Pediatr Blood Cancer 2013;60:336–337
BRIEF REPORT
Histopathology of Retinoblastoma: Does Standardization
Make a Difference in Reporting?
Sabyasachi Sengupta, DO, DNB,1 Subramanian Krishnakumar, MD,2 Tarun Sharma, MS,
Lingam Gopal, MS, FRCS,3 and Vikas Khetan, DO, DNB1*
The International Retinoblastoma Staging Working Group
(IRSWG) provided guidelines for tissue handling of eyes enucleated
for retinoblastoma. We adopted these guidelines from January 2009.
Reviewing histopathology slides between January 2006 and
December 2010 we found significantly more number of eyes with
massive choroidal, optic nerve, and anterior segment invasion by
Key words: histopathology; IRSWG; retinoblastoma; standardization
INTRODUCTION
Retinoblastoma is the commonest malignant intraocular tumor
in children [1]. Recent advances in the management of retinoblas-
toma have lead to dramatic improvement in survival rates al-
though mortality is still as high as 50% in the developing
nations [2,3]. Intracranial spread and systemic metastasis are the
main causes for mortality in patients with retinoblastoma. Reduc-
tion in the rate of systemic tumor dissemination by identification
of high-risk histopathologic factors after enucleation followed by
appropriate adjuvant chemotherapy may help improve survival.
Pathologic processing and interpreting histological character-
istics requires standardized protocols for universal agreement. To
address this issue, a group of global experts formed the Interna-
tional Retinoblastoma Staging Working Group (IRSWG) and pro-
vided guidelines for tissue handling and evaluation of prognostic
risk factors in retinoblastoma [4].
We have recently adopted the protocol as per the guidelines of
the IRSWG; to process the enucleated specimen. The purpose of
the current study is to compare the histopathologic results in
terms of identifying high-risk tumor characteristics before and
after introduction of this standardized protocol.
MATERIALS AND METHODS
The study was approved by the institutional review board of
Vision Research Foundation, Sankara Nethralaya, and adheres to
the tenets of the declaration of Helsinki. Histopathologic records
of all eyes that underwent enucleation for intraocular retinoblas-
toma from January 2006 to December 2010 were drawn up from a
database maintained at the department of ocular pathology and
reviewed retrospectively. The study period was chosen as all
slides were available for review. All the histopathological slides
during the study period were review by two experienced ocular
pathologists. Eyes with extrascleral spread and orbital disease on
histopathology and those that received pre-enucleation chemo-
therapy were excluded from the study. As a practice pattern, we
perform enucleation for groups D and E retinoblastoma (Interna-
tional Classification for Intraocular Retinoblastoma) [5] when it is
unilateral and for the eye with group E retinoblastoma when it is
bilateral.
Before the IRSWG protocol (pre-IRSWG), a pupil–optic nerve
(PO) section and a transverse section of the optic nerve from the
ß 2012 Wiley Periodicals, Inc.
DOI 10.1002/pbc.24357
Published online 12 October 2012 in Wiley Online Library
(wileyonlinelibrary.com).
FRCS, MBA,
1
tumor cells in the post-IRSWG era. The guidelines provided by the
IRSWG; in addition to being standardized; recognize more eyes with
histopathologic risk factors compared to techniques applied in the
past. This may translate to more children receiving adjuvant chemo-
therapy and contribute to reduced mortality rates in the future.Pediatr
Blood Cancer 2013;60:336–337. ß 2012 Wiley Periodicals, Inc.
surgical margin from each case were submitted for histopatholo-
gy. High-risk features evaluated included the presence and extent
of optic nerve invasion and the presence, location, and extent of
uveal invasion. Choroidal invasion was reported as percentage of
total choroidal thickness invaded. After 2009, we adopted the
IRSWG protocol described in detail elsewhere [4]. In summary,
we used the PO section, two calottes in the form of bread loafs
showing additional choroidal surface and one block containing the
surgical margin of the optic nerve. More than 3 mm choroidal
invasion was considered as significant (massive).
Clinical data subject to review were limited to information
included on the laboratory accession forms that accompanied
the enucleated eyes. Data were entered into Microsoft excel sheets
and all statistical analysis was performed using commercially
available statistical package (SPSS for Windows, version 14,
SPSS, Chicago, IL). Chi-squared test was used to compare pro-
portions of histopathological characteristics before and after ap-
plication of the IRSWG protocol. All P-values were considered
statistically significant when the values were <0.05.
