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Histopathological Parameters in Pterygia and Significant Clinical Correlations
Histopathological Parameters in Pterygia and Significant Clinical Correlations
Abstract
Purpose: To evaluate the clinical and histopathological parameters of pterygium to determine significant
correlations between parameters that can affect management strategies.
Methods: A total of 47 pterygia were clinically examined and excised for histopathological evaluation of
epithelial and stromal changes. Some samples were immunostained with P53 (a protein of 53 kilodalton
used as dysplastic epithelial marker), CD20 (CD/cluster of differentiation, are group of surface receptors
providing targets for cellular immunophenotyping, CD20 as a B lymphocyte marker), CD 3 (as T lymphocyte
marker) or vascular endothelial growth factor (VEGF/as vascular marker).
Results: Most patients were male (59.6%). Cosmetic complaints (83%), grade II redness (61.7%), grade 2
extension (63.8), and associated astigmatism of <2.5 D (83%) were observed. Histopathological features included
solar elastosis (100%), squamous hyperplasia (83%), increased stromal vascularity with hemorrhage (76.6%),
and lymphocytic stromal infiltration, perivascular distribution, and mild epithelial lymphocytic exocytosis in
72.3%, 74.5%, and 70.2% of cases, respectively. Other changes included goblet cell hyperplasia (31.9%), prominent
epithelial pigmentation (48.9%), and, most importantly, epithelial atypia (53.2%). Clinical redness was significantly
correlated with vascularity, epithelial hyperplasia, and lymphocytic stromal infiltration; lymphocytic stromal
infiltration was also significantly correlated with pterygium extension and with low astigmatism.
Conclusion: The inflammatory response was mild in most cases and the density was not significantly
correlated with any clinical parameter. Vascularity was related to clinical redness. Treatment with anti‑VEGF
may be beneficial, even for grade 1 pterygia that are not dominantly fibrotic.
How to cite this article: Reda AM, Shaaban YM, Saad El‑Din SA.
DOI:
10.4103/jovr.jovr_31_17 Histopathological parameters in pterygia and significant clinical
correlations. J Ophthalmic Vis Res 2018;13:110-8.
110 © 2018 Journal of Ophthalmic and Vision Research | Published by Wolters Kluwer - Medknow
Clinicopathological Study on Pterygia; Reda et al
irradiation is suspected to be the most important factor sections passed through the corneal side as well as
for its development.[2] Primary pterygium is diagnosed the canthus side. Tissue blocks were processed, cut,
by the presence of a wing of thick, reddish, fibrovascular and stained with hematoxylin and eosin stain. Some
growth encroaching on the cornea. The nasal side is more samples were immunostained with P53 (protein of 53
commonly affected than the temporal side.[3] kilodalton as dysplastic epithelial marker), CD 20 (cluster
Surgical excision is mostly performed for cosmetic of differentiation as B lymphocyte marker), CD 3
reasons. However, large pterygia may obscure the (cluster of differentiation as T lymphocyte marker) or
visual axis and may cause astigmatism,[4] and surgery is vascular endothelial growth factor (VEGF, as vascular
an important treatment option for primary pterygium. marker). The following epithelial and stromal parameters
Complications of surgery include recurrence; recurrence were recorded.
rates of 15% to 75% in cases of simple excision, and Assessment of epithelial atypia was done by
of approximately 6% in free flap surgery have been examination of H&E stained sections. Then, four
reported.[5,6] Moreover, many patients refuse surgery cases only from our studied cases, (one showed no
and request conservative treatment that primarily epithelial atypia, one showed reactive epithelial atypia,
targets the red appearance of the congested pterygium. one showed immature squamous metaplasia and
For cases with just cosmetic complain (redness), and finally one showed mild looking epithelial dysplasia
refuse surgery, medical treatment is the best choice and with no moderate or severe epithelial dysplasia
essential.[7] were found in our studied cases), were subjected
In the present study, we assessed all clinical variables to P 53 (used as marker for epithelial dysplasia)
to determine the histopathological changes that occur in immunohistochemical study (A mouse monoclonal
pterygium, and correlated them to determine the best antibody, concentrated 1:400 clone Bp53‑12, Sigma‑Adrich
management strategy for each patient. Lab., California, USA). Assessment of the positive nuclei
was done. All studied cases were showing positive
METHODS nuclear stain at basal cell layer and some suprabasal
keratinocytes, thus P53 immunostaining was not further
A total of 47 cases of primary pterygium were examined applied on the other studied cases as it didn’t add value.
