Delayed Enucleation of Eyes with Advanced Intraocular Retinoblastoma Due

to Pre-enucleation Treatment Increases Metastatic Death

Junyang Zhao, MD1, Meirong Wei, MD2, Guohua Liu, MD3, Zhao Xun Feng, BSc4, Carlos E.

Solarte5 MD, FRCSC, Bin Li, MD6, Yizhuo Wang, MD7, Chengyue Zhang, MD1, Brenda L.

Gallie, MD, FRCSC4, 8-9

Authors’ affiliations

1 Department of Ophthalmology, Beijing Children’s Hospital, Capital Medical University,

National Center for Children’s Health, China.

2 Department of Ophthalmology, LiuZhou Maternal and Child Health Care Hospital, China.

3 Department of Ophthalmology, Qilu Children’s Hospital of Shan Dong University, China.

4 Department of Ophthalmology and Vision Sciences, Hospital for Sick Children, Toronto,

Ontario, Canada.

5 Department of Ophthalmology and Visual Sciences, University of Alberta, Edmonton, Alberta,

Canada.

6 Department of Ophthalmology, Beijing Tongren Hospital, Capital Medical University, China

7 Department of Pediatric, Beijing Tongren Hospital, Capital Medical University, China.

8 Departments of Molecular Genetics and Medical Biophysics, Faculty of Medicine, University

of Toronto, Toronto, Ontario, Canada.

9 Division of Visual Sciences, Toronto Western Research Institute, Toronto, Ontario, Canada.

Corresponding author: Junyang Zhao, 56 Nanlishi Lu, Xicheng District, Beijing, China,

100045 zhaojunyang@163.com

Running Head: Delayed Enucleation of Advanced Retinoblastoma Increases Death

Word count: /3000 words

1

Numbers of figures and tables: 1 consort diagram, 3 figures and 3 tables and 3 online only

figures

Key Words: retinoblastoma, enucleation, chemotherapy, pathology

2

Purpose

Advanced intraocular retinoblastoma can be cured by primary enucleation. With recent advances

in treatment modalities, there is a trend toward trial salvage which can delay enucleation. We

hypothesized that delayed enucleation over a defined length of time would increase metastatic

death.

Methods

A multicenter retrospective review of 554 eyes primarily and secondarily enucleated for groups

D and E retinoblastoma (IIRC). One eye enrolled per child. Treatment groups were compared for

histopathology and disease-specific survival.

Results

Primarily enucleated eyes had median ≤ 0.1 month from diagnosis to enucleation. In comparison,

pre-enucleation chemotherapy delayed enucleation (group D: median 8.4 months; group E:

median 2.8 months). Disease-specific survival was lower with prolonged delay between

diagnosis and enucleation. Delay > 3.5 months (group D) and > 2 months (group E) were

associated with diminished survival (group D: p = .02; group E: p = .02). Eyes treated with pre-

enucleation chemotherapy had fewer high-risk histopathology (pT3/pT4) than primarily

enucleated eyes (group D: p = .04; group E: p = .003). Children with group E eyes and 1 to 3

cycles of pre-enucleation chemotherapy had no difference in survival as children with primary

enucleation, but those with ≥ 4 cycles had lower survival than children primarily enucleated (p

= .03). Children with high-risk eyes (pT3/pT4) after pre-enucleation chemotherapy had

diminished survival than children with high-risk eyes primarily enucleated (p = .002).

Conclusion

3

A B S T R A C T

We showed that there is a window of opportunity in which enucleation is most effective and no

amount of pre-enucleation chemotherapy confer any survival benefit. Timely enucleation, < 3.5

months (group D) and < 2 months (group E), minimizes the risk of metastatic death.