RESULTS
A total of 283 enucleations were performed for retinoblastoma
during the 5-year study period. Out of these, 152 were performed
in the pre-IRSWG era and 131 were done in the post-IRSWG era.
Six eyes were enucleated after chemotherapy in the post-IRSWG
group (due to radiologic evidence of optic nerve involvement) and
hence were excluded from the study. We found no eyes with
histological evidence of extrascleral spread during the study
1
Shri Bhagawan Mahavir Vitreoretinal Services, Sankara Nethralaya,
Chennai, India; 2L&T Department of Ocular Pathology, Vision Re-
search Foundation, Sankara Nethralaya, Chennai, India; 3Department
of Ophthalmology, National University Health System, Singapore
Conflict of interest: Nothing to declare.
This article has been presented at the All India Ophthalmic Society
Annual Conference held at Cochin, India on February 3, 2012.
*Correspondence to: Dr. Vikas Khetan, DO, DNB, Shri Bhagawan
Mahavir Vitreoretinal Service, Sankara Nethralaya, No. 18 College
Road, Chennai 600 006, India. E-mail: drkhetan@yahoo.com,
drvk@snmail.org
Received 17 July 2012; Accepted 10 September 2012

TABLE I. Comparative Analysis of Baseline Demographic and
Clinical Data Between the Pre- and Post-IRSWG Groups
Pre-IRSWG Post-IRSWG
Category (n ¼ 152) (n ¼ 125) P-value
Age (Mean
SD) 13
4 months 14
3 months 0.43
Gender (Males) 102 64 0.23
Tumor
Group D 53 (34.8%) 48 (38.4%) 0.54
Group E 99 (65.2%) 77 (61.6%) 0.42
Leucocoria 57% 53% 0.33
period. The two groups were comparable in terms of the demo-
graphic and clinical data (Table I). Table II shows a comparison of
high-risk histopathological characters found during evaluation of
the specimens in the two groups. Significant choroidal invasion
(>3 mm choroidal invasion) was seen in 32 out of 67 eyes (48%)
with choroidal invasion in the post-IRSWG group. Invasion of any
portion of the optic nerve, especially the post-laminar optic nerve,
was also detected in significantly more number of eyes in the
post-IRSWG group. However, both groups had similar number of
eyes with tumor cells invading the surgical cut end of the optic
nerve.
DISCUSSION
Histopathologic features of an advanced tumor govern the
most crucial part of disease management, that is, post-enucleation
adjuvant chemotherapy. It is now generally agreed that massive
choroidal infiltration, post-laminar optic nerve invasion, invasion
of the optic nerve to transection, sclera infiltration, and extra-
scleral extension are the risk factors predictive of metastasis [6–
8]. Presence of any of these factors mandate administration of six
cycles of adjuvant chemotherapy with vincristine, etoposide, and
carboplatin [9].
The IRSWG in 2009, proposed guidelines to standardize this
process and urged future publications to validate the superiority of
the proposed consensus criteria over practices adopted in the past.
However, to the best of our knowledge, no study has achieved this
purpose till date.
We found a difference between the pre- and post-IRSWG
protocols in which the latter was able to identify significantly
more number of choroidal/optic nerve/anterior segment invasions.
Gupta et al. [10] recently reported choroidal invasion in 40% of
specimens and post-laminar optic nerve invasion in 17% out of
a total of 142 eyes in an Indian setting. Similarly, Kashyap et al.
[11] reported choroidal invasion of 43% including 22% with
massive choroidal invasion and post-laminar optic nerve invasion
in 17% out of a total of 326 eyes. Both of these Indian studies
have utilized the standardized protocol proposed by the ISRWG.
