Clinical predictors at diagnosis of low histopathologic risk features in unilateral cT2b (Group D)

retinoblastoma at diagnosis

Stephanie N. Kletke, MD1, Zhao Xun Feng, BSc2, Lili-Naz Hazrati, MD, PhD, FRCPC3, Brenda L. Gallie,

MD, FRCSC1,2,4, Sameh E. Soliman, MD2,5

Authors’ Affiliations

1 Department of Ophthalmology and Vision Sciences, University of Toronto, Toronto, Canada;

2 Department of Ophthalmology and Vision Sciences, The Hospital for Sick Children, Toronto, Canada;

3 Department of Pediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Canada;

4 Departments of Molecular Genetics and Medical Biophysics, University of Toronto, Toronto, Canada;

5 Department of Ophthalmology, Faculty of Medicine, Alexandria University, Alexandria, Egypt.

Corresponding Author: Sameh E. Soliman, 555 University Avenue, Room 7265, Toronto, Canada,

M5G 1X8. samehelsayedsoliman@yahoo.com

Running Head: Low-risk Histopathology in Unilateral cT2b Retinoblastoma

Word count: 2691/3000 words

Number of Figures and Tables: /51 figure, 3 tables and 2 supplementary files.

Keywords: unilateral retinoblastoma; Group D; histopathology; cancer; primary enucleation;, vitreous

seeds.

Word Count for Original Clinical Science Research: (Excluding title page, abstract, tables, references,

acknowledgements, contributions) max 3000; Abstract max 250 words; References max 35

At a glance (324/35)

Retrospective review of 38 primarily enucleated unilateral cT2b/Group -D retinoblastoma eyes

showed that visible optic nerve, macular sparing and <1 quadrant of retinal detachment predicted 100%

probability of low-risk histopathology, with visible optic-nerve, macular sparing and <1-quadrant of

retinal detachment rendering them safer forsupporting trial ocular salvage decision.

2

Abstract (25047/250)

Background/Aims: Whether Attempted eye salvage for unilateral cT2b (Group D) retinoblastoma

may increases risk of tumor spread compared to primary enucleation is debated. Identification of clinical

features predictive of low histopathologic risk may would guide potentially safe trial salvage decision.

Methods: A retrospective review of eyes primarily enucleated for unilateral cT2b retinoblastoma

(2008-2018) was conducted. Clinical features (intraocular pressure, optic nerve obscuration, macular

involvement, tumor seeding and serous retinal detachment (RD) >1 quadrant (RD)), histopathological

findings, and dates of metastasis and death were reviewed. Primary outcome was high-risk (HR)

histopathology (pT3/pT4) versus low-risk (LR) (pT1/pT2) (8th Edition American Joint Committee on

Cancer). histopathology. Clinico-pathologic correlations was were evaluated.

Results: Histopathology diagnosed 4/38–10.5% HR and 34/38–89.5% LR eyes. HR eyes demonstrated

massive choroidal invasion (4/38–10.5%), and or trans-scleral, extraocular and retrolaminar optic nerve

invasion (1/38–2.6%). Clinical findings included macular involvement (31/38–82%), optic nerve

obscuration (278/38–714%), and RD (28/38–74%). The probability that an eye had HR histopathology

was 13% (4/28) with macular involvement, 1114% (4/31) with optic nerve obscuration, and 14% (4/28)

with RD. The probability that an eye hadof LR histopathology was 100% with macular sparing (7/7),

91% with optic nerve visibility (10/10) and 100% with <1 quadrant of RD (10/10). One child (who with

lacked all 3 clinical LHR predictive features) and had HR histopathology (pT3a) developed metastases

and died; other children are alive and well (mean follow-up 65 months).

Conclusion: Trial salvage decision is suitablepotentially just for All uUnilateral cT2b eyes that

showseyes thatwith show mMacular sparing, optic- nerve visibility and <1 quadrant of RD were highly

predictive of LR . These clinical signs does not predict had LR histopathology when enucleated at

diagnosisoutcomes but only LR histopathology at diagnosis, suggesting that eye salvage could be safely

attempted. . in unilateral cT2b eyes, enabling identification of eyes suitable for likely safe trial salvage.

