Improved visual outcome in familial retinoblastoma with late preterm or early term delivery after prenatal RB1 mutation identificationComment by Sameh Gaballah: Cover LetterInclude a cover letter and complete contact information for the corresponding author (affiliation, postal/mail address, e-mail address, and telephone and fax numbers) and whether the authors have published or submitted any related papers from the same study (see Duplicate/Previous Publication or Submission)Manuscript File FormatsFor submission and review, the acceptable manuscript file format is Microsoft Word Do not submit your manuscript in pdf formatUse 10-, 11-, or 12-point font size, double-space text, and leave right margins unjustified (ragged)Title PageThe title page should be the first page of your main manuscript file It should include a manuscript title; the full names, highest academic degrees, and affiliations of all authors (if an author’s affiliation has changed since the work was done, the new affiliation also should be listed); name and complete contact information for corresponding author; authors’ contributions and conflict of interest disclosures; and word count (not including abstract, acknowledgment, or references)At a GlanceThis feature provides a quick bulleted synopsis of an article’s findings Please provide 3 to 5 very brief bulleted points (data should be included) of the major take-away messages of your paper, starting with a brief statement indicating the purpose of your research Focus on primary and significant findings Do not over emphasize secondary or nonsignificant outcomes

Sameh E. Soliman, MD; Helen Dimaras, PhD; Vikas Khetan, MB, BS; Elise Héon, MD, FRCSC; Helen S. L. Chan, MB, BS, FRCSC; Brenda L. Gallie, MD, FRCSC

This work was partly presented as an oral presentation in the Research Day of the Department of Ophthalmology and Visual Sciences of the University of Toronto in Toronto, 29 May 2015 Presented by Sameh Soliman

Corresponding Author: 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

Authors’ Affiliations:

Departments of Ophthalmology & Vision Sciences, (Sogyliman, Dimaras, Héon , Gallie) and Division of Hematology/Oncology, Pediatrics (Chan), Hospital for Sick Children, Toronto, Canada; Division of Visual Sciences, Toronto Western Research Institute, Toronto, Canada (Dimaras, Héon , Gallie); Ophthalmology Department, Faculty of Medicine, Alexandria University, Egypt (Soliman); Sankara Nethralya Hospital, Chennai, India (Khetan); Departments of Pediatrics (Chan), Molecular Genetics and Medical Biophysics (Gallie) and Ophthalmology (Dimaras, Héon, Gallie), University of Toronto, Toronto, Ontario, Canada.

Author contributions:

Drs. Soliman and Gallie had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Soliman, Dimaras, Gallie, Khetan

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

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

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

Statistical analysis: Soliman, Dimaras, Gallie

Study supervision: Chan, Héon, Gallie

Financial Support: None

Conflict of Interest: No conflicting relationship exists for any author

Running head: Early delivery of familial retinoblastoma

Address for reprints: Dr. Brenda Gallie at the Department of Ophthalmology and Vision Sciences, the Hospital for Sick Children, 525 University Avenue, Toronto, ON M5G 2L3, Canada

Word count: 3183 3359 /3000 words

Numbers of figures and tables: 1 3 figures and 2 tables

Key Words: prenatal retinoblastoma, retinoblastoma gene mutation, RB1, molecular testing, late pre-term delivery, near-term delivery, amniocentesis

