Clinical predictors 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: 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 (35/35)

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

100% probability of low-risk histopathology with visible optic-nerve head, macular sparing and <1-

quadrant of serous retinal detachment rendering them safer for trial ocular salvage.

Abstract (245/250)

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

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

features predictive of low histopathologic risk may 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 >1 quadrant (RD)), histopathological findings,

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). Clinico-pathologic

correlation was 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 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% with macular involvement, 11% with optic nerve obscuration, and 14% with RD. The

probability that an eye had LR histopathology was 100% with macular sparing, 91% with optic nerve

visibility and 100% with <1 quadrant of RD. One child (with lack all 3 clinical LHR predictive features)

and HR histopathology (pT3a) developed metastases and died; other children are alive and well (mean

follow-up 65 months).

Conclusion: Trial Salvage is potentially safe for unilateral cT2b eyes that shows clinical signs of

mMacular sparing, optic nerve visibility and <1 quadrant of RD were highly predictive ofgiven their high

probability for LR histopathology. in unilateral cT2b eyes, enabling identification of eyes suitable for

likely safe trial salvage.

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)(3-8) or systemic

chemotherapy(9) with focal consolidation has been suggested.(10) However, primary enucleation is an

effective and safe option to minimize risk of extraocular extension and metastasis. In Canada, the

National Retinoblastoma Strategy Guidelines for Care published in 2009 recommend enucleation of

affected unilateral Group D eyes.(11)

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

including intravitreal chemotherapy (IVC),(12-15) IAC,(3-8) periocular chemotherapy,(16) and tumor

endoresection via pars plana vitrectomy (PPV).(17) 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 low histopathologic risk, in an attempt to guide potentially safe trial 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 (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 guidelines(11)) through

February 2018 were evaluated. Exclusion criteria included unilateral retinoblastoma of any other staging,

primarily enucleated cT2a/cT2b eyes in bilateral cases, or cT2a/cT2b 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 with the proposed treatment,

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.(18)

Invasion of 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”.(18) 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.

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 clinical features (macular involvement, optic nerve obscuration or RD)features

to haved HR histopathology. Negative predictive value was defined as the probability that an eye with

low-risk clinical features (macular sparing, optic nerve visibility and <1 quadrant of RD) had to have 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 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 primarily enucleated Group D eyes ofeyes of 38 children (mean presenting age 21

months, 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 in all eyes. Tumor

involved the macula in 31/38–82%. Children with macular involvement were diagnosed 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 presence inseeding 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 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 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 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 histopathologic

evidence of anterior segment involvement (pT2b). There was one case (2.6%) of trans-scleral and

extraocular extension (pT4). Table 2 summarizes the histopathologic features of the studied population.

Summary of 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 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 table 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

was yet untested due to economic causes. The two tumor RB1 pathogenic variants were identified in

34/37 eyes. The three eyes were designated (Hx) as the germline status 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 pathogenic variant (3/7), low penetrance RB1 pathogenic variant (3/7) and 13-q deletion

syndrome (1/7). The children that show high risk were H1 (1 extraocular, pT4, Figure 1A), Hx (2 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

1 year following retinoblastoma diagnosis.(19) He 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 died 18 months after metastasis diagnosis, despite focal radiotherapy. The other children in

this cohort are alive and well. None of the children were lost to follow-up.

Discussion

The International Intraocular Retinoblastoma Classification (IIRC) 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).(20) In 2010, the 7th Edition AJCC defined clinical and

pathological staging for overall prognosis.(21) The 8th Edition TNMH was recently updated based on

evidence-based data,(2) and serves as the current gold standard for retinoblastoma staging. IIRC Group E

eyes shows advanced intraocular tumors as phthisis bulbi (cT3a), anterior 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 any vitreous and/or subretinal seeding (cT2b). High-risk histopathologic features predictive

of increased metastatic risk are defined as pT3/pT4 following enucleation, and include massive choroidal

invasion,(22, 23) retrolaminar invasion of the optic nerve head with or without a positive margin,(22, 24)

scleral invasion and extraocular extension (8th Edition AJCC, Table 1).(2)

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

prevent extraocular tumour dissemination 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.(39). Multiple modalities, including systemic chemotherapy, IAC, IVC,

periocular chemotherapy and PPV have been suggested for eye salvage. However, primary enucleation is

a safe option, allowing an early return to normal life,(28) fewer interventions and EUAs,(37) less

socioeconomic impacts, as well as histopathological review. This is the accepted practice in many centers

for cT3 (IIRC Group E) eyes and probably most cT2b eyes (IIRC group D). The dilemma of parental

refusal of enucleation justifying treatment for these advanced eyes depend 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 lead to delayed diagnosis of metastasis if HR features are

not identified timely. Systemic chemoreduction should also be considered cautiously, as pre-enucleation

chemotherapy may downstage pathological findings of extraocular disease.(38) With no evidence from

randomized controlled trials to guide management of cT2a/cT2b eyes, the clinician must consider the

impact of years of trial salvage for an eye with limited visual potential on quality of life for the child and

family.

