While I cared for RB patients 20 to 25 years ago I am no longer current on all this. It seems to me that you are looking at patients who are screened pre-natally and delivered early. I do not recall or know or know if it is known when tumors typically develop but I think we already know that all are not present prenatally. If they were, we could do one screening and if there are no tumors not do further follow up. So, some develop before birth, some around the time of birth and some after birth. Now, I read:"Expectant management of familial retinoblastoma (prenatal RB1 mutation detection and planned earlytermdelivery) achieved more often no tumors at birth, earlier tumor detection, less invasive therapies andbetter visual outcome, compared to conventional postnatal screening."Well, if you look earlier, of course there will be fewer tumors, earlier detection and since presumably tumors found will be smaller, less invasive therapies. I guess there might be better vision outcomes but maybe not. Anyway, given what we know about RB, it seems your study is not needed unless we did not already know that some tumors are not present in utero. My comments in part are silly and I think I do not mean to detract from your study but perhaps I am suggesting that you reword the main points to speak more to what new here you are finding and why it may matter. Of course, I suspect it is appropriate to add that prospective validation will be needed?


Abstract (348353/350 words)

OBJECTIVE To compare overall outcomes of postnatal screening for children within familial

retinoblastoma conventionally screened postnatal, compared toto prenatal those expectantly

managedscreening with prenatal RB1 mutation identification and early term delivery.

DESIGN A retrospective, observational study.

PARTICIPANTS Twenty children 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 within

one week of birth who had postnatal RB1 testing. Expectant Cohort 2 consisted of infants identified by

amniocentesis to carry their relative’s known RB1 mutant allele, who were scheduled for early term

delivery (36-38 weeks gestation). All children received Ttreatment for eye tumors 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.

4


Reviewer 2:Should 'enucleation and external beam irradiation' be 'enucleation or external beam irradiation'?Sameh: changed to and/or.


Reviewer 2:Should 'age at first tumor each eye' be 'age at first tumor in each eye'?Sameh: done


Reviewer 1:Under Participants, the location of the study should be transferred to the methods section.Sameh: Done as suggested


Reviewer 2:Should 'postnatal' be 'post-natally'?We think Postnatal is more correct.


Reviewer 2:does the term 'familial retinoblastoma' suggest that the patient does have the malignancy? Sameh: YES, if family history of retinoblastoma. We would include in the post-natal screening all the children that has family history and no RB1 mutation and never developed a tumor.


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 or A0 (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.

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.

5


Reviewer 2:Since conclusions are inferences from results, they could be given in the present tense.Sameh: done as suggested


Reviewer 2:When reporting outcomes, the follow-up times should be reported. Sameh: added


Reviewer 2:When reporting p values, it is conventional to write the statistical method used, before the p value, with a comma. Sameh: Done as suggested.


Reviewer 2:It is not conventional to report percentages when the numbers are so small.


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 a germline 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.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.

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 (conventional screening), including under anesthesia.10 Early diagnosis when tumors are small and

treatable with less invasive therapies is thought to optimize salvage of the eye and vision.5,6,10 We have

managed familial retinoblastoma by screening the fetus for the RB1 mutation of the proband parent. If the

6


Reviewe 2:'including under anesthesia' could be 'under general anesthesia'.


Reviewer 2:In the Introduction, I'm not sure about the terms 'expectant management' and 'conventional screening'. They do not mean anything, to me at least, without having to read the text. This is really about pre-term vs full-term delivery. Screening, testing etc are means to an end (i.e., early induction of labor).


Reviewe 2:'tumor detection and management' Patients are managed and tumors are treated.


Reviewer 2:'making treatment of these tumors difficult' is not necessary, as it is not treatment that is difficult but conservation of vision. Sameh: Removed as suggested.


Reviewer 2:'eye where' should be 'eye, where'.Sameh Done


Reviewer 2:'retinoblastoma tumor(s)' could be 'retinoblastoma(s)', since these are tumors.Sameh: Changed as suggested


Reviewer 2:'(50% of children) is out of place, without the word 'affecting'.