Abstract ( /350)Comment by Sameh Gaballah: AbstractsInclude a structured abstract of no more than 350 words for reports of original data and meta-analyses For other major manuscripts, include an unstructured abstract of no more than 200 words that summarizes the objective, main points, and conclusions of the article Abstracts are not required for Editorials, Viewpoints, and some special featuresAll reports of original data, systematic reviews, and meta-analyses should be submitted with structured abstracts as described below No information should be reported in the abstract that does not appear in the text of the manuscriptAbstracts for Reports of Original Data:Reports of original data should include an abstract of no more than 350 words using the headings listed below For brevity, parts of the abstract may be written as phrases rather than complete sentences Each section should include the following content:Importance: The abstract should begin with a sentence or 2 explaining the clinical (or other) importance of the study questionObjective: State the precise objective or study question addressed in the report (eg, “To determine whether…”) If more than 1 objective is addressed, the main objective should be indicated and only key secondary objectives stated If an a priori hypothesis was tested, it should be statedDesign: Describe the basic design of the study State the years of the study and the duration of follow-up If applicable, include the name of the study (eg, the Framingham Heart Study) As relevant, indicate whether observers were masked to patient groupings, particularly for subjective measurementsSetting: Describe the study setting to assist readers to determine the applicability of the report to other circumstances, for example, general community, a primary care or referral center, private or institutional practice, or ambulatory or hospitalized careParticipants: State the clinical disorders, important eligibility criteria, and key sociodemographic features of patients The numbers of participants and how they were selected should be provided (see below), including the number of otherwise eligible individuals who were approached but refused If matching is used for comparison groups, characteristics that are matched should be specified In follow-up studies, the proportion of participants who completed the study must be indicated In intervention studies, the number of patients withdrawn because of adverse effects should be given For selection procedures, these terms should be used, if appropriate: random sample (where random refers to a formal, randomized selection in which all eligible individuals have a fixed and usually equal chance of selection); population-based sample; referred sample; consecutive sample; volunteer sample; convenience sampleNote: For accepted manuscripts, the above 3 sections are usually combined during the editing process (as "Design, Setting, and Participants"), but for manuscript submission these sections should be kept separateIntervention(s) for Clinical Trials or Exposure(s) for observational studies: The essential features of any interventions or exposures should be described, including their method and duration of administration The intervention or exposure should be named by its most common clinical name, and nonproprietary drug names should be usedMain Outcome Measure(s): Indicate the primary study outcome measurement(s) as planned before data collection began If the manuscript does not report the main planned outcomes of a study, this fact should be stated and the reason indicated State clearly if the hypothesis being tested was formulated during or after data collection Explain outcomes or measurements unfamiliar to a general medical readershipResults: The main outcomes of the study should be reported and quantified, including baseline characteristics and final included/analyzed sample Include absolute numbers and measures of absolute risks (such as increase/decrease or absolute differences between groups), along with confidence intervals (for example, 95%) or Pvalues Approaches such as number needed to treat to achieve a unit of benefit may be included when appropriate Measures of relative risk also may be reported (eg, relative risk, hazard ratios) and should include confidence intervals Studies of screening and diagnostic tests should report sensitivity, specificity, and likelihood ratio If predictive value or accuracy is reported, prevalence or pretest likelihood should be given as well All randomized clinical trials should include the results of intention-to-treat analysis, and all surveys should include response ratesConclusions and Relevance: Provide only conclusions of the study that are directly supported by the results, along with implications for clinical practice or health policy, avoiding speculation and overgeneralization Indicate whether additional study is required before the information should be used in usual clinical settings Give equal emphasis to positive and negative findings of equal scientific merit Also, provide a statement of relevance indicating implications for clinical practice or health policy, avoiding speculation and overgeneralization The relevance statement may also indicate whether additional study is required before the information should be used in clinical settings

Importance: The abstract should begin with a sentence or 2 explaining the clinical (or other) importance of the study questionComment by Sameh Soliman: Don’t forget

IMPORTANCE Prenatal Sameh???RB1 Mutation detection made prediction of familial retinoblastoma more accurate and if combined with earlier delivery would have an impact on tumor size and overall treatment outcome.

OBJECTIVE: State the precise objective or study question addressed in the report (eg, “To determine whether…”) If more than 1 objective is addressed, the main objective should be indicated and only key secondary objectives stated If an a priori hypothesis was tested, it should be stated

OBJECTIVE To determine overall outcomes of infants with familial retinoblastoma diagnosed prenatally and delivered early term or late preterm compared to infants diagnosed postnatally.

Design: Describe the basic design of the study. State the years of the study and the duration of follow-up. If applicable, include the name of the study (eg, the Framingham Heart Study) As relevant, indicate whether observers were masked to patient groupings, particularly for subjective measurements

DESIGN A retrospective, observational study of children born between 1 June 1996 and 1 June 2014 with familial retinoblastoma cared for at Hospital for Sick Children, followed till April 2015.

Setting: Describe the study setting to assist readers to determine the applicability of the report to other circumstances, for example, general community, a primary care or referral center, private or institutional practice, or ambulatory or hospitalized care

SETTING This study was conducted at the academic institutional retinoblastoma referral center.