We observed very similar rates of choroidal and post-laminar
optic nerve invasion in our study during the post-IRSWG
era. As our institute is a referral centre for retinoblastoma and
receives cases from all over the country, our data, along with
the two studies mentioned above, represent a large proportion
of retinoblastoma eyes from India and suggest that a much higher
Pediatr Blood Cancer DOI 10.1002/pbc
Validation of IRSWG Protocol for Retinoblastoma 337
TABLE II. Comparison of Histopathological Characteristics
Between Pre- and Post-IRSWG Groups
Pre-IRSWG Post-IRSWG
Category (n ¼ 152) (n ¼ 125) P-value
Any invasion 80 (52.6%) 92 (73.6%) <0.001
Choroidal invasion 50 (33.1%) 67 (53.6%) <0.001
Choroidal invasion >3 mm Unknown 32 (25.6%) —
Optic nerve invasion 44 (28.9%) 62 (49.6%) <0.001
Post-laminar invasion 14 (9.2%) 21 (16.8%) <0.001
Surgical cut end invasion 3 (2%) 2 (1.6%) 0.23
Anterior segment invasion 17 (11.2) 34 (27.2%) <0.001
Combined invasion 28 (18.4%) 52 (41.6%) <0.001
proportion of enucleated eyes have high-risk histopathologic
factors in India compared to western literature over the past
decade.
In accordance with the histopathological results from the post-
ISRWG era seen in our study, more patients received adjuvant
chemotherapy over the past 2 years compared to previous years.
The actual clinical significance of the IRSWG protocol will be
vindicated by improvement in 5-year survival rates and reduction
in tumor recurrences as well. For this purpose, it would be pru-
dent to compare the survival rates between the two groups at a
later date.
The strength of our study is the substantial sample size and
strict adherence to the standardized protocol for tissue processing.
Although, the histopathologic evaluation was done on different
samples, all enucleated eyes belonged to either group D or E
retinoblastoma making comparisons possible. Also, it is practical-
ly not possible to section eyes for both protocols at the same time.
The drawbacks are the retrospective nature and the unavailability
of 5-year survival data at the present time.
In conclusion, the IRSWG protocol helped to identify high-
risk histopathologic factors in significantly more number of
eyes with retinoblastoma compared to the older tissue processing
techniques. Our study compared two different protocols for
histopathologic evaluation of retinoblastoma and validated the
guidelines provided by the IRSWG. This may improve overall
patient survival but requires further study in the future.
REFERENCES
1. Devesa SS. The incidence of retinoblastoma. Am J Ophthalmol 1975;80:263–265.
2. MacCarthy A, Birch JM, Draper GJ, et al. Retinoblastoma: Treatment and survival in Great Britain
1963 to 2002. Br J Ophthalmol 2009;93:38–39.
3. Canturk S, Qaddoumi I, Khetan V, et al. Survival of retinoblastoma in less-developed countries impact
of socioeconomic and health-related indicators. Br J Ophthalmol 2010;94:1432–1436.
4. Sastre X, Chantada GL, Doz F, et al. Proceedings of the consensus meetings from the International
Retinoblastoma Staging Working Group on the pathology guidelines for the examination of enucleated
eyes and evaluation of prognostic risk factors in retinoblastoma. Arch Pathol Lab Med 2009;133:1199–
1202.
5. Shields CL, Shields JA. Basic understanding of current classification and management of retinoblasto-
ma. Curr Opin Ophthalmol 2006;17:228–234.
6. Shields CL, Shields JA, Baez KA, et al. Choroidal invasion of retinoblastoma: Metastatic potential and
clinical risk factors. Br J Ophthalmol 1993;77:544–548.
7. Shields CL, Shields JA, Baez K, et al. Optic nerve invasion of retinoblastoma. Metastatic potential and
clinical risk factors. Cancer 1994;73:692–698.
8. Kopelman JE, McLean IW, Rosenberg SH. Multivariate analysis of risk factors for metastasis in
retinoblastoma treated by enucleation. Ophthalmology 1987;94:371–377.
9. Honavar SG, Singh AD, Shields CL, et al. Post-enucleation adjuvant chemotherapy in high-risk
retinoblastoma. Arch Ophthalmol 2002;120:923–931.
10. Gupta R, Vemuganti GK, Reddy VA, et al. Histopathologic risk factors in Retinoblastoma in India.
Arch Pathol Lab Med 2009;133:1210–1214.
11. Kashyap S, Meel R, Pushker N, et al. Clinical predictors of high risk histopathology in retinoblastoma.
Pediatr Blood Cancer 2012;58:356–361.