Journal of Cancer Therapy, 2013, 4, 1443-1447
Published Online November 2013 (http://www.scirp.org/journal/jct)
http://dx.doi.org/10.4236/jct.2013.49172
Open Access JCT
1443
Efferocytosis in Retinoblastoma
Dipankar Das*, Harsha Bhattacharjee, Kasturi Bhattacharjee, Prerana S. Tahiliani,
Pankaj Bhattacharyya, Manabjyoti Barman, Sumita Sarma Barthakur, Panna Deka,
Apurba Deka, Rajashree Paul
Sri Sankaradeva Nethralaya, Guwahati, India.
Email: *dr_dasdipankar@yahoo.com
Received October 25th, 2013; revised November 17th, 2013; accepted November 24th, 2013
Copyright © 2013 Dipankar Das et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Retinoblastoma has varied presentations. As age advances in retinoblastoma, the undifferentiated tumour is the common
appearance. Apop tosis in retinoblastoma in an untreated case can give us a clue about the tumour biology. Effero cytosis
in intraocular tu mour is a new concept which was seen in an enucleated eyeball specimen in a 4-year-old boy. Effective
efferocytosis can be conceptualized for new pharmacodynamics application for anti-cancer drugs. We present a rare
case of retinoblastoma with efferocytosis in retinal pigment epith elium and interesting apoptotic ch anges.
Keywords: Retinoblastoma; Apoptosis; Efferocytosis; Vesicles; Undifferentiated
1. Introduction
Retinoblastoma is the common intraocular tumour in
chi ld r en [1 ] . Th e survival of children with retinoblastoma,
if diagnosed early, has gone up to 95% in advanced cen-
ters [1,2]. Apoptosis and programmed cell death in these
tumours are important observations to understand the
tumour biology [2-4]. It is always a challenge to explore
apoptosis for und erstanding the cancer treatment [2-7].
Interesting pathological observations of apoptotic ch-
anges in a unilateral, undifferentiated and untreated case
of retinoblastoma were seen where nuclear fragmentation
and apoptotic vesicle arrangement were realized and
macrophages in the process showed their phagocytic ac-
tions on the apoptotic vesicles. A new phenomenon of
cell biology called “efferocytosis” was documented for
the first time in the retinoblastoma.
2. Case Report
A 4 years old male child reported to a tertiary institu te of
northeast India with a complaint o f white pupillary reflex
in the right eye (OD) noted by his parents for past 15
days. On examination, his vision in the OD was percep-
tion of light positive and 6/6, N6 in the left eye (OS).
Anterior segment findings were normal in OD and on
dilated fundus examination of the OD, a portion of the
white mass with calcified extension was seen with asso-
ciated retinal detachment Figure 1. The case was clini-
cally diagnosed Group E retinoblastoma of the OD. B
scan ultrasound of the OD showed a superior temporal
mass with adjoining calcification and calcified area had
high reflectivity on A-scan. There was no extra ocular
extension noted on the ultrasound. Computed tomogra-
phy scans of brain and orbit revealed retinoblastoma
mass confined to the right eye ball without any obvious
extra ocular extension. Enucleation of the OD with sili-
con ball implantation was done and examination under
anesthesia was carried out in the OS in the same sitting.
The enucleated specimen was sent to the ocular pathol-
ogy laboratory of the institution where it was fixed over-
night.
2.1. Gross Description
The enucleated eyeball measured anterior-posteriorly
22.07 mm, horizontally 23.18 mm, vertically 22.63 mm;
cornea measured horizontally 11.52 mm, vertically 10.83
mm; pupil measured 5.17 mm in diameter. Optic nerve
measured 10.10 mm in length and 3.25 mm in diameter
along with meninges. White pupillary reflex was seen.
White calcified area measuring 12.37 mm by maximum
height 9.14 mm was seen separately from freshly looking
main tumour mass measuring 18.21 mm × 11.53 mm
Figure 2. Lens was pushed forward. There was retinal
*Corresponding a uthor.
Efferocytosis in Retinoblastoma
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Figure 1. Fundus examination of the right eye showing a
portion of the white mass with calcified extension and asso-
ciated retinal detachme nt.
Figure 2. Showing white calcified area seen separately from
freshly looking main tumour mass and the lateral calottes
with cut section of distal end of optic nerve.
detachment seen superiorly. Sclera seemed to be normal.
By bread loaf technique, lateral calottes were cut and
submitted. Transillumination defect was seen in the vit-
reous cavity. Eyeball was sectioned vertically. Cut end of
the optic nerve was submitted separately and examined.
2.2. Microscopic Description
The enucleated eyeball showed normal corneal layers. A
focus of tumour was seen behind the iris and in one of
the de tach ed angles Figure 3. Portion of lens was no rma l.
