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

retinoblastoma

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: 2389/3000 words

Number of Figures and Tables: 1 figure, 2 tables and 2 supplementary files.

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

seeds.

At a glance (32/35)

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

that visible optic nerve, macular sparing or <1 quadrant of retinal detachment predicted 100% low-risk

histopathology, supporting trial ocular salvage.

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Abstract (250/250)

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

increase risk of tumor spread compared to primary enucleation. Identification of clinical features

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

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), histopathological findings,

and dates of metastasis and death were reviewed. Primary outcome was high-risk (HR) (pT3/pT4) versus

low-risk (LR) (pT1/pT2) (8th Edition American Joint Committee on Cancer) histopathology. Clinico-

pathologic correlations 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%), or trans-scleral, extraocular and retrolaminar optic nerve

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

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

13% (4/28) with macular involvement, 14% (4/31) with optic nerve obscuration, or 14% (4/28) with RD.

The probability of LR histopathology was 100% with macular sparing (7/7), optic nerve visibility (10/10)

and <1 quadrant of RD (10/10). One child who lacked all 3 clinical LR predictive features and had HR

histopathology (pT3a) developed metastases and died; other children are alive and well (mean follow-up

65 months).

Conclusion: All unilateral cT2b eyes with macular sparing, optic-nerve visibility and <1 quadrant of

RD had LR histopathology when enucleated at diagnosis, suggesting that eye salvage could be safely

attempted.

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Introduction

Unilateral retinoblastoma staged Group D by the International Intraocular Retinoblastoma

Classification (IIRC)1 and cT2 by the 8th Edition American Joint Committee on Cancer (AJCC) TNMH

(tumor, node, metastasis and heritable trait) staging,2 poses a management challenge. Attempted eye

salvage using primary intra-arterial (IAC)3-7 or systemic chemotherapy8 (both with focal consolidation) is

now commonly suggested. However, primary enucleation is an effective and safe option to minimize risk

of extraocular extension and metastasis. The Canadian National Retinoblastoma Strategy Guidelines for

Care published in 2009 recommend enucleation of affected unilateral Group D eyes.9

Recently, multiple treatment modalities are 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 is whether attempted eye salvage increases the risk

of extraocular tumor dissemination. Our aim was to identify clinical features of primarily enucleated

unilateral cT2 (Group D) eyes that predict at diagnosis low histopathologic risk, in order to guide “safe”

trial eye salvage.

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

Children diagnosed with unilateral Group D (cT2) 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

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evaluated. Exclusion criteria included unilateral retinoblastoma of any other clinical stage, bilaterally

affect children, and cT2 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 serous retinal

detachment (RD), parental consent 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

under the retinal pigment epithelial (RPE) 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 identified. Enucleated eyes were retrospectively staged by the 8th Ed. AJCC

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

Outcome Measures

The primary outcome was the presence of high-risk (HR) histopathology (pT3/pT4), versus low-risk

(LR) histopathology (pT1/pT2).2 High-risk histopathologic features included massive choroidal invasion,

retrolaminar invasion of the optic nerve head, scleral invasion and extraocular extension.

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Clinicopathologic correlation was evaluated. Positive predictive value was the probability that certain

clinical features would predict HR histopathology. Negative predictive value was the probability that

certain clinical features would predict LR histopathology.

Statistical Analysis

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

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

Thirty-eight (Supplementary table 1) primarily enucleated Group D eyes of 38 children (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

At presentation, all eyes had normal IOP. Vitreous seeding was present in all eyes. Tumor involved the

macula in 31/38–82%. Children with macular involvement tended to be younger at diagnosis than

children with macular sparing (mean 20 vs 28 months, respectively) (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.

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

performed during the staging EUA for all except 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 under the RPE 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 histopathologic evidence of

anterior segment involvement (pT2b). One eye (2.6%) had trans-scleral and extraocular extension (pT4).

High-Risk Pathology 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 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.

Clinicopathologic 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 Table 2). 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

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

with macular sparing, 100% with optic nerve visibility or 100% with <1 quadrant of RD. (Table 2)

Molecular analysis

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

(Hx) was untested. The two tumor RB1 pathogenic variants were identified in 34/37 eyes [H0*16 (27/38–

71%) and H1 (7/38–18%)] and 3 eyes were Hx (heritability cannot be verified). H1 children showed

mosaicism for the RB1 pathogenic variant (3/7), low penetrance RB1 pathogenic variant (3/7) and 13q

deletion syndrome (1/7). The children that showed high-risk pathology were H1 (1 pT4 eye, Figure 1A),

HX (2 pT3a eyes, one died, Figure 1C and 1D) and H0*16 (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. Bony metastases were found 1 year following

retinoblastoma diagnosis.17 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. The child

died 18 months after metastasis diagnosis, despite focal radiotherapy. The other children in this cohort are

alive and well. No child was lost to follow-up.