4

Retinoblastoma is the most common primary malignant intraocular tumor of the eye.1 It accounts

for 4% of all pediatric malignancies affecting one in every 16,000 to 18,000 live births.2-4 If

discovered early, retinoblastoma is one of the most treatable cancer. The International Intraocular

Retinoblastoma Classification (IIRC) (2005) staged tumor from group A (very low risk) through

E (very high risk) to predict outcome following systemic chemotherapy and consolidation focal

therapy.5 IIRC predicts ≥ 90% chemotherapy success for salvage of groups A, B and C eyes.6

Even advanced retinoblastoma (groups D and E) confined to the eye can be cured by simple

enucleation. However, of concern is that delayed enucleation may increase risk of extraocular

tumor extension which has limited curative options and high mortality rate.7,8

Unlike other pediatric cancers, rigorous controlled clinical trial in retinoblastoma is missing

for a multitude of reasons including: low incidence of disease to interest pharmaceutical industry,

complexity with bilateral disease and vision preservation as a competing outcome measure to

cure of cancer.1 Therefore, multi-center collaboration in research is essential to advance evidence

for clinical management of retinoblastoma. This study reviewed 600 children enucleated for

advanced retinoblastoma and is an expansion of our publication in 2011 which identified a delay

> 3 months from diagnosis to enucleation increases mortality for children with group E eyes.8

Since then, there has been an increasing shift toward salvage of group D eyes by use of systemic

chemotherapy9 and lately intravitreal chemotherapy,10-12 intra-arterial chemotherapy (IAC),13-15

periocular chemotherapy,16 and tumor endoresection via pars plana vitrectomy.17 Trial eye

salvage with any of these modalities can delay enucleation. Our aim is to re-evaluate the risk of

delayed enucleation for both groups D and E eyes. Secondary objective is to study the

5

INTRODUCTION

relationship between pre-enucleation systemic chemotherapy and histopathologic risk and their

effect on long-term patient survival.

Data Collection and Ethics

This was a retrospective study of all IIRC groups D and E children who had primary and

secondary enucleation at 29 Chinese treatment centers between January 17, 2006 and December

26, 2010. The final follow-up date accounted in this study was December 13, 2017. Clinical

information collected include age at diagnosis, sex, disease laterality (unilateral/bilateral),

clinical staging of diseased eyes at diagnosis, date of enucleation, neoadjuvant and adjuvant

treatments, last follow-up date, evidence of extraocular extension and date of death. IIRC (2005)

staging5 were used at time of data collection and retrospective review of data did not permit

conversion to the newer cTNM staging by the 8th Edition American Joint Committee on Cancer

(AJCC).18 The study was approved by the ethics boards of Beijing Children’s Hospital, Beijing

Tongren Hospital, Liuzhou Maternal and Child Health Care Hospital and Qilu Children’s

Hospital of Shan Dong University in accordance with the Declaration of Helsinki.

Eligibility

Of 600 children, none had clinical evidence of extraocular disease or metastasis at diagnosis

by lumbar puncture, bone marrow aspiration, computed tomography and/or magnetic resonance

imaging. For analysis of effect of delayed enucleation on mortality, excluded were children died

from chemotherapy toxicity and children with bilateral advanced retinoblastoma (D/D, D/E, E/D

or E/E). For analysis of effect of pre-enucleation chemotherapy (cycles or presence/absence) on

mortality, in addition to the above exclusion criteria, children with less than one complete

6

METHODS

chemotherapy cycle (< 3 weeks) or those with salvage therapy other than systemic chemotherapy

were secondarily excluded (Figure 1).

Histopathologic Assessment

All enucleated eyes were pathologically staged initially using the 7th Edition AJCC pTNM19

based on review of hematoxylin and eosin-stained slides. Microscopic slides included multiple

section levels of the mid-globe, both calottes and surgical margin of optic nerve. Evaluated

features included the presence and extent of tumor invasion into the optic nerve, anterior

chamber, choroid and sclera. A second independent reviewer updated the histopathology staging

to 8th Ed. AJCC pTNM18 via review of pathology reports and representative microscopic

sections. For analysis, histopathologic risk was condensed into two groups: low-risk (pT1 and

pT2) and high-risk (pT3 and pT4). High-risk features include massive choroidal invasion (≥ 3

mm), scleral invasion, episcleral invasion, retrolaminar optic nerve invasion and invasion of

optic nerve surgical margin. Table 1 summarizes the 8th Ed. AJCC pathological staging.