Reviewer 2:Page 6. Should 'commonly' be 'usually'?Sameh: yes and done


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)11 with full retinal examination on day 1. Further management is

conventional screening and treatment. We hypothesized that earlier diagnosed retinoblastoma tumors will

would be smaller and easier to treat.

The aim of this study it performWe present a retrospective comparative review of expectant prenatal

genetic diagnosis vs versus conventional postnatal management of children with familial retinoblastoma.

We show that children with expectant management had earlier detection and treatment of small tumors,

lower treatment 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 the Declaration of Helsinki. Family privacy was preserved by eliminating

any personal identifiers during data collection. Data collected for children with familial retinoblastoma

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

7


Reviewer 1:The first paragraph should indicate if this study conformed to the Tenets of the Declaration of Helsinki and what privacy protections were afforded these families.


Reviewer 1:The last sentence of the introduction is a conclusion. It should be phrased that, "The purpose of this study is to examine …..


Reviewer 2:It is not conventional to summarize the results at the end of the introduction.Sameh: we deleted the statement with results.


Reviewer 2:The aim is not clearly stated.


Reviewer 2:'We hypothesized that earlier diagnosed retinoblastoma tumors will be smaller and easier to treat.' Should 'will' be 'would'?Sameh: changed as suggested


first examination; age and location of first tumor(s) in each eye; treatments used; International Intraocular

Retinoblastoma Classification (IIRC)12 of each eye; Tumor Node Metastasis (TNM)13 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 IIRC 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 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 postnatal (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

8


Reviewer 2:'Useful vision is defined' should be 'was defined'. Sameh: Done


Reviewer 2:'in an eye' is ambiguous.Sameh, changed its position


Reviewer 2:'in an eye' is ambiguous.


Reviewer 2:'Active treatment duration' is not 'counted' but 'measured'.Sameh: Done


Reviewer 1:Page 8, line 2: Dr. Gallie was a member of the 8th edition, AJCC TNM(H) staging committee. Perhaps, this "new" staging system could be used for this study as it directly impacts patient outcomes and survival.


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 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 retinoblastoma14 (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.

9


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,15,16 #5)

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

2a, Figure 1). Of Cohort 1 children, 63% (5/8) and 25% (3/12) of Cohort 2 had tumor in at least one eye

at birth (Table 2b, Figure 1). At birth, 38% of eyes (6/16) in Cohort 1 had a visually threatening tumor

(IIRC12 Group B 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 (IIRC12 Group B), and tumors

detected later were smaller and not threatening vision, as previously described (Figures 1, 3).17,18 The

median age of diagnosis of first tumor of 15 IIRC12 Group B eyes (threatening optic nerve and fovea, and

10


Reviewer 2:'Of Cohort 1 children, 63% (5/8) and 25% (3/12) of Cohort 2 had tumor in at least one eye at birth (Table 2b, Figure 1).' This sentence needs re-writing and should be supported by statistics, like the next sentence and elsewhere.


Reviewer 2: Either the mean or the median days should be reported, according to whether or not the distribution is skewed.


Reviewer 2: 'Eventually' is usually at the beginning of the sentence.


6 with at least 1 tumor >3 mm) was 9 days, tending younger than the 92 days for 24 IIRC12 Group A eyes

(tumors < 3 mm and away from optic nerve and fovea). The corrected age at first tumor diagnosis for

these eyes showed the same tendency (medians 7 and 60 days respectively).

At initial tumor diagnosis, 2/8 (25%) children in Cohort 1 had IIRC12 Group A/A or A/0 eyes,

compared to 8/12 (67%) in Cohort 2 (P=0.02, Fisher exact test) (Figure 1, Table 2b). At first diagnosis,

tumors were not threatening vision (IIRC12 Group A) in 7/16 (50%) Cohort 1 eyes, compared to 17/24

(71%) Cohort 2 eyes, (Table 2a). Vision-threatening tumor (Group B or worse) were present at first

diagnosis in 38% of Cohort 1 compared to 17% of Cohort 2 (Figure 1, Table 2a).