Participants: State the clinical disorders, important eligibility criteria, and key sociodemographic features of patients The numbers of participants and how they were selected should be provided (see below), including the number of otherwise eligible individuals who were approached but refused If matching is used for comparison groups, characteristics that are matched should be specified In follow-up studies, the proportion of participants who completed the study must be indicated In intervention studies, the number of patients withdrawn because of adverse effects should be given For selection procedures, these terms should be used, if appropriate: random sample (where random refers to a formal, randomized selection in which all eligible individuals have a fixed and usually equal chance of selection); population-based sample; referred sample; consecutive sample; volunteer sample; convenience sample

PARTICIPANTS: All children with familial retinoblastoma treated at SickKids were included. All children remain under care at the Hospital for Sick Children.

Intervention(s) for Clinical Trials or Exposure(s) for observational studies: The essential features of any interventions or exposures should be described, including their method and duration of administration The intervention or exposure should be named by its most common clinical name, and nonproprietary drug names should be used

EXPOSURE(S) Infants shown on amniocentesis to carry the parent’s RB1 mutant allele were planned for early pre-term or late preterm delivery (36-37 weeks of gestation), compared to normal term delivery (40 weeks of gestation) and postnatal RB1 testing. All children received treatments for eye tumors.

Main Outcome Measure(s): Indicate the primary study outcome measurement(s) as planned before data collection began If the manuscript does not report the main planned outcomes of a study, this fact should be stated and the reason indicated State clearly if the hypothesis being tested was formulated during or after data collection Explain outcomes or measurements unfamiliar to a general medical readership

MAIN OUTCOME MEASURES The hypothesis prior to data collection was that preterm delivery of infants at near 100% risk of bilateral retinoblastoma safely optimizes visual outcome and minimizes use of invasive treatments. Primary study outcome measurements were gestational age, age at first tumor, eye classification and staging, treatments given, visual outcome, number of anesthetics, pregnancy or delivery complications, and estimated overall cost of care.

RESULTS: The main outcomes of the study should be reported and quantified, including baseline characteristics and final included/analyzed sample Include absolute numbers and measures of absolute risks (such as increase/decrease or absolute differences between groups), along with confidence intervals (for example, 95%) or Pvalues. Approaches such as number needed to treat to achieve a unit of benefit may be included when appropriate. Measures of relative risk also may be reported (eg, relative risk, hazard ratios) and should include confidence intervals. Studies of screening and diagnostic tests should report sensitivity, specificity, and likelihood ratio. If predictive value or accuracy is reported, prevalence or pretest likelihood should be given as well… All randomized clinical trials should include the results of intention-to-treat analysis, and all surveys should include response rates.

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 because of risk for retinoblastoma and 3 were born spontaneously preterm. Immediate postnatal examination revealed vision-threatening tumors in 25% (3/12) of infants diagnosed prenatal to carry the family’s RB1 mutation, compared to 67% (6/9) of those diagnosed postnatal. All patients eventually developed tumors in both eyes. Good vision was maintained in all patients diagnosed prenatal; 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 worse visual outcome. One child in the postnatal RB1 mutation detection developed extraocular disease and still under active treatment.

Conclusions and Relevance: Provide only conclusions of the study that are directly supported by the results, along with implications for clinical practice or health policy, avoiding speculation and overgeneralization. Indicate whether additional study is required before the information should be used in usual clinical settings. Give equal emphasis to positive and negative findings of equal scientific merit. Also, provide a statement of Relevance indicating implications for clinical practice or health policy, avoiding speculation and overgeneralization. The relevance statement may also indicate whether additional study is required before the information should be used in clinical settingsComment by Gallie Brenda: SAmeh, add this to below….

CONCLUSIONS AND RELEVANCE: Prenatal diagnosis of retinoblastoma followed by late preterm and near-term delivery had shown a decrease in eyes with tumor at birth and a better visual outcome when compared to those with full term delivery with no complications related to preterm delivery. facilitated expedient intervention and optimized outcomes.

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

Children with RB1 germline alleles may already have retinoblastoma tumor(s) at birth, which are often in the posterior pole of the eye where they threaten vision.4-8 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 compromise vision. Most of these children are bilaterally affected, with either simultaneous or sequential detection of tumors.4,7 Later developing tumors tend to will develop more tumors in the first year of life, which tend to be located peripherally. The child is bilaterally affected in either simultaneous or sequential involvement. 4,7 Low penetrance mutations (10% of families)3 and mosiacism result in fewer tumors and more unilaterally affected children9. The timing of first tumors after birth has not been studied.