Statistical Analysis

Sex, age at diagnosis, time from diagnosis to enucleation, follow-up since diagnosis,

systemic chemotherapy or not before enucleation, histopathologic risk (high vs low risk),

systemic chemotherapy or not after enucleation and IIRC classification (group D or E) were

summarized using frequency/percentage for categorical variables and median/range for

continuous variables. Baseline characteristics of children with and without pre-enucleation

chemotherapy were compared using Pearson’s chi-squared test for categorical variables and

Mann-Whitney U test for continuous variables. Receiver-operating characteristic (ROC) analysis

was used to determine true positive rate (TPR) and false positive rate (FPR) for all possible cut

off values of time from diagnosis to enucleation in relation to disease-specific survival (DSS)

7

(Supplement 1). For any given threshold, TPR is the proportion of dead children correctly

predicted, while FPR is the proportion of living children wrongly predicted to be dead. The

thresholds with the highest TPR were identified, among which the one with the lowest FPR was

selected. TPR was prioritized in threshold selection given the greater importance of correctly

identifying children at risk of metastasis. Thresholds were used for placing children into

categorical groups for survival analysis. Kaplan-Mier (KM) survival analysis was associated to

log-rank test for testing survival equality. Cox proportional-Hazards model was used

additionally to calculate survival equality and hazard ratio between sub-groups. Length of DSS

was measured from date of diagnosis to date of metastatic death. Patients alive were censored at

last follow up. All P-values reported are two sided and less than 0.05 indicated significance. All

analysis was performed using STATA version 15.1 (Stata Corporation) and SPSS Version 25

(IBM Corp).

Patient Clinical Information

A total of 202 group D and 352 group E eyes from 554 children (335 males and 219

females) were included, with a median age of 23 months (range, 1 to 122 months) at diagnosis.

Non-studied eyes of bilateral children were IIRC group A in 27 cases, group B in 32 cases and

group C in 20 cases. Enucleation was the primary treatment for 275 eyes, while 279 eyes were

treated initially with systemic chemotherapy prior to enucleation (Table 2). 29 children also

received other salvage treatments in addition to systemic chemotherapy: IAC in 18 cases, PPV

endoresection in 6 cases, plaque radiotherapy in 3 cases, 1 case of stem cell transplantation and 1

case of immunotherapy. Sex and age at diagnosis were not significantly different between

children with and without pre-enucleation systemic chemotherapy. The medium time from

8

RESULTS

diagnosis to enucleation were ≤ 0.1 months for primarily enucleated groups D and E eyes. In

comparison, pre-enucleation chemotherapy significantly delayed enucleation (group D median

0.1 vs 8.4 months, P < .001; group E median 0-day vs 2.8 months, P < .001). Group E eyes with

pre-enucleation chemotherapy had longer follow-up than group E eyes primarily enucleated

(median 93 vs 84 months, P = .03). Higher proportion of primarily enucleated eyes had high-risk

histopathology (pT3 and pT4) than eyes treated with chemotherapy (group D 21% vs 11%, P

= .04; group E 34% vs 20%, P = .003). Fewer children with pre-enucleation chemotherapy than

primarily enucleated children received post-enucleation chemotherapy (group D, 14% vs 51%, P

< .001; group E, 47% vs 62%, P = .03).

Pre-enucleation Chemotherapy

Median pre-enucleation chemotherapy cycles were 4 (range, 1 to 15 cycles). The

chemotherapy regimens used were carboplatin, etoposide/teniposide, and vincristine. Eyes were

removed when tumors progressed with no possibility of useful vision.

Patients Died from Retinoblastoma Metastasis

Of 22 children who died from metastasis 6 had group D and 16 had group E eyes, among

which 14 received pre-enucleation chemotherapy (median, 5.5 cycles) and 11 received post-

enucleation chemotherapy (median, 3 cycles) (Table 3). The median time from diagnosis to

enucleation was 3.7 months (range, 0 to 42.3 months). Histopathologic examination identified 5

eyes as low-risk and 17 eyes as high-risk. pTNM staging were: 4 pT1 [18%], 1 pT2b [5%], 2

pT3a [9%], 4 pT3b [18%], 2 pT3c [9%] and 9 pT4 [41%].

Primary Outcomes

ROC analysis identified time from diagnosis to enucleation > 3.5 months (group D) and > 2

months (group E) as cut offs (Supplement 2 and 3). For survival analysis, group D eyes were

9

placed into two groups: eyes enucleated < 3.5 months and > 3.5 months from time of diagnosis.

Group E eyes were grouped into eyes enucleated < 2 months and > 2 months from time of

diagnosis (Figure 2).