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Introduction

Unilateral retinoblastoma staged as Group D by the International Intraocular Retinoblastoma

Classification (IIRC)1 and as cT2a or cT2b by the 8th Edition American Joint Committee on Cancer

(AJCC) TNMH (tumor, node, metastasis and heritable trait) staging,2 poses a management challenge of

international debate. Attempted eye salvage using primary intra-arterial (IAC)3-7 or systemic

chemotherapy8 (both with focal consolidation) has beenis now commonly suggested. However, primary

enucleation is an effective and safe option to minimize risk of extraocular extension and metastasis. In

Canada, The Canadian National Retinoblastoma Strategy Guidelines for Care published in 2009

recommend enucleation of affected unilateral Group D eyes.9

Recently, multiple treatment modalities have beenare suggested to improve success of eye salvage,

including intravitreal chemotherapy (IVitC),10-12 IAC, , 3-7 periocular chemotherapy,13 and tumor

endoresection via pars plana vitrectomy (PPV).14 The primary concern with such modalities is whether

attempted eye salvage increases the risk of extraocular tumor dissemination. Our aim was to identify

potential clinical features of primarily enucleated unilateral cT2a/cT2b (Group D) eyes predictive ofthat

can predict at diagnosis low histopathologic risk at diagnosis, in an attemptorder to guide “potentially

safe” trial eye salvage decision.

Methods

Study Design

A retrospective, non-comparative, single institutional observational study was conducted in

accordance with the guidelines of the Declaration of Helsinki. Institutional Research Ethics Board

approval was obtained.

Eligibility

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Children diagnosed with unilateral Group D (cT2a or cT2b) retinoblastoma managed with primary

enucleation of the affected eye at the Hospital for Sick Children (SickKids), Toronto, Canada between

January 2008 (following submission and implementation of the Canadian guidelines9) through February

2018 were evaluated. Exclusion criteria included unilateral retinoblastoma of any other clinical stageing,

primarily enucleated cT2a/cT2b eyes in bilaterally affect children cases, or and cT2a/cT2bcT2 eyes that

were secondarily enucleated following trial salvage.

Data Collection

Clinical and Radiological Features

Medical records, including fundus photographs from examinations under anesthesia (EUA), were

reviewed for age at diagnosis and enucleation, laterality, clinical features at presentation (intraocular

pressure (IOP), tumor seeding, optic nerve obscuration, macular involvement and >1 quadrant of serous

retinal detachment (RD) , either > or) <1 quadrant), parental agreement consent with to the proposed

treatment, eye staging by IIRC, molecular genetic analysis, follow-up duration, adjuvant treatments

received, metastasis and death. Eyes were retrospectively staged by the 8th Ed. AJCC TNMH.2 Baseline

magnetic resonance imaging (MRI) or computed tomography (CT) of the brain and orbit were reviewed.

Histopathologic Features

Histopathology reports and representative slides were reviewed for all children. Presence of choroidal

invasion was documented as “none”, “focal [<3 mm]” or “massive [>3 mm in maximum diameter]”,

based on consensus definitions from the International Retinoblastoma Staging Working Group.15 Invasion

of under the sub-retinal pigment epithelial (sub-RPE) space but not through Bruch’s membrane was

identified. Optic nerve invasion was categorized as “none”, “prelaminar”, “retrolaminar but not to the

optic nerve resection margin” and “tumor at the transected end”.15 Scleral invasion, anterior segment

involvement and extraocular disease were also identified. Enucleated eyes were retrospectively staged by

the 8th Ed. AJCC pTNM.2 Table 1 summarizes the 8th Ed. AJCC pathological staging.

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Outcome Measures

The primary outcome was the presence of high-risk (HR) histopathology, defined as( pT3 or pT4),

versus low-risk (LR) histopathology, defined as( pT1 or pT2) (8th Ed. AJCC).2 High-risk histopathologic

features included massive choroidal invasion, retrolaminar invasion of the optic nerve head, scleral

invasion and extraocular extension.