A basophilic tumour mass in the vitreou s cavity was seen
which was endophytic in nature with necrosis and calci-
fication. The tumour was undifferentiated with intra scle-
ral tumour invasion was seen focally Figure 4. There
were unusual pigmented macrophages seen in the main
tumour mass and also in the area of scleral invasion Fig-
ure 5. The pigmented macrophages were seen tracking
from the main tumour mass to the scleral invasion Fig-
ure 6. Cells contained undifferentiated, pleomorphic and
basophilic retinoblastoma cells and vesicular (apoptotic)
bodies Figure 7. The macrophages showed engulfment
of vesicular bodies Figure 8. Some cells showed apop-
tosis in the tumour mass where fragmentation of nuclear
component were seen along with vesicle formation Fig-
ure 9. Retinal pigment epithelium (RPE) showed the
phenomenon of efferocytosis where macrophages were
seen engulfing apoptotic vesicles Figure 10. The vesicles
(efferosome) were in different shapes and sizes noted in
the RPE Figure 11. There was no extra scleral involve-
ment and no tumour invasion was seen in the attached
optic nerve. Lateral callotes showed full thickness chor-
oidal involvement. Portion of the cut end of the optic
nerve did not show tumour invasion.
Figure 3. (HE, ×200) Showing a focus of tumour behind the
iris and in one of the detached angles.
Figure 4. (HE, ×100) Showing focal intra scleral tumour
invasion.
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Efferocytosis in Retinoblastoma 1445
Figure 5. (HE, ×400) Showing presence of unusual pig-
mented macrophages in the main tumour mass and also in
the area of scleral invasion.
Figure 6. Showing the pigmented macrophages tracking
from the main tumour mass to the scleral invasion.
Figure 7. (HE, ×400) Showing undifferentiated, pleomor-
phic and basophilic retinoblastoma cells and vesicular (apo-
ptotic) bodies.
Figure 8. (HE, ×400) Showing the macrophages in the proc-
ess of engulfing the vesicular bodies.
Figure 9. (HE, ×400) Showing some cells in apoptosis in the
tumour mass where fragmentation of nuclear components
seen along with vesicle formation.
Figure 10. (HE, ×400) Showing retinal pigment epithelium
(RPE) and the phenomenon of efferocytosis where macro-
phages were seen engul f ing apoptotic vesicles.
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Figure 11. (HE, ×400) Showing vesicles (efferosome) in dif-
ferent shapes and sizes noted in the RPE.
3. Discussion
Efferocytosis is derived from the Latin word “effere” for
“to bury”, and it is the process by which the dead cells
are removed by macrophage phagocytic cells [3-5]. Dur-
ing efferocytosis, the cell membranes of phagocytic cell
engulf the apoptotic cells forming a large fluid filled
vesicle containing the dead cells [3-6]. The ingested vesi-
cle is called efferosome and this process is similar to
macropinocytosis [3,4]. Documentation of efferocytosis
in retinal pigment epithelial cells in retinob lastoma speci-
men of human eye was shown for the first time in the
scientific literature. Apoptosis or programmed cell death
is orderly and genetically controlled form of cell death
which can be seen in the tumour or when chemotherapy
induces such change in the tumour mass [5,6]. In a mor-
phological logic, it differs from necrosis in which cellular
shrinkage and chromatin condensation occur, followed
by fragmentation of nuclear components and apoptotic
vesicle formation which are cleared by phagocytosis
without damaging the neighboring tissues [5,6,8]. We
had seen that in our case where nuclear fragmentation
and apoptotic vesicle arrangement and the macrophages
in the process engulfed the vesicular bodies, some of the
vesicles were also seen in the RPE where they were en-
gulfed by the macrophages and thus effective efferocyto-
sis was recognized. These have major pharmacodynamic
applications for the anticancer drugs. The first step of
any anticancer drug is the interaction of cellular target [5-
9]. As we have seen in our case, there were two groups
of cells in the tumour mass and the portion where it had
spread i.e. sclera. One group of cells was typical undif-
ferentiated retinoblastoma cells which were basophilic
nucleated cells with scanty cytoplasm and the other
group was the vesicular cells. So the targeted chemo-
therapy needs to focus on the vesicular apoptotic cells
which can have local effect and then they will be also
phagocytosed by the RPE cells. Thus, the effective drug
will be delivered in the tumour mass and then eliminated
out as efferosome during the process. In nutshell, cells
that undergo apoptosis form the second step where the
efficacy of anticancer drugs was mediated by those tar-
geted apoptotic vesicles. In larger study, the extent of
expression of various oncogenes such as P-53 [9], bcl-2,
bax, c-myc and others and exploring the endogenous
factors will benefit the understanding of cancer biology
of retinoblastoma. It is always a challenge for pharma-
cological research to explore apoptosis by modulating the
extrinsic and intrinsic regulators in a positive and nega-
tive direction in order to improve the efficacy of anti-
cancer treatment.