between 2015 and 2016. Histories including age, sex, For statistical analysis, each case was classified as with
occupation, duration, and complaint (cosmetic or visual) or without epithelial atypia.
were taken. Assessment of squamous epithelial hyperplasia
Prior to surgery, all patients underwent a complete whether mild, moderate or severe was done. For
ocular examination including slit lamp biomicroscopy and statistical purposes, it was categorized as absent or
photography. Ocular examinations included measurement present. The number of goblet cells was determined,
of best corrected visual acuity according to the Snellen and was classified as few cells or prominent cells
visual acuity chart. Astigmatism was assessed using a (goblet cell hyperplasia). All samples were examined
Topcon RM‑8000B autorefractometer (Tokyo, Japan). for the presence and severity of lymphocytic exocytosis.
The pterygium width at the limbus was measured in In all cases, epithelial lymphocytic exocytosis was
millimeters using a slit lamp. Pterygia were graded determined as mild to moderate.
according to size and extent of corneal involvement, as Epithelial pigmentation in basal cells and in suprabasal
follows: grade 1: between the limbus and a point midway keratinocytes was considered absent (if none or few) or
between the limbus and pupillary margin; grade 2: head present (if prominent along basal keratinocytes or in
of the pterygium reaching the pupillary margin; and suprabasal keratinocytes. Some authors define this as
grade 3: crossing the pupillary margin.[7] Pterygia were primary acquired melanosis of the conjunctiva;[9] others
also graded for severity of redness, as follows: grade I: no use the term conjunctival hypermelanosis.[10]
redness or faint pinkish hue; grade II: scattered areas of All cases were examined for evidence of stromal solar
moderate redness; and grade III: significant and diffuse elastosis. They were also examined for the presence
redness.[8] Exclusion criteria were previous medical and relative amounts stromal vascularity and fibrosis.
treatment for pterygium including topical steroids In the included cases, vascularity and fibrosis were
or non‑steroidal anti‑inflammatory drugs, previous classified into two groups. One group showed prominent
conjunctival surgery, conjunctival cicatricial disease, vascularity that overwhelming the fibrosis, and the other
systemic autoimmune disease, and untreated dry eye group showed no significant difference between fibrosis
disease. All patients were treated using the same surgical and vascularity.
technique (excision and graft). Written informed consent All cases were examined for stromal inflammation by
was obtained from all participants before surgery. considering both pattern (mild perivascular, or diffuse)
The obtained tissues were preserved in 10% formalin. and type of inflammatory cells: chronic (lymphocytes
Each specimen was cut longitudinally through the with or without plasma cells) or mixed (lymphocytes
head and the body of the pterygium. Thus, in all cases, with neutrophils). All cases were also examined
Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018 111
Clinicopathological Study on Pterygia; Reda et al
for hemorrhage and siderophages. For any case ranged from 4 to 60 months (mean, 20.7 ± 16.1). The
showing siderophage‑like cells, two more sections were pterygium extension over the cornea was grade 1 in
taken from the specimen. One section was treated in 9 cases (19.1%), grade 2 in 30 cases (63.8%), and grade 3
ethanolic picric acid to remove any suspected formalin in 8 cases (17%). The severity of redness was reported
pigment,[11] and the other was stained using Perl’s stain as grade I in 4 cases (8.5%), grade II in 29 cases (61.7),
(Thermo Scientific Waltham, Massachusetts, USA). This and grade III in 14 cases (17%). The width ranged
parameter was classified as absent or present. from 1.4 to 7.8 mm (mean, 2.9 ± 1.6 mm). Cases were
Some cases were included in an immunohistochemical divided into two groups (<4 mm width, 39 cases, 83%)
study using CD20 (monoclonal mouse antihuman and (>4 mm width, 8 cases, 17%). Eight cases had
antibody, clone L26, concentrated 1:50, Abcam astigmatism >2.5 D; 39 cases had astigmatism <2.5 D.