It is recommended that infants with a family history of retinoblastoma be screened as soon as possible after birth and repeatedly for the first few years of life, including under anaesthesia, aiming at early diagnosis when tumors are small and easy to treattreatable with less invasive therapies for salvage of the with oculareye and visualion salvage.6,7,10

Preterm birth is defined as a live birth occurring before completion of 37 weeks gestation. 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). 11,12 The main concern with late preterm or early term delivery is its reported effect on neurological and cognitive development and later school performance in children with a wide range of indications for early delivery, ,13-15 but visual dysfunction from a larger macular tumor can may risk cause similar neurocognitive defects due to blindness16, although this has not been studied. despite never studied in a comparative manner.

We now present the first report of outcomes 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 resulted in early allowed detection and treatment of small , early tumors, resulting in lower treatment morbidity, and better tumor control and visual outcome, compared to children born full term at 39-40 weeks.

MethodsStudy Design

Research ethics board approval (REB approval number 1000028725) was obtained from The Hospital for Sick Children (SickKids) for 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. Data collected 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 mutaion; age and location of first and all subsequent tumor (s) in each eye; treatments used; number of anaesthetics; International Intraocular Retinoblastoma Classification17 of each eye (IIRC); Tumor Node Metastasis (TNM) staging for eyes and child10; treatment duration; 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.18Comment by Gallie Brenda: I though we went decimal???We went 1-LogMAR. We collected LOGMAR. And then used 1-LoGMAR.

The corrected age for gestation at birth for each child was calculated (taking 39 weeks as full term). Vision threatening tumors were defined as in close to optic nerve or macular area (IIRC17 Group B or worse). Treatments will were be devidedsummarized as into focal therapies (Laser therapy, cryotherapy and periocular subtenon’s injection of chemotherapy) and or systemic therapies (systemic chemotherapy or stereotactic external beam irradiation). Treatment burden (defined by the impact of treatmnet course on general health and development of the child and potential impact on the his family) was evaluated 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), and iv) occurrence of extraocular disease. (Figure 1). Treatment success was defined as avoidance of enucleation or external beam irradiation or extraocular disease. Good Acceptable visual outcome was defined as visual acuity > 20/200 (>0 in 1-LogMAR scale or <1 in LogMAR) (cut edge of legal blindness). A legally blind child is defined as best eye visual acuity < 20/200 (<0 in 1-LogMAR scale). Comment by Gallie Brenda: Anything better than blind is GOOD???Sameh: I tried stratifying them into good (>0.5), Acceptable (0-0.5), Ambulatory (or legally blind) less than 0. Only 4 eyes and no patients are present in the Acceptable group. All are either >0.5 or legally blind. I think we can change the word Good into Acceptable and say that a subset has very good vision.Comment by Gallie Brenda: I think ??? 1-LogMAR is DECIMAL???? See methods.Sameh: No. 1-LogMar is not equivalent to decimal. Please see table in excel titled LOGMAR. A detailed comparison between different VA outcomes is present.

Statistics

Basic descriptive statistics (student t-test, chi square test (when all cell frequencies are more than 5), Fisher exact test (when any cell frequency is less than 5), Mann Whitney test and Mood’s median test) were used for statistical comparisons between patients who underwent prenatal testing and preterm delivery (Cohort 1) and those who were diagnosed post-natal (Cohort 2). Correlations and Kaplaen- Meyer survival graphs were plotted using Microsoft Excel 2007.

ResultsPatient Demographics

The records ofTwenty-one 21 children with familial retinoblastoma children were reviewed (11 males, 10 females) were eligible for this study (Supplementary Table 1). Diagnosis for Cohort 1 (9 children) 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) ADDIN EN.CITE ADDIN EN.CITE.DATA 19 or spontaneous delivery (#8). The Twelve 12 children (57%) (Cohort 2) were prenatally diagnosed to carry an their family’s 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, who was neverhad not been tested and understood that her children had no risk since she was unilaterally affected. Cohort 1 children (#1-9) were tested postnatal for their family’s RB1 mutation on by blood; Cohort 2 children (#10-21) were tested prenatal on by 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. At birth, null RB1 mutations resulted in nNo tumors were detected at birth (IIRC17 Group 0) in 7/15 (47%) infants and 17/30 (57%) eyes with null RB1 mutations;, and low penetrance mutations resulted in and inno tumors in 5/5 (100%) infants and 10/10 (100%) 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 84, median 39 days), than those with low penetrance mutations (mean 135, median 120 days) (P=0.03*, Phi=0.38, Mood’s median test). However, the gestational age at first tumor for those with null mutations (mean 71, median 33 days) tended to be was younger but was not significantly different, than for those with low penetrance mutations (mean 111, median 81 days) (P=0.06, Phi=0.32, Mood’s median 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 detectable diagnosis tumor is unknown.) (table 1c).