An association of delayed enucleation with diminished survival was identified. Survival was

lower if enucleation of group D eyes were delayed > 3.5 months from diagnosis (48-month DSS,

93% vs 100%; P = .02). No child with group D eye enucleated within 3.5 months died. For

children with group E eyes, survival was lower if enucleation were delayed > 2 months (48-

month DSS 97% vs 90%; HR = 3.10; P = .02).

Secondary Outcomes

After secondary exclusion, 510 eyes (177 group D and 333 group E) were studied for the

effect of pre-enucleation chemotherapy cycles on survival (Figure 3). Group E eyes that received

1 to 3 cycles of systemic chemotherapy had no difference in survival as primarily enucleated

eyes (48-month DSS, 97% vs 96%; Hazard ratio = 0.77; P = .74). However, those with four or

more cycles of chemotherapy had diminished survival (48-month DSS, 86% vs 96%; HR = 3.35;

P = .03). For children with group D eyes, 4 with pre-enucleation chemotherapy died, while all

primarily enucleated children survived. However, the number is too few for survival analysis.

Of enucleated eyes with low histopathologic risk (pT1 and pT2), children with pre-

enucleation chemotherapy or not had no difference in survival (48-month DSS, 99% vs 99%;

Hazard ratio = 0.94; P = .95; Figure 4). Despite similar adverse histopathology (primary vs

secondary enucleation % pT4, 15% vs 25%, P = .255), high-risk eyes that received systemic

chemotherapy had lower survival than high-risk eyes primarily enucleated (48-month DSS, 59%

vs 93%; HR = 5.05; P = .002).

Exploratory Outcomes

10

Of 554 children studied, 248 received systemic chemotherapy after enucleation, with a

median of 3 cycles (range, 1 to 15 cycles; Supplement 4). Of 440 low-risk children (pT1 and

pT2), 150 received post-enucleation chemotherapy and 16 of 114 high-risk children (pT3 and

pT4) did not receive post-enucleation chemotherapy. Low-risk children with and without post-

enucleation chemotherapy had no difference in survival (48-month DSS, 99% vs 99%; Hazard

ratio = 0.49; P = 0.52). High-risk children with post-enucleation chemotherapy had better

survival than high-risk children without post-enucleation chemotherapy (48-month DSS, 89% vs

49%; Hazard ratio = 0.21; P < 0.001).

Although advanced intraocular retinoblastoma is relatively rare in developed countries, it is the

most common presentation in the developing world. In low to mid-income Asian countries,

groups D and E disease constitutes 78% to 89% of all intraocular retinoblastoma cases.20,21 Multi-

center collaboration enabled us to accrue the largest dataset of enucleated groups D and E eyes.

Family and physician may choose to use pre-enucleation systemic chemotherapy for various

reasons including the desire for eye salvage, parental resistance to enucleation and the belief that

chemotherapy would lower risk of metastasis.

Particularly relevant today because of shifting emphasis toward salvage therapy, our study

highlights the risk to life with prolong delay to enucleation. We showed that delayed enucleation

from diagnosis > 3.5 months for group D eyes and > 2 months for group E eyes increase risk of

metastasis. Given reported salvage rate of 47% to 95% for group D eyes from centers

worldwide,15,22 trial salvage of group D eyes during the first 3.5 months after diagnosis may be

justified. Group E eyes usually present with irreversible ocular damage and high probability of

adverse histopathology;23-26 immediate enucleation is the safest option. Retinoblastoma team can

11

DISCUSSION

take advantage of the 2-month grace period to counsel parents who are resistant to enucleation of

group E eyes.

The AJCC pTNM staging classifies the risk of metastasis based on extent of tumor invasion

and guides post-enucleation treatment. But, despite having the same advanced histopathology

staging (pT3 and pT4), children who received pre-enucleation chemotherapy had diminished

survival compare to primarily enucleated children. High-risk histopathology after pre-

enucleation chemotherapy may be an indicator that the tumor is chemotherapy-resistant.

Therefore, treatment with the same regimen of chemotherapy (VEC) post-enucleation may be

ineffective at clearing off resistant microscopic metastasis. Our study underscores the important

of not solely relying on pTNM staging for post-enucleation management especially when pre-

enucleation chemotherapy was used.