Clinico-pathologic correlation was evaluated. Positive predictive value was defined as the probability

that an eye with high-risk certain clinical features (macular involvement, optic nerve obscuration or

RD)features towould havedpredict HR histopathology (i.e. HR clinical features predicting HR

histopathology). Negative predictive value was defined as the probability that an eye with low-risk certain

clinical featuress (macular sparing, optic nerve visibility and <1 quadrant of RD) had to havewould

predict LR histopathology (i.e. LR clinical features predicting LR histopathology).

Secondary outcomes included the proportion of eyes for which salvage therapy may have been

considered as an alternative to enucleation based on standard of care in 2018, as determined by senior

author review (B.L.G., S.E.S.).

Statistical Analysis

Results were summarized using frequency/percentage for categorical variables and mean, median, and

standard deviation and range for continuous variables. Groups were compared using Fisher’s exact test

for categorical variables and Student’s t-test for continuous variables. All P-values reported were two-

sided and significance was judged at the 5% level. All analyses were performed using SPSS Version 25

(IBM Corp).

Results

Demographic and Clinical Features

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Thirty-eight primarily enucleated Group D eyes ofeyes of 38 children (mean presenting age mean 21

months, range 2 – 48) with unilateral retinoblastoma were included (63% right, 37% left). All eyes were

staged cT2b (8th Edition AJCC).2 Based on high-quality molecular genetic analysis, heritable trait was HX

(4/38–11%), H0 (27/38–71%) and H1 (7/38–18%). H1 children showed mosaicism for the RB1

pathogenic variant (3/7), low penetrance RB1 pathogenic variant (3/7) and13-q deletion syndrome (1/7).

At presentation, all eyes had within normal IOP. Vitreous seeding was seen present in all eyes. Tumor

involved the macula in 31/38–82%. Children with macular involvement were tended to be younger at

diagnosisdiagnosed earlier than children with macular sparing (mean 20 vs 28 months, respectively),

though this difference was not significant (p=0.09). The optic nerve was obscured in 28/38–74% and RD

(>1 quadrant) was present in 28/38–74%. Retinal detachment impaired accurate assessment of subretinal

seeding in some eyes. The presence of macular involvement, optic nerve obscuration and RD were

positively correlated [macula and optic nerve (p=0.01), macula and RD (p< 0.001), optic nerve and RD

(p=0.002)]. Four of 28 eyes with optic nerve obscuration demonstrated possible optic nerve enhancement

on baseline imaging of the brain/orbit. There were no radiological cases of extraocular or intracranial

involvement.

The median interval from diagnosis to enucleation was 4 days (range, 0 – 14). Primary enucleation

occurred was performed during the staging EUA for all children, with the exception of one child, for

whom enucleation was delayed due to low partial thromboplastin time. All parents consented to

enucleation as the primary treatment.

Histopathologic Features

Choroidal involvement included “none” (26/38–68.4%), “focal” (8/38–21.1%), and “massive”

(4/38–10.5%). Six eyes (15.8%) demonstrated tumour cells under the in the sub-RPE space without

invasion of Bruch’s membrane. Optic nerve involvement included “none” (10/38–26.3%), “prelaminar

invasion” (27/38–71.1%), and “retrolaminar invasion but not to the optic nerve resection margin” (1/38–

2.6%). There were no cases of tumor involvement of the resected margin. One eye (2.6%) demonstrated

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histopathologic evidence of anterior segment involvement (pT2b). There was One case eye (2.6%) of had

trans-scleral and extraocular extension (pT4). Table 2 summarizes the histopathologic features of the

studied populationstudy eyes.

Summary of High-Risk Pathology Eyes High-Risk Eyes (Figure 1)

Histopathology review identified 4/38–10.5% HR eyes and 34/38–89.5% LR eyes. HR eyes

demonstrated massive choroidal invasion (4/38–10.5%), and and trans-scleral, extraocular and

retrolaminar optic nerve invasion (1/38–2.6%). Mean age at diagnosis was not significantly different for

children with HR versus LR eyes (p>0.05). Presenting signs included leukocoria (3/4–75%) and

strabismus (1/4–25%). Baseline MRI brain and orbits showed no evidence of optic nerve, extraocular or

intracranial involvement in children with HR eyes. There was no evidence of metastases at presentation.