Stage Classification of Tumors Eyes at Birth

Thirty-three percent (3/9) of Cohort 1 and 75% (9/12) of Cohort 2 were free of visible tumor in either eye at birth (Table 1a, Figure 1) (p=0.09). We assumed that child #8 had tumor at birth since he had a group D IIRC17 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 IIRC17 Group A eye of child #8, as above (Table 1b).

All patients eventually developed tumors in both eyes regardless of whether their RB1 mutation was full or low penetrance. Tumors emerged at a younger age first in the macular and peri-macular region (IIRC17 Group B), as previously described20. The median gestational age of diagnosis of 14 IIRC17 B eyes (all threatening optic nerve and fovea, 6 also >3 mm) was 38 days, tended to be younger than of the 103 days for 26 IIRC17 A eyes (< 3mm and away from optic nerve and fovea) was 103 days, and of 14 IIRC17 B eyes (all threatening optic nerve and fovea, 6 also >3 mm) was 38 days, which is younger reflecting the early development of visually threatening tumors (P=0.32, Phi=-0.19, Mood’s median test).

Bilateral IIRC17 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 compared to 8/12 (67%) in Cohort 2 (p=0.009*, Fisher exact test) (Table 2a). IIRC17 Group A was the initial diagnosis of 9/18 (50%) eyes in Cohort 1, and compared to 15/22 (77%) eyes in Cohort 2 (p=0.33, Table 2a). One eye was an IIRC17 D eye and presented at age of 3 months (child#8).

Treatment Course

All infants were frequently examined from birth onwards (except child #8 who presented at age 3 months) as per the National Retinoblastoma Strategy Guidelines for Care.10 If there were no tumors at birth, each child was examined awake every week for 1 month, every 2 weeks for 2 months. After 3 months of age, the children will havehad an examination under general anesthesia (EUA) every two2-4 weeks. If there was any tumor at birth, the children will havehad EUAs every two 2-4 weeks till until control of tumors. Cohort 1 patients were treated with focal therapy (all), chemotherapy (4), stereotactic radiation (2), and enucleation of one eye (45) (Supplementary Table 1, Figures 1). Cohort 2 patients were treated with focal therapy (all); later systemic chemotherapy using vincristine, carboplatin, etoposide and cyclosporine (Toronto protocol)(5), enucleation of one eye and stereotactic radiation (1) (Figure 1). Comment by Gallie Brenda: Define the interval…? (mean, median, range of intervals…… of EUAS???done

The Treatment burden showed no statistical significant difference between Cohort 1 and 2 ina any of the four parameters tested. The median active treatment duration was 458 days (0-2101 days) in Cohort 1, compared to 447 days (0-971 days) in Cohort 2 (p=1, Mood’s median test). Treatment by focal therapy alone (avoidance of systemic chemotherapy or EBRT) was possible in 4/9 (44%) of Cohort 1 and 7/12 (58%) of Cohort 2 (P=0.67, Fisher exact test) (Table 2b). The median number of EUAs in cohort 1 is 25 (range 18-81) and for cohort 2 is 29 (range 20-41) EUAs (p=1, Mood’s median test). One child (11%) from Cohort one 1 had developed extraocular orbital disease and still under active treatment (P=0.4, Fisher exact test) (table 2b). Comment by Gallie Brenda: DEFINITION??? How calculated? What are the numbers? Definition of burden is now added in methods. It will not be in numbers unless we want to develop a score but we will assess it indirectly through the four mentioned parameters in the methods.

OutcomesComment by Gallie Brenda: IS THIS NOT PART OF TREATMENT (INTERVENTION) IMPACT SCORE?

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. Follow up (years) was Overall mean Follow up (mean, median) of was overall 8, 5.6 years (median 5.6); years; Cohort 1, mean follow up was 8.4, years (median 5.6), years; and Cohort 2, mean follow up was 7.6, years (median 5.8 ), years (Supplementary Table 1).