Our case was a 4-year-old boy with unilateral, undif-
ferentiated retinoblastoma. Clinical picture and gross
specimen showed two distinct zones of involvement. One
was calcified area and the other was a fresh active grey-
ish coloured mass with retinal detachment. We presumed
that patient might have small endophytic lesion earlier
which spontaneously regressed and later on, there was
recurrence. The biology of such mixed tumour with that
situation could also be explained even for the apoptotic
change. The patient had no history of previous chemo-
therapy. Tumour necrosis, calcification and obvious in-
tra-scleral spread might be due to the aggressive nature
of recurrence. There was an interesting macrophagic
change seen in the sclera where the tumour was invaded.
Definite track of macrophagic migration was noted to the
sclera from the tumor mass. The pigmented macrophages
were larger from the normal pigmented cells seen in and
around that area of sclera and the tumour. Whether
macrophages traveled from the mass to the invaded tis-
sue before actual basophilic tumor cells migration or
macrophages really carried the tumour antibodies or pro-
teins to the distant areas in retinoblastoma will be the
subject of interest for the researcher in future.
4. Acknowledgements
The authors are indebted to: 1) Sri Kanchi Sankara
Health & Educational Foundation (SKSH&F); 2) Arjun
Mandal, Optometrist, Agartala, Tripura, India; 3) Mr.
Tarit Das, FM, Sri Sankaradeva Nethralaya, Guwahati,
Assam, India for their help and assistance.
REFERENCES
[1] C. L. Shields, A. Mashayekhi, H. Demirci, A. T. Mead-
ows and J. A. Shields, “Practical Approach to Manage-
ment of Retinoblastoma,” Archives of Ophthalmology,
Vol. 22, No. 5, 2004, pp. 729-735.
http://dx.doi.org/10.1001/archopht.122.5.729
[2] R. C. Eagle Jr., “High-Risk Features and Tumour Differ-
entiation in Retinoblastoma: A Retrospective Histopatho-
Open Access JCT
Efferocytosis in Retinoblastoma
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1447
logic Study,” Archives of Pathology & Laboratory Medi-
cine, Vol. 133, 2009, pp. 1203-1209.
[3] A. H. Wellie, J. F. R. Kerr and A. R. Currie, “Cell Death:
The Significance of Apoptosis,” International Review of
Cytology, Vol. 68, 1980, pp. 251-306.
http://dx.doi.org/10.1016/S0074-7696(08)62312-8
[4] R. S. Scott, E. J. Mcmohan, S. M. Pop, E. A. Reep, R.
Caricchio, P. L. Cohen, H. S. Earp and G. K. Matsun-
shine, “Phagocytosis and Clearance of Apoptotic Cells Is
Mediated by MER,” Nature, Vol. 411, No. 6834, 2001,
pp. 207-211. http://dx.doi.org/10.1038/35075603
[5] A. H. De Cathelineau and P. H. Henson, “The Final Step
in Programmed Cell Death: Phagocytes Carry Apoptotic
Cells to the Grave,” Essays in Biochemistry, Vol. 39,
2003, pp. 105-117.
[6] R. W. Vandivier, P. M. Henson and I. S. Douglas, “Burr-
ing the Dead: The Impact of Failed Apoptotic Cell Re-
moval (Efferocytosis) on Chronic Inflammatory Lung
Disease,” Chest, Vol. 129, No. 6, 2006, pp. 1673-1682.
http://dx.doi.org/10.1378/chest.129.6.1673
[7] S. J. Garalei, K. A. McPhillips, S. C. Frasch, et al., “Cell
Surface Calreticulin Initiates Clearance of Viable or Apo-
ptotic Cells through Transactivation of LRP on Phago-
cyte,” Cell, Vol. 123, No. 2, 2005, pp. 321-334.
http://dx.doi.org/10.1016/j.cell.2005.08.032
[8] M. H. Cesan, K. Pegan, A. Spes and B. Turk, “Lysosomal
Pathways to Cell Death and Their Therapeutic Applica-
tion,” Experimental Cell Research, Vol. 318, No. 11,
2012, pp. 1245-1251.
http://dx.doi.org/10.1016/j.yexcr.2012.03.005
[9] J. S. Lee, Y. J. Kim, C. L. Kim and G. M. Lee, “Differen-
tial Induction of Autophagy in Caspase 3/7 Down Regu-
lating and Bcl-2 Overexpressing Recombinant CHO Cells
Subjected to Sodium Butyrate Treatment,” Journal of
Biotechnology, Vol. 161, No. 1, 2012, pp. 34-41.
http://dx.doi.org/10.1016/j.jbiotec.2012.05.011