Clinico-pathologic Correlation

Optic nerve obscuration was not significantly associated with retrolaminar optic nerve invasion in this

cohort (p=1.000). Macular involvement was not significantly associated with massive choroidal invasion

(p=0.557) or scleral invasion (p=1.000). Serous RD was not significantly associated with massive

choroidal invasion (p=0.287) or scleral invasion (p=1.000, Supplementary Ttable 1). None of the eyes

showing enhanced optic nerve on the MRI scanning at presentation had retrolaminar nerve invasion

(p=1).

The probability that an eye had HR histopathology was 13% with macular involvement, 14% with

optic nerve obscuration, and 14% with RD. The probability that an eye had LR histopathology was 100%

with macular sparing, 100% with optic nerve visibility and 100% with <1 quadrant of RD. (Table 3)

Molecular analysis

Molecular genetic testing was performed on tumor samples from 37 enucleated eyes while one eye

(Hx) was yet untested due to economic causes. The two tumor RB1 RB1 pathogenic variants were

identified in 34/37 eyes [H0* (27/38–71%) and H1 (7/38–18%)]. andThe three3 eyes were designated

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(Hx () as the germline statushHeritability cannot be verified). and one had hypermethylation of the RB1

premotor in blood cells. In retrospective designation by the 8th ed. AJCC, heritable trait was HX (4/38–

11%), H0 (27/38–71%) and H1 (7/38–18%). H1 children showed mosaicism for the RB1 RB1 pathogenic

variant (3/7), low penetrance RB1 RB1 pathogenic variant (3/7) and 13-q deletion syndrome (1/7). The

children that showed high riskhigh-risk pathology were H1 (1 extraocular, pT4 eye, Figure 1A), HXx (2

pT3a eyes, pT3a, one died, Figure 1C and 1D) and H0* (pT3a, Figure 1B).

Follow-up, Metastasis and Death

At mean follow-up of 65 months, one child (2.6%) with all three clinical HR predictive features

and HR histopathology (pT3a) developed metastases and died. The child was diagnosed with Bony

metastases were found 1 year following retinoblastoma diagnosis.16 He The child received six cycles of

systemic chemotherapy, autologous bone marrow transplant and focal radiation. While ocular pathology

was initially interpreted as LR, internal retrospective review identified an area of massive choroidal

invasion. Metastatic surveillance remained negative until 1 year later, when intracranial dural-based

metastases were identified on MRI. He The child died 18 months after metastasis diagnosis, despite focal

radiotherapy. The other children in this cohort are alive and well. None of theNo children were was( lost

to follow-up.

Discussion

The International Intraocular Retinoblastoma Classification (IIRC) (2005) introduced in 2005 staged

eyes clinically as Group A (very low risk) through E (very high risk) to predict outcomes following

systemic chemoreduction and focal therapy.1 A modification was proposed in 2006 (International

Classification of Retinoblastoma, ICRB). In 2010, the 7th Edition AJCC defined clinical and pathological

staging for overall prognosis. The 8th Edition TNMH was recently updated based on evidence-based data

from an international survey,2 and now serves as the current gold standard for retinoblastoma staging.

IIRC Group E eyes shows advanced intraocular tumorsfeatures such as phthisis bulbi (cT3a), anterior

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segment tumor invasion (cT3b), rubeosis irides with neovascular glaucoma (cT3c), hyphema and/or

massive vitreous hemorrhage (cT3d) and or aseptic orbital cellulitis (cT3e). IIRC Group D eyes have

intraocular tumor with significant RD (cT2a) and/or seeding (any vitreous and/or subretinal, seeding

(cT2b). High-risk histopathologic features of the enucleated eye predictive of increased metastatic risk are

defined as pT3/pT4 following enucleation, andwhich include massive choroidal invasion,17,18 retrolaminar

invasion of the optic nerve head with or without a positive margin,17,19 scleral invasion and extraocular

extension (T8th Edition AJCC, Table 1).2

The mainfirst goal of treatment for advanced unilateral retinoblastoma is to save the child’s life and

prevent extraocular tumour dissemination; secondary goal is to followed by saving a seeing eye. The

concept of salvage of a blind eye for cosmesis is no longer justified, given the improved implant and

prosthesis movement with myoconjunctival enucleation.20. Multiple modalities, including Systemic

chemotherapy, IAC, IVC, periocular chemotherapy and PPV have been suggested for eye salvage.