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 80% (12/15

eyes) were within the posterior pole.

Treatment Course

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

Strategy Guidelines for Care.10 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 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,

11


Reviewer 2:In the following phrase 'with focal therapy (all)' the word 'all' is confusing.


Reviewer 2:'The corrected age at first tumor diagnosis for these eyes showed the same tendency (medians 7 and 60 days respectively).' I cannot understand this sentence.


etoposide and cyclosporine (Toronto protocol)19 {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 (all); 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 and 7/12 (58%) of Cohort 2 (Table 2b).

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

days (0-971 days) in Cohort 2. The median number of EUAs was equal: 29 (range 18-81) Cohort 1 and 29

(range 20-41) Cohort 2.

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. Follow

up (mean, median) was 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, age of Cohort 1 was mean 10 years (median 10, range 3-19) and Cohort 2

mean 9 years (median 9, range 3-16).

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

33% (3/8) of Cohort 1 and 92% (11/12) of Cohort 2 patients (P=0.02, Fisher exact test) (Table 2b).

Kaplan Meier 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

12


Reviewer 2:Kaplan Meier ocular survival rates of 62% and 92% are meaningless unless a timepoint is given.


Reviewer 2:The following sentence could do with re-writing 'Neither enucleation nor external beam irradiation were required (defined as treatment success) in 33% (3/8) of Cohort 1 and 92% (11/12) of Cohort 2 patients (P=0.02, Fisher exact test) (Table 2b).' as 'neither enucleation nor external beam irradiation' is confusing. Better to write 'Treatment success occurred in…'


Reviewer 2:In Outcomes, only median should be reported if follow-up times were skewed, as they usually are.


Reviewer 2:Many comparisons are not supported by statistics (e.g., active treatment duration).


features20-22 in the enucleated eye and received adjuvant treatment. All children are presently alive without

metastases.

Children were legally blind in 12% (1/8) of Cohort 1 and 0% of Cohort 2 (Table 1a). 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 1a). 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.

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.

This approach allowed treatment of small tumors as they emerged, resulting in better ocular and visual

outcomes and 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 outweighs the risks associated with induced late

13


Reviewer 2:The structure of the Discussion could be improved.


Reviewer 1:The discussion is too long and needs to be focused. It should only repeat the most important results, contrast them with the literature, discuss the weaknesses of the work as well as the strengths and end with directions for the future. The latter should include how genetic staging will affect our management. That said, this reviewer found a great deal of interesting and important information in the discussion.


Reviewer 2:General comments: This is an interesting and useful article, which suggests that the birth of individuals with high risk for retinoblastoma should be induced early. It would be useful if the disease stage/number/size of tumors could be correlated with the gestational age/weeks of pregnancy when the labor is induced.


Reviewer 2:'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.' This needs a p value.


Reviewer 2:The last sentence should read 'All children were alive without metastasis at the close of the study.'


preterm delivery (Figure 1). 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 to identify 97% of the germline mutations in bilaterally affected probands and ~15% of

unilateral probands who carry a germline gene mutation.3,9,24 When the proband's unique mutation is

known, molecular testing of family members can determine which relatives also carry the mutation and

are at risk to develop retinoblastoma and other cancers. We report 12 infants identified in utero by

molecular testing to carry the mutant RB1 allele of a parent. The tested infants who did not inherit their

family’s mutation (not shown) required no surveillance.