Neither enucleation nor external beam irradiation were required (defined as treatment success) in 44% of Cohort 1 and 92% of Cohort 2 (P=0.046*, Fisher exact test). Kaplan Meier ocular survival for Cohort 1 was 6762% compared to 92% for Cohort 2 (Figure 2). All children from both Cohorts are still alive; , only one child from Cohort one 1 is still under active treatment.

Visual outcomes were good acceptable for 50% of eyes in Cohort 1 and 92% of eyes in Cohort 2 (P=0.014*, Fisher exact test). Children were legally blind (visual acuity less than 20/200 using both eyes) in 22% of Cohort 1 and 0% of Cohort 2 (p=0.017*, Fisher exact test). Seventy one percent of eyes (17/24) of cohort 2 had final visual acuity better than 20/40 compared to 50% (9/18) of eyes in cohort one.Comment by Gallie Brenda: DEFINITION?? VA > 20/200 written in methods. But not clear with respect to LogMar or 1-LogMAR.Sameh: in methods it is written in both Snellen and 1-LogMAR

Treatment success (avoidance of enucleation and/or stereotactic radiation) and good vision per eye was documented 50% (9/18) of Cohort 1 and 88% (21/24) of Cohort 2 (p=0.014*, Fisher exact test) (Table 2b, Figure 1). A negative correlation was found between gestational age and final visual outcome (r=-0.24) with better visual outcome in earlier deliveries. (Figure 3).

Discussion

In the first study of its kind, we report that prenatal molecular diagnosis of familial retinoblastoma and elective late-preterm or early term delivery allowed monitoring for, and treatment of, tumors as they emerged, which resulted in better ocular and visual outcomes and less severe medical interventions in very young children. This data illustrates that for infants with close to 100% risk of retinoblastoma in both eyes because they carry an RB1 mutant allele, the risk of vision and eye loss despite intensive therapies, outweighs the risks associated with induced late preterm delivery (Figure 1). Consistent with previous reports,5 67% of children with a germline gene mutation already had tumors at full term birth. This reducedReduction to 25% when the germline mutation is was prenatally detected and earlier delivery (late preterm or early term) was plannedaccomplished.

It is practical to identify 96% of the germline mutations in bilaterally affected probands and to identify the >15% of unilateral probands who carry a germline gene mutation.3,9,21 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 to develop retinoblastoma. We report on 12 infants identified by in utero molecular testing to carry the mutant RB1 allele of a parent. The 50% of tested infants who did not inherit their family’s mutation require no surveillance, can be born at full term and do not need examinations to detect tumors, since they are at no greater risk of developing retinoblastoma than the general population.

Without molecular information, repeated retinal examination is recommended for all first degree relatives until age 7 years, the first 3 years under general anesthesia.10 Multiple studies now suggest deleterious effects of multiple general anesthetics in early infancy on the neurocognitive development of the child.22-24 Such repeated clinical screening also imposes psychological and financial burden on the children and families. Identification by early molecular RB1 testing of the children who are not at risk and require no clinical intervention cost significantly less than direct costs than clinical screening for tumors.18,25

Optimal treatment for retinoblastoma includes combined therapeutic modalities to optimize vision and minimize treatment morbidity, while achieving tumor control. However, retinoblastoma treatment in the first 3 months of life is a challenge since these young children may not have sufficient renal function for full dose systemic therapies. In our study child #9, who had a tumor at 36 weeks gestation large enough to detectfor detection by obstetrical ultrasound, showed drug-resistant tumor following reduced-dose chemotherapy, ultimately requiring enucleation of one eye.19 The onlyGood treatment options at this age are limited to are focal therapy (laser and cryotherapy) and periocular chemotherapy.26

The earliest tumors commonly involve the macular or paramacular region, dangerously riskingthreatening 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 threaten damage the optic nerve or and central vision. Systemic chemotherapy effectively shrinks tumors such that focal therapy can be applied with minimal visual damage. Systemic chemotherapy in neonates has other associated morbiditiesis difficult due to the unknowns of immature liver and kidney function to metabolize the drugs increasing the potential of severe adverse effects. The . We recognize the conventional recommendation is to either reduce chemotherapy dosages by 50%, particularly for infants in the first three months of life,30 or administer a single agent carboplatin chemotherapy 26; but the partial doses set up for development of multidrug resistance proteins in the tumor cells that promote chemotherapeutic drug efflux from tumor cells preventing drug accumulation in tumor cells, making later recurrences difficult to treat. 31-33 The development of Periocular topotecan for treatment of small-volume retinoblastoma 34 also may increase the effectiveness ofassisted 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 al{al 7 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 retinoblastoma (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).7,35 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%).36,37 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),13-15 but visual dysfunction from a larger macular tumor can cause similar neurocognitive defects due to blindness16 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 outcome{outcome 38 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 pregnancy{pregnancy.39 There have been two prior reports indicating pre-natal molecular testing for retinoblastoma; in one, the fetus sibling of a proband was found not to carry the sibling’s mutation{mutation 40, 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.