However, Primary enucleation is the a safest and lest costly option, allowing an early return to normal

life,20 fewer interventions and EUAs,21 lessless socioeconomic impacts,22 as well asand histopathological

review of disease extent to guide further therapy. This is the accepted practice in many centersPrimary

enucleation is current practice for cT3 (IIRC Group E) eyes and probably mostmany cT2b eyes (IIRC

Ggroup D). In our cohort all parents accepted our recommendation for primary enucleation. Parental

acceptance of enucleation depends on the treating physician and how the parents are counselled.

Potential for useful vision is important but salvage of a unilateral blind eye for cosmesis may not be

justified, given good prosthesis movement with myoconjunctival enucleation.23 The dilemma of parental

refusal of enucleation justifying treatment for these advanced eyes dependdilemma of justifying salvage

treatment for advanced eyes on parental refusal of enucleation depends on the treating physician and how

the parents are counselled. In our cohort we did not face parental refusal in any of our enucleated eyes.

Prolonged attempts at globe salvage could may lead to delayeddelay diagnosis of HR features pointing to

potential subclinical metastasis. metastasis if HR features are not identified timely.24 Systemic

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chemoreduction should also be considered cautiously, as Pre-enucleation chemotherapy may downstage

pathological findings, delay enucleation and block recognition of HR diseaseextraocular disease.24 With

no randomized controlled trial evidence from randomized controlled trials to guide management of

cT2a/cT2b eyes, the clinician and family must can consider balance the impact of potential years of trial

salvage with hidden risks,24 for against an eye with limited visionual potential and on quality of life for

the child and family.

Systemic chemotherapy, IAC, IVitC, periocular chemotherapy and PPV are available for attempted

eye salvage. The success of intravitrealIVitC chemotherapy into controlling vitreous disease (cT2b,

Group D),10-12 justifies trial salvage for a unilateral cT2b as long as risk metastatic risk is minimal. a main

clinical feature of cT2b (IIRC Group D) eyes, together with theThe availability of more published

literature on IAC lacks definitive research,5 that previously posed a dilemma, asand IAC chemotherapy

delivered focallyonly to the eye that mayisis unlikely ineffective not protectto treat against hidden

escaped tumortumour cells that have escaped the affected eye.5 made the trial salvage attempt for a

unilateral cT2b justified as long as the tumor is less likely to spread beyond the eye. Clinical features at

presentation predictive of HR histopathology include older age, symptoms >6 months, hyphema,

pseudohypopyon, orbital cellulitis, secondary glaucoma and buphthalmos.25-27 Furthermore, exophytic

growth pattern, tumor thickness >15 mm and vitreous hemorrhage predict optic nerve invasion,19 and iris

neovascularization is associated with choroidal invasion.18,28 However, theseThese characteristics

predominantly describe cT3 (Group E) eyes, and are not relevant when considering the treatment ofnot

cT2b eyes. Yousef et al29 concluded that clinical staging alone (TNM 7th ed., IIRC or Reese Ellseworth

classification) is insufficient to predict HR histopathology. This encouraged us to study the individual

clinical findings as predictors of HR histopathology. rather than the whole eye staging.

The idealA useful predictive reproducible clinical finding need to be a reproducible objective

findingwould be easily pickedidentified at theintital staging EUA, the time of the decision of trial salvage

vs primary enucleation.. Furthermore, it canneedneeds to be picked clinically and not dependingbased on

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an investigation that might be unavailable as an MRI or genetic testing. In our analysis We excluded

subjective findings such as presenting complaint, or duration of symptoms, debatablesubjective eye

findings such as pattern of tumor growth (endophytic, exophytic or mixed) and presence of subretinal

seeding under detached retina., together with unavailable findings at diagnosis as RB1 pathogenic variant

status.