In this study, children with pre-enucleation chemotherapy had fewer adverse histopathology

but no improved survival compared to primarily enucleated children. This confirms hypothesis

from our previous publication that pre-enucleation chemotherapy masks histopathologic risk.8

The fewer high-risk histopathology also explains the finding of fewer children with pre-

enucleation chemotherapy than primarily enucleated children received post-enucleation

chemotherapy.

Our study also confirmed finding by others that post-enucleation chemotherapy should be

reserved to children with high-risk histopathology.27-29 For children with low-risk eyes, post-

enucleation chemotherapy exposes them to toxicity without any survival benefit. In this study,

two children died from chemotherapy toxicity. They both had post-enucleation chemotherapy

despite pT1 and pT2b histopathology. Post-enucleation chemotherapy may have been given to

low-risk children out of fear of masked high-risk features. Our study now demonstrates that

12

chemotherapy to low-risk children does not improve survival. Five children died from tumor

metastasis despite low-risk histopathology. These eyes all had focal choroidal invasion or

pre-/intra-laminar invasion but not both. The worse survival of these children compared to

children with neither of these features prompts further study into the risk profile of

histopathologic features. Another possibility is that adverse features were present in these eyes

but weren’t in the studied sections.

In summary, our study confirmed the danger of delayed enucleation in children with

advanced intraocular retinoblastoma. We showed that no amount of pre-enucleation systemic

chemotherapy confers any survival benefit. Instead, delay to enucleation > 3.5 months (group D)

and > 2 months (group E) from systemic chemotherapy increases risk of death from metastasis.

Primary enucleation remains the method that best achieves cure while prolong eye salvage risks

life of the child.

The author(s) indicated no potential conflicts of interest.

Conception and design: Junyang Zhao, Meirong Wei, Guohua Liu, Zhao Xun Feng, Carlos E.

Solarte, Brenda L. Gallie

Administrative support: Bin Li

Provision of study materials or patients: Junyang Zhao, Meirong Wei, Guohua Liu

Collection and assembly of data: Junyang Zhao, Meirong Wei, Guohua Liu, Zhao Xun Feng,

Yizhuo Wang, Chengyue Zhang, Brenda L. Gallie

13

AUTHORS’ DISCLOSURE OF CONFLICTS OF INTEREST

AUTHOR CONTRIBUTIONS

Data analysis and interpretation: Junyang Zhao, Zhao Xun Feng, Carlos E. Solarte, Brenda L.

Gallie

Manuscript writing: All authors

Final approval of manuscript: All authors

14

1. Dimaras H, Corson TW, Cobrinik D, et al: Retinoblastoma. Nat Rev Dis Primers 1:15021, 2015

2. Gallie BL, Budning A, DeBoer G, et al: Chemotherapy with focal therapy can cure intraocular retinoblastoma without radiotherapy. Arch Ophthalmol 114:1321-8, 1996

3. Shields CL, Shields JA: Basic understanding of current classification and management of retinoblastoma. Curr Opin Ophthalmol 17:228-34, 2006

4. Broaddus E, Topham A, Singh AD: Incidence of retinoblastoma in the USA: 1975-2004. Br J Ophthalmol 93:21-3, 2009

5. Murphree AL: Intraocular retinoblastoma: the case for a new group classification. Ophthalmology Clinics of North America 18:41-53, 2005

6. Shields CL, Mashayekhi A, Au AK, et al: The International Classification of Retinoblastoma predicts chemoreduction success. Ophthalmology 113:2276-80, 2006

7. Gunduz K, Muftuoglu O, Gunalp I, et al: Metastatic retinoblastoma clinical features, treatment, and prognosis. Ophthalmology 113:1558-66, 2006

8. Zhao J, Dimaras H, Massey C, et al: Pre-enucleation chemotherapy for eyes severely affected by retinoblastoma masks risk of tumor extension and increases death from metastasis. J Clin Oncol 29:845-51, 2011

9. Chan HS, Gallie BL, Munier FL, et al: Chemotherapy for retinoblastoma. Ophthalmol Clin North Am 18:55-63, viii, 2005

10. Berry JL, Shah S, Bechtold M, et al: Long-term outcomes of Group D retinoblastoma eyes during the intravitreal melphalan era. Pediatr Blood Cancer 64, 2017