The success of intravitreal chemotherapy in controlling vitreous disease, a main clinical feature of

T2b (IIRC Group D) eyes, together with the availability of more published literature on IAC, that

previously posed a dilemma, as chemotherapy delivered focally that may not protect against tumor 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.(29, 40, 41) Furthermore, exophytic growth pattern, tumor

thickness >15 mm and vitreous hemorrhage predict optic nerve invasion,(24) and iris neovascularization

is associated with choroidal invasion.(23, 42) However, these characteristics predominantly describe cT3

(Group E) eyes and are not relevant when considering the treatment of cT2b eyes. Yousef et al concluded

that clinical staging alone (TNM 7th ed., IIRC or Reese Elseworth classification) is insufficient to predict

HR histopathology. This encouraged us to study the individual clinical findings as predictors rather than

the staging.

The ideal predictive clinical finding need to be a reproducible objective finding easily picked at

the staging EUA, the time of the decision of trial salvage. Furthermore, it canneed to be picked clinically

and not depending on an investigation that might be unavailable as an MRI or genetic testing. In our

analysis we excluded subjective findings as presenting complain or duration of symptoms, debatable

findings 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 harbor HR histopathologic features

following primary enucleation.(25-31) However, the literature is heterogeneous and limited by non-

consensus in defining HR histopathology features, variable classifications, and inclusion of primarily and

secondarily enucleated eyes, children with unilateral and bilateral disease, as well as eyes with no

indication of 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%, 114%, and 14%, respectively). However, macular sparing, visibility

of the optic nerve and <1 quadrant of RD had 100% predictive value for the presence of LR

histopathology, suggesting that an eye with these three clinical features may undergo cautious trial

salvage.

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

presentation, 75% had macular involvement, 91% had optic disc obscuration and 97% had RD, compared

to 82%, 74% and 74%, respectively in our cohort. They report absence of vitreous seeds as a sole

significant predictor of HR based on p=0.42.(30) 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 for our collective

samples (78 eyes, table 3), absence of vitreous seeds lost its significance (p=0.05) other factors showed a

lower p-value than reported yet insignificant. 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 71% probability of having LR histopathology (Table X3). Jesse et al 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 (43). This goes in accordance with our 100% probability for LR with visible optic nerve in

our cohort.

Early detection of HR histopathologic features is important and when present, warrants

metastatic surveillance and adjuvant therapy. Postenucleation adjuvant treatments, including

systemic chemotherapy, significantly reduce metastatic events from 24% to 4%, particularly in the

presence of massive choroidal and retrolaminar invasion.(32) Furthermore, post-enucleation

adjuvant VEC was associated with no metastatic events for 51 high-risk ICRB(20) Group E eyes

(mean 66 month follow-up).(33) However, the specific indications for adjuvant therapy are debated,

(34) with some groups suggesting good prognosis for isolated choroidal or retrolaminar optic nerve

invasion and negative margins without adjuvant treatment.(35, 36) 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.

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

histopathological features of the included eyes.

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-risk in unilateral cT2b eyes, and may predict which advanced eyes are suitable for trial

salvage. Given the widespread management debate of unilateral cT2a/cT2b eyes, there is a need for

robust, multicentre collaborative studies involving a larger group of children to further assess these

clinico-pathologic correlations.

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Tables

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

Edition.

Table 2. Histopathologic features of the studied eyes.

Table 3. Combined analysis of Fabian et al (30) 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, 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. He underwent six cycles of vincristine, etoposide, carboplatin (VEC) and

cyclophosphamide, followed by orbital irradiation.

(B) Left, 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 she received four

cycles of VEC.

(C) Left, RetCam 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, 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 (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. He was diagnosed with dural-based

metastases 1 year later. Despite radiotherapy, the child died 18 months after presentation with metastases.