Approximately 2–33% of Group D eyes are expected to harborharbour HR histopathologic features

following in primary enucleationenucleated eyes.20,27,29-33 However, the heterogeneous literature is

heterogeneous and limited by non-consensus in defining HR histopathology features, variable

classifications, and inclusion of primarily and secondarily enucleated eyes and, children with unilateral

and bilateral disease, as well asand eyes with no staging indication ofrecorded staging. In our cohort of

primarily enucleated unilateral cT2b eyes, 10.5% had HR histopathology. Macular involvement, optic

nerve obscuration and >1 quadrant of RD had low predictive value for HR histopathology (13%, 14%,

and 14%, respectively). However, the combination of macular sparing, visibility of the optic nerve and <1

quadrant of RD had 100% predictive value for the presence of LR histopathology, suggesting that such an

eye with these three clinical features may undergois appropriate for cautious trial salvage.

.Fabian et al32 reported on 40 primarily enucleated IIRC Group D eyes (all cT2b, 37 unilateral). At

presentation, 95% (38/40) had macular involvement, 95% (38/40) had optic disc obscuration and 97%

had RD, compared to 82%, 74% and 74%, respectively in our cohort. They reported absence of vitreous

seeds as athe sole significant predictor of HR based on p=0.42.32 Small sample sizes renders tests of

significance inaccurate as one extra entry can shift the p-value significantly which lead us to use the

predictive values rather than then tests of significance to interpret our data. We ran significance test forIn

our study of our collectiveall samples (78 eyes, table 3), absence of vitreous seeds lost itswas not

significantcance (p=0.05) and other factors showed a lower p-value than reported yet insignificantby

others. When we applied predictive values, we had the same results of 100% predictive of LR

histopathology if the optic nerve is seen and the fovea is not involved. Absence of vitreous seeds showed

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71% probability of having LR histopathology (Table X3). Berry et al 33 reported a retrospective review of

IIRC Group D/E eyes and found that 15% of eyes with optic nerve obscuration at presentation (69/102)

had postlaminar invasion, while 0% with visible optic nerve at diagnosis (33/102) had postlaminar

invasion following primary enucleation, suggesting a possible clinico-pathologic association 33. This goes

in accordance with our 100% probability for LR with visible optic nerve in our cohort.

The decision of trial ocular salvage is taken at time of diagnosis making any clinical predictors of

histopathologic risk features valuable in the process of decision making. However, tumor response to

therapies might change or downstage the initial histopathology risk.24 Our proposed clinical predictors are

to be used at initial diagnosis and no evidence is available suggesting their usefulness in recurrent or

refractory cases decision makingdecision-making. These predictors are not used to predict outcomes but

only predict histopathology risk at diagnosis. Eyes with LR at diagnosis are not guaranteed to remain LR

if unresponsive to initial treatment and the decision of further trying salvage should be based on an

interplay of intended outcomes, metastatic risks, treatment morbidity, socioeconomic impacts and visual

potential taking into consideration that the other eye is perfectly normal. Early detection of HR

histopathologic features is important and when present, warrants metastatic surveillance and

adjuvant therapy. Post-enucleation adjuvant treatments, including systemic chemotherapy,

significantly reduce metastatic events from 24% to 4%, particularly in the presence of massive

choroidal and retrolaminar invasion.34 Furthermore, post-enucleation adjuvant VEC was

associated with no metastatic events for 51 high-risk ICRB35 Group E eyes (mean 66 month follow-

up).36 However, the specific indications for adjuvant therapy are debated,37 with some groups

suggesting good prognosis for isolated choroidal or retrolaminar optic nerve invasion and negative

margins without adjuvant treatment.38,39 In our cohort, the child who developed metastasis did not

receive post-enucleation adjuvant therapy, as massive choroidal invasion was only identified

following retrospective review. The other three children with HR histopathology received adjuvant

treatments and are alive and well at last follow-up.

13

The limitations of this study include its retrospective design and relatively small sample size.