References

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

18.Richter S, Vandezande K, Chen N, et al. Sensitive and efficient detection of RB1 gene mutations enhances care for families with retinoblastoma. Am J Hum Genet. 2003;72(2):253-269.

19.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.

20.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.

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36.Akolekar R, Beta J, Picciarelli G, Ogilvie C, D'Antonio F. Procedure-related risk of miscarriage following amniocentesis and chorionic villus sampling: a systematic review and meta-analysis. Ultrasound in obstetrics & gynecology : the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 2015;45(1):16-26.

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Table 1: Occurrence of tumors at birth according to the type of RB1 mutation (Null vs Low penetrance mutation) in the whole child (table 1a), eyes (table 1b) and prevalence of bilateral IIRC group A tumors (table 1c).

Table 1a

Table 1b

Table 1c

Children with tumors at birth

Eyes with tumors at birth

Children with A/A eyes at first tumors

(child #8 included)

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

(child #8 included)

YES

NO

total

% NO

YES

NO

total

% NO

YES

NO

total

% NO

Null RB1 mutation

9

7

16

44%

14

17

31

55%

6

9

15

60%

Cohort 1

5

2

7

9

6

15

1

6

7

Cohort 2

3

5

8

5

11

16

5

3

8

Low penetrance RB1 mutation

0

5

5

100%

0

10

10

100%

3

2

5

40%

Cohort 1

0

1

1

0

2

2

0

1

1

Cohort 2

0

4

4

0

8

8

3

1

4

Total

 

 

21

 

 

 

41

 

 

 

21

 

FET

p=0.04*

p=0.02*

p=0.61

Table 2: Outcome parameters for both groups per eye (table 2a) and per child (table 2b) and their level of significance.

Table 2a: Outcome parameters per eye

Postnatal RB1 test (n=18)

Prenatal RB1 test (n=24)

P value

 

No

%

No

%

Tumor(s) at birth

10

0.56

5

21%

0.027*

Treatment success

11

0.61

22

92%

0.025*

Ocular salvage

13

0.72

23

96%

0.07

Visual Outcome

 

 

 

0.014*

Good vision

9

50%

21

88%

 

Poor Vision

9

50%

3

12%

 

Table 2b: Outcome parameters per child

Postnatal RB1 test (n=9)

Prenatal RB1 test (n=12)

P value

 

No

%

No

%

Tumor(s) at birth

6

67%

3

25%

0.087

IIRC AA at first tumor

1

11%

8

67%

0.009*

Treatment burden

 

 

 

 

0.67

Focal therapy only

4

44%

7

58%

 

Systemic chemotherapy

5

56%

5

42%

 

Treatment success

3

33%

11

92%

0.002*

Ocular salvage

4

44%

11

92%

0.046*

Extra-ocular disease

1

11%

0

0%

0.4

Visual Outcome

 

 

 

 

0.017*

Good vision

7

78%

12

100%

 

Blind

2

22%

0

0%

 

Figure 1: Schematic representation of each child in Cohort 1 (postnatal RB1 detection) and Cohort 2 (prenatal RB1 detection) from delivery until time of first tumor, IIRC at first tumor per eye, treatment burden (focal, systemic chemotherapy, or radiation treatment). Number of EUAs, visual acuity at last follow up and follow up duration.

Figure 2: Kaplan Meyer curves of treatment success showing a significant treatment success in the prenatal RB1 detection group versus the postnatal RB1 detection group.

Figure 3: A correlation between the Visual acuity at last follow up (1-LogMAR) at Y axis and the gestational age at delivery in weeks on X.axis showing a negative correlation.