The earliest tumors commonly involve the perimacular region, threatening loss of central vision,

while tumors that develop later are usually peripheral, with less visual impact.17,18,25 Macular and

perimacular tumors 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 detection by obstetrical ultrasound, which developed drug-resistance following reduced-dose

chemotherapy as a newborn,14 ultimately requiring enucleation at the age of 14 years. 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

14


Reviewer 2:'due to the unknowns of immature liver and kidney function to metabolize the drugs, increasing the potential of severe adverse effects' - sentence can be improved.


recommendation is to either reduce chemotherapy dosages by 50%, particularly for infants in the first

three months of life,26 or administer a single agent carboplatin chemotherapy.27 However, reduced doses

carry risk of selecting for multidrug resistance in the tumor cells, making later recurrences difficult to

treat.28-30 Periocular topotecan for treatment of small-volume retinoblastoma31 may increase the

effectiveness of focal therapy without facilitating resistance. Intra-arterial chemotherapy is not feasible in

such tiny babies.

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 diagnosis, 70% had

retinoblastoma with vision threatened in 41% of eyes; 41% had eye salvage failure (required radiation

and/or enucleation); one child developed metastasis; and 74% achieved vision >20/100. In comparison,

Cohort 2 showed 17% vision threatening tumors, 8% eye salvage failures and 88% vision >20/100.

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

sibling of a proband was found not to carry the sibling’s mutation;32 and in the other, 2 of 5 tested fetuses

of a mosaic proband were born without the parental mutation.33

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

internationally accepted convention for retinoblastoma.6,34 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%).35,36 We show that planned late preterm/early term delivery (36-38 weeks gestation) for those

15


Reviewer 2:In the next paragraph, 'indicated' is the wrong verb.


Reviewer 2:'Cohort 2 showed 17% vision threatening tumors, 8% eye salvage failures and 88% vision >20/100.' This sentence is poorly written and confusing.


Reviewer 2:The Imhof data on page 15 do not tally with data in the first para of page 14.


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 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,40 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.41

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

unilateral probands.42 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.43 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

16


Reviewer 1:The second sentence of the last paragraph on page 16 is poorly written.


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 retinoblastoma, as a potential stem cell source in later anti-cancer

therapy.

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 management 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.

17


Editorial office:Please secure written permission from person(s) mentioned in the acknowledgment section.


Reviewer 2:The Discussion should discuss the weaknesses of the study.


Reviewer 1:General Comments:This is a well-written manuscript, describing research on an important subject, written by known experts in the field. It suffers from the weaknesses inherent in its structure (small single center, retrospective observational study)


References

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

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

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

Organization;2012.

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

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

13. The 7th edition AJCC staging system for eye cancer: an international language for ophthalmic

oncology. Archives of pathology & laboratory medicine. 2009;133(8):1197-1198.

14. 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.

18


Reviewer 1:References: 10 and 13 do not have authors or study groups listed.


15. 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.

16. 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.

17. 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.

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

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

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

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

20. 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.

21. 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.

22. 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.

23. Abramson DH, Mendelsohn ME, Servodidio CA, Tretter T, Gombos DS. Familial retinoblastoma:

where and when? Acta Ophthalmol Scand. 1998;76(3):334-338.

24. 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.

25. 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.

26. 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.

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

2012;17(4):239-242.

28. 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.

19

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


29. 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.

30. 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.

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

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

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

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

33. 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.

34. 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.

35. 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.

36. 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.

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

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

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

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

Genet. 2016;In Press.

43. 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.

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Table 1. 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

21

Sameh Gaballah, 2016-07-01,

Reviewer 2:It is conventional to report p values to 3 decimal places. Why is there an asterisk next to only one p value in Table 1?


Editorial office:Remove embedded tables in manuscript file; upload as separate Word files (1 table per file).


Table 2a: 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*

22


Reviewer 2:'Combined treatment success and acceptable vision' is, with respect, clumsy. Perhaps treatment success could be defined to include acceptable vision.


Table 2b: 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.

23


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).

24


Reviewer 2:In Figure 4, the statistical test should accompany the p value.


Reviewer 1:Wants to delete this fig.


Reviewer 2:In Figure 1, it may have been better to have gestational age throughout the x axis as the split confuses matter. For example, age of 1 month means different times according to the gestational age at birth.


Editorial office:Upload all figures in tif or eps format.


Reviewer 2:The figures are fuzzy and of poor quality.

Documents

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