However, our inclusion criteria of only unilateral, primarily enucleated Group D eyes were

stringent, achieving a homogenous study population. Another limitation is the low rate of positive

events, which limits statistical analysis of associations between clinical findings and

histopathologicalhistopathologic features of the included eyes. Furthermore, our analysis is at the point

of diagnosis and does not offer any data on progress of the histopathologic risk with different treatments

Conclusion

In summary, 10.5% of primarily enucleated unilateral cT2b (Group D) eyes had HR

histopathology. Macular sparing, optic nerve visibility and <1 quadrant of RD at presentation were highly

predictive of low-riskLR in unilateral cT2b eyes, and may predict which advanced eyes are suitable for

trial salvage decision. HR histopathology was found in10.5% of primarily enucleated unilateral cT2b

(Group D) eyes. Given The widespread management debate of on management of unilateral cT2a/cT2b

eyes, there is a need forwould be solved by robust, multicentre collaborative studies involving a larger

group ofmany children to further assessestablish these clinico-pathologic correlations.

References

Tables

Table 1. Summary of American Joint Committee on Cancer (AJCC) pathological staging 8th

Edition.

Table 2. Histopathologic features of the studied eyes.

14

Table 3. Combined analysis of Fabian et al 32 and current sample showing significance versus

probability assessment.

Supplementary Table 1. Significance of association between clinical findings and histopathologic

features of enucleated eyes.

Figure Legend

Figure 1. (A) Left, wide-angle RetCam fundus photograph of child 1 showing a right multilobulated

tumor with overlying serous retinal detachment (RD) and subretinal seeding. Middle left,

Histopathological section under low magnification through the optic nerve demonstrating extra-scleral

and post-lamina cribrosa invasion (arrows), but not to the optic nerve resection margin (pT4). Middle

right, high magnification of trans-scleral and extra-scleral invasion (arrow). Right, High magnification

showing retrolaminar invasion (arrow). Whole-body MRI (WBMRI), lumbar puncture (LP) and bilateral

bone marrow aspirate (BMA) were negative for malignancy. HeThe child underwent six cycles of

vincristine, etoposide, carboplatin (VEC) and cyclophosphamide, followed by orbital irradiation.

(B) Left, wide-angle RetCam fundus photograph of child 2 demonstrating a large inferior tumor with

overlying RD, vitreous and subretinal seeds. The optic nerve was obscured. Middle and right,

Histopathological sections under low and intermediate magnification showing massive choroidal invasion

(asterisk) beyond the confines of the retinal pigment epithelium (arrow), with no evidence of scleral

invasion (pT3a). There was prelaminar optic nerve invasion. LP and BMA were negative for malignancy

and shethe child received four cycles of VEC.

(C) Left, RetCamwide-angle fundus photograph of child 4 demonstrating a large tumor obscuring the

nerve and macula, with associated hemorrhage, RD and diffuse vitreous seeding. There was no anterior

segment extension evident on ultrasound biomicroscopy. Histopathology was confirmed to be massive

(pT3a). The child received systemic adjuvant chemotherapy.

15

(D) Left, wide-angle RetCam fundus image of child 3 demonstrating a large tumor with associated

RD, subretinal and focal vitreous seeding. There was no visualization of the optic nerve and the macula

was involved. Initial review was consistent with low-risk histopathology. One year later the child

presented with fever and pain, and WBMRI identified a paraspinal tumor. Molecular analysis confirmed

metastasis.16s (Racher 2016). Bilateral BMA were involved with tumor cells. MRI showed no orbital or

intracranial disease and LP was negative. Internal review of the ocular pathology, including further

choroidal sections, showed an area of massive choroidal invasion (pT3a). The child received 6 cycles of

VEC and cyclosporine, followed by autologous bone marrow transplant and focal irradiation. HeThe

child was diagnosed with dural-based metastases 1 year later. Despite radiotherapy, the child died 18

months after presentation with metastases.

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9. Canadian Retinoblastoma S. National Retinoblastoma Strategy Canadian Guidelines for Care: Strategie therapeutique du retinoblastome guide clinique canadien. Can J Ophthalmol. 2009;44 Suppl 2:S1-88.

10. Munier FL, Gaillard MC, Balmer A, et al. Intravitreal chemotherapy for vitreous disease in retinoblastoma revisited: from prohibition to conditional indications. Br J Ophthalmol. 2012;96(8):1078-1083.

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