Postnatal RB1 detection0.02.04.06.08.010.012.014.016.018.020.022.024.026.028.030.032.034.036.038.040.046.052.058.064.01.00.9444444444444450.9444444444444450.9444444444444450.9444444444444450.8888888888888890.7777777777777780.7777777777777780.7777777777777780.7777777777777780.7777777777777780.7777777777777780.7222222222222220.7222222222222220.7222222222222220.7222222222222220.7222222222222220.7222222222222220.7222222222222220.6666666666666670.6666666666666670.6666666666666670.6666666666666670.6111111111111110.611111111111111Prenatal RB1 detection0.02.04.06.08.010.012.014.016.018.020.022.024.026.028.030.032.034.036.038.040.046.052.058.064.01.00.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.9166666666666660.916666666666666

37.037.036.037.037.037.040.040.040.040.040.028.032.037.037.037.037.038.040.028.032.034.034.036.036.037.037.036.037.038.036.040.036.039.039.037.036.036.040.040.040.037.01.31.11.01.01.01.01.01.01.01.01.01.01.00.90.90.90.90.90.90.90.90.80.80.80.80.80.6000000000000020.50.50.20.00.0-0.3-0.3-0.3-0.5-1.0-1.0-1.0-1.0-1.0-1.0

Gestational Age

20/20; 20/2528 29 30 31 32 33 34 35 36 37 38 39 401 2 3 4 5 6 7 8 9 10 20/20; 20/200320/20; E120/20; 20/20 820/20; 20/2520/30; 20/6020/600; 20/60910E;20/304

E(OS)

20/20,E 1120/15; 20/1020/50; 20/201320/20; 20/2515

Postnatal RB1testPrenatal RB1test

5.6 18 7.1 18 14.8 5.2 12.8 9.5 8.8 4.36.4

FU (y)

Spontaneous birth Induced birth Birth to first tumor

monthsweeks

E(OS)

20/25; 20/25173.26E;20/252.7

VA (OD, OS)

2 20/200*3.75

IIRC (OD, OS)

A, AC, BA, BA, BA, BB, A7A, B20/30; 20/302.8

(OS)

D, AB, BE; 20/20

E(OD)

2.4

(OU)

E(OD)

E; 20/40015.5 12141618192021A, A15.5 B, AB, B

(OU)

E(OD)

4.9B, BA, AA, AA, AB, BA, A20/25; 20/100B, B20/125; 20/25A, A2.3

EUAs

25 41 24 22 21 3336 30302431 20304318 81 41 282123 22 20/20; 20/253.8 A, A

E(OD)

M

E

Focal Therapy Chemotherapy Radiotherapy Enucleation MMetastasis

Gestational Age

20/20; 20/25

28 29 30 31 32 33 34 35 36 37 38 39 40

1 2 3 4 5 6 7 8 9 10

20/20; 20/200

3

20/20; E

1

20/20; 20/20

8

20/20; 20/25

20/30; 20/60

20/600; 20/60

9

10

E; 20/30

4

E(OS)

20/20, E

11

20/15; 20/10

20/50; 20/20

13

20/20; 20/25

15

Postnatal RB1 test

Prenatal RB1 test

5.6

18

7.1

18

14.8

5.2

12.8

9.5

8.8

4.3

6.4

FU (y)

Spontaneous birth

Induced birth

Birth to first tumor

months

weeks

E(OS)

20/25; 20/25

17

3.2

6

E; 20/25

2.7

VA (OD, OS)

2

20/200*

3.7

5

IIRC (OD, OS)

A, A

C, B

A, B

A, B

A, B

B, A

7

A, B

20/30; 20/30

2.8

(OS)

D, A

B, B

E; 20/20

E(OD)

2.4

(OU)

E(OD)

E; 20/400

15.5

12

14

16

18

19

20

21

A, A

15.5

B, A

B, B

(OU)

E(OD)

4.9

B, B

A, A

A, A

A, A

B, B

A, A

20/25; 20/100

B, B

20/125; 20/25

A, A

2.3

EUAs

25

41

24

22

21

33

36

30

30

24

31

20

30

43

18

81

41

28

21

23

22

20/20; 20/25

3.8

A, A

E(OD)

M

E

Focal Therapy

Chemotherapy

Radiotherapy

Enucleation

M

Metastasis

Figure 1. Tumor timing, therapy and outcomes. Patients in each of the postnatal and prenatal retinoblasotma detection shown by gestational age at birth, international intraocular retinoblastoma classification (IIRC), treatment at time of first tumor occurrence and subsequently, final visual outcome and total follow-up time.

9