Discussion

The International Intraocular Retinoblastoma Classification (IIRC) (2005) staged eyes clinically

Group A (very low risk) through E (very high risk) to predict outcomes following systemic

chemoreduction and focal therapy.1 The 8th Edition TNMH was based on evidence from an international

survey,2 now the gold standard for retinoblastoma staging. Advanced intraocular disease includes IIRC

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Group E [with phthisis bulbi (cT3a), anterior segment tumor invasion (cT3b), rubeosis irides with

neovascular glaucoma (cT3c), hyphema and/or massive vitreous hemorrhage (cT3d) and aseptic orbital

cellulitis (cT3e)] and IIRC Group D eyes [with significant RD (cT2a) and/or seeding (vitreous and/or

subretinal, cT2b)]. High-risk histopathologic features of the enucleated eye predictive of increased

metastatic risk are pT3/pT4, which include massive choroidal invasion,18,19 retrolaminar invasion of the

optic nerve head with or without a positive margin,18,20 scleral invasion and extraocular extension (Table

1).2

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

extraocular tumour dissemination; secondary goal is to save a seeing eye. Primary enucleation is the

safest and lest costly option, allowing an early return to normal life,21 fewer interventions and EUAs,22

less socioeconomic impact,23 and histopathologic review of disease extent to guide further therapy.

Primary enucleation is current practice for cT3 (IIRC Group E) eyes and many cT2b eyes (IIRC Group

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.24 Prolonged attempts at

globe salvage may delay diagnosis of HR features pointing to potential subclinical metastasis. Pre-

enucleation chemotherapy may downstage pathological findings, delay enucleation and block recognition

of HR disease.25 With no randomized controlled trial evidence to guide management of cT2 eyes, the

clinician and family can balance the impact of potential years of trial salvage with hidden risks,25 against

vision potential and 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 IVitC to

control vitreous disease10-12 justifies trial salvage for a unilateral cT2b as long as metastatic risk is

minimal. The literature on IAC lacks definitive research5 and IAC chemotherapy delivered only to the eye

is unlikely to treat hidden escaped tumour.

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Clinical features at presentation reported to predict HR histopathology predominantly describe cT3

(Group E) eyes, not cT2b eyes and include older age, symptoms >6 months, hyphema, pseudohypopyon,

orbital cellulitis, secondary glaucoma and buphthalmos.26-28 Furthermore, exophytic growth pattern, tumor

thickness >15 mm and vitreous hemorrhage predict optic nerve invasion,20 and iris neovascularization is

associated with choroidal invasion.19,29 Yousef et al30 concluded that clinical staging alone (TNM 7th ed.,

IIRC or Reese Ellsworth classification) is insufficient to predict HR histopathology. This encouraged us

to study the individual clinical findings as predictors of HR histopathology. A useful predictive

reproducible clinical finding would be easily identified at initial staging EUA, the time of decision of trial

salvage vs primary enucleation. We excluded subjective findings such as presenting complaint, duration

of symptoms, pattern of growth (endophytic, exophytic or mixed) and presence of subretinal seeding

under detached retina.

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

primary enucleated eyes.21,28,30-34 However, the heterogeneous literature is limited by non-consensus in

defining HR histopathology features, variable classifications, inclusion of primarily and secondarily

enucleated eyes and children with unilateral and bilateral disease, and eyes with no staging recorded. In

our cohort of primarily enucleated unilateral cT2b eyes, 10.5% had HR histopathology. Macular

involvement, optic nerve obscuration or >1 quadrant of RD had low predictive value for HR

histopathology (13%, 14%, or 14%, respectively). However, macular sparing, visibility of the optic nerve

or <1 quadrant of RD had 100% predictive value for LR histopathology, suggesting that such an eye is

appropriate for cautious trial salvage.

Fabian et al33 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 the sole significant predictor of HR based on p=0.42.33 Small sample sizes renders tests of

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significance inaccurate as one extra entry can shift the p-value significantly. We used predictive values

rather than significance tests to interpret our data. In our study of all samples (78 eyes, table 2), absence

of vitreous seeds was not significant (p=0.05) and other factors showed a lower p-value than reported by

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

histopathology if visible optic nerve and non-involved fovea. Absence of vitreous seeds showed 71%

probability of having LR histopathology. Berry et al34 reported that at diagnosis, 15% of eyes with optic

nerve obscuration (69/102), and 0% with visible optic nerve (33/102) had post-laminar invasion following

primary enucleation, suggesting a possible clinico-pathologic association. This goes in accordance with

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

Our proposed clinical predictors are to be used at initial diagnosis and no evidence is available to

suggest their usefulness in decision-making for recurrent or refractory cases. These predictors do not

predict outcomes as LR eyes at diagnosis are not guaranteed to remain LR if unresponsive to initial

treatment. The decision of further trial 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.

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 histopathologic 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

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

LR, and may predict which advanced eyes are suitable for trial salvage decision. HR histopathology was

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found in10.5% of primarily enucleated unilateral cT2b (Group D) eyes. The widespread debate on

management of unilateral cT2 eyes, would be solved by robust, multicentre collaborative studies

involving many children to establish clinico-pathologic correlations.

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Tables

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

Table 2. Predictive values versus significance (p-value) in analysis of Fabian et al 33 and current

sample.

Supplementary Table 1. Clinicopathologic features of the whole studied sample (n=38)

Supplementary Table 2: Significance of association between clinical findings and histopathologic

features of enucleated eyes.

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Figure Legend

Figure 1. (A) Left, wide-angle 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. The child underwent six cycles of vincristine, etoposide, carboplatin

(VEC) and cyclophosphamide, followed by orbital irradiation.

(B) Left, wide-angle 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 the child received

four cycles of VEC.

(C) Left, wide-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.

(D) Left, wide-angle 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.17 Bilateral BMA were involved with tumor cells. MRI showed no orbital or intracranial

15

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. The child was

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

presentation with metastases.

16