11. Munier FL, Gaillard MC, Balmer A, et al: Intravitreal chemotherapy for vitreous disease in retinoblastoma revisited: from prohibition to conditional indications. Br J Ophthalmol 96:1078-83, 2012

12. Munier FL, Soliman S, Moulin AP, et al: Profiling safety of intravitreal injections for retinoblastoma using an anti-reflux procedure and sterilisation of the needle track. Br J Ophthalmol 96:1084-7, 2012

13. Munier FL, Mosimann P, Puccinelli F, et al: First-line intra-arterial versus intravenous chemotherapy in unilateral sporadic group D retinoblastoma: evidence of better visual outcomes, ocular survival and shorter time to success with intra-arterial delivery from retrospective review of 20 years of treatment. Br J Ophthalmol, 2016

14. Yousef YA, Soliman SE, Astudillo PP, et al: Intra-arterial Chemotherapy for Retinoblastoma: A Systematic Review. JAMA Ophthalmol, 2016

15. Shields CL, Manjandavida FP, Lally SE, et al: Intra-arterial chemotherapy for retinoblastoma in 70 eyes: outcomes based on the international classification of retinoblastoma. Ophthalmology 121:1453-60, 2014

16. Mallipatna AC, Dimaras H, Chan HS, et al: Periocular topotecan for intraocular retinoblastoma. Arch Ophthalmol 129:738-45, 2011

17. Zhao J, Li Q, Wu S, et al: Pars Plana Vitrectomy and Endoresection of Refractory Intraocular Retinoblastoma. Ophthalmology 125:320-322, 2018

15

18. Mallipatna A, Gallie BL, Chévez-Barrios P, et al: Retinoblastoma, in Amin MB, Edge SB, Greene FL (eds): AJCC Cancer Staging Manual. New York, NY, Springer, 2017, pp 819-831

19. Finger PT, Harbour JW, Murphree AL, et al: Retinoblastoma, in Edge SB, Byrd DR, Carducci MA, et al (eds): AJCC Cancer Staging Manual. New York, NY, Springer, 2010, pp 561-568

20. Chawla B, Jain A, Seth R, et al: Clinical outcome and regression patterns of retinoblastoma treated with systemic chemoreduction and focal therapy: A prospective study. Indian J Ophthalmol 64:524-9, 2016

21. Gao J, Zeng J, Guo B, et al: Clinical presentation and treatment outcome of retinoblastoma in children of South Western China. Medicine (Baltimore) 95:e5204, 2016

22. Berry JL, Jubran R, Kim JW, et al: Long-term outcomes of Group D eyes in bilateral retinoblastoma patients treated with chemoreduction and low-dose IMRT salvage. Pediatr Blood Cancer 60:688-93, 2013

23. Kaliki S, Shields CL, Rojanaporn D, et al: High-risk retinoblastoma based on international classification of retinoblastoma: analysis of 519 enucleated eyes. Ophthalmology 120:997-1003, 2013

24. Kaliki S, Srinivasan V, Gupta A, et al: Clinical features predictive of high-risk retinoblastoma in 403 Asian Indian patients: a case-control study. Ophthalmology 122:1165-72, 2015

25. Yousef YA, Al-Hussaini M, Mehyar M, et al: Predictive Value of Tnm Classification, International Classification, and Reese-Ellsworth Staging of Retinoblastoma for the Likelihood of High-Risk Pathologic Features. Retina 35:1883-9, 2015

26. Wilson MW, Qaddoumi I, Billups C, et al: A clinicopathological correlation of 67 eyes primarily enucleated for advanced intraocular retinoblastoma. The British journal of ophthalmology 95:553-8, 2011

27. Khan SS, Ning H, Wilkins JT, et al: Association of Body Mass Index With Lifetime Risk of Cardiovascular Disease and Compression of Morbidity. JAMA Cardiol, 2018

28. Chantada GL, Fandino AC, Guitter MR, et al: Results of a prospective study for the treatment of unilateral retinoblastoma. Pediatr Blood Cancer 55:60-6, 2010

29. Chantada G, Fandino A, Davila MT, et al: Results of a prospective study for the treatment of retinoblastoma. Cancer 100:834-42, 2004

16