Scientific Support, San Jose, California, USA), CD3 The epithelial changes included epithelial atypia
(monoclonal rabbit antihuman antibody, concentrated in 53.2% of cases ranging from reactive atypia with
1:100, Abcam Scientific Support, USA), and immature squamous metaplasia (21 cases) to mild
VEGF (monoclonal mouse antihuman antibody, clone dysplasia (4 cases); no moderate or severe dysplasia was
VG1, concentrated 1:100, Thermo Fisher Scientific, observed. Epithelial hyperplasia was observed in 83.0%
Waltham, Massachusetts, USA) to assess the type of of cases, and goblet cell hyperplasia was observed in
lymphocytes in the infiltrate and the expression of this 31.9% of cases. Mild epithelial lymphocytic exocytosis
factor in the blood vessels of the lesion. The cases included was observed in 70.2% of cases; the moderate form was
in the CD20 and CD3 immunohistochemical studies observed in 29.8% of cases. Stromal inflammation had
were selected based on the presence of stromal and a perivascular pattern in 74.5% of cases and a diffuse
epithelial lymphocytosis (12 cases with diffuse stromal pattern in 25.5% of cases. Immunohistochemical studies
inflammation, 2 additional cases with moderate epithelial revealed that most lymphoid cells in all 14 cases were
lymphocytic exocytosis; 14 cases total). Cases that were B lymphocytes with moderate epithelial lymphocytic
included in VEGF testing were selected based on the exocytosis, and diffuse stromal inflammation was
presence of both clinical grade 1 pterygium (9 cases) and observed in 29.78% of cases. Prominent epithelial
clinical grade 3 pterygium (8 cases) as a trial to correlate pigmentation was present in 48.9% of cases [Figure 1].
the expression of this marker with the pterygium grade. Stromal solar elastosis was evident in all cases.
Prominent stromal vascularity (vascularity is more
dominant than fibrosis) was observed in 76.6% of cases;
Statistical Analysis
there was no significant difference between vascularity
The clinical and histopathological parameters were and fibrosis in 23.4% of cases. Stromal vascularity
analyzed to determine any correlation between them. was further analyzed by an immunohistochemical
Statistical pre‑coded data were entered into Microsoft study using VEGF in 17 cases that were clinically
Excel 2010 for Windows (Microsoft Corp., Redmond, grade 1 (9 cases) or grade 3 (8 cases), and was found to be
WA, USA). Data were then transferred to IBM SPSS expressed in all lesional blood vessels. The inflammation
Statistics for Windows, Version 23.0 (IBM Corp., was predominantly chronic in 72.3% and mixed in 27.7%
Armonk, NY, USA) for analysis. Quantitative variables of the studied cases. Hemorrhage was present in 76.6%
were presented as the mean and standard deviation (SD); of cases. Hemorrhage with sidrophages was present
qualitative variables were presented as the frequency in 7 cases that were further examined using Perl’s
and percentage. Groups were compared using the stain [Figure 2].
Mann‑Whitney test for quantitative variables and Few histopathological changes were significantly
the Chi square and Fisher’s exact tests for qualitative correlated with clinical redness. They included epithelial
variables. P values < 0.05 were considered statistically hyperplasia (P = 0.039), prominent vascularity over
significant, and values < 0.01 were considered highly fibrosis (P < 0.001), and the chronic inflammatory
significant. response (P < 0.001) [Table 1 and Figure 3]. A correlation
of the extension of the pterygium and of astigmatism
RESULTS with all histopathological variables revealed
a significant reverse correlation with the chronic
This study included 47 patients: 28 males (59.6%) inflammatory response (P = 0.033, P = 0.028, respectively)
and 19 females (40.4%). The ages ranged from 25 to [Tables 2, 3 and Figure 4].
46 years (mean, 34.3 ± 5.3 years). Patients were divided A significant positive correlation was found
into two age groups: 25‑35 years (34 patients, 72.3%) and between astigmatism and the following: pterygium
36‑45 years (13 patients, 27.7%). Occupations included 40 with grade III redness (P = 0.011), pterygium with
outdoor jobs (85.1%) and 7 indoor jobs (14.9%). Thirty‑nine grade 3 corneal extension (P < 0.001), pterygium
patients (83%) had cosmetic complaints; 8 patients (17%) width >4 mm (P < 0.001), and longer pterygium
had visual complaints. The duration of the pterygium duration (P = 0.026) [Table 4].
112 Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018
Clinicopathological Study on Pterygia; Reda et al
a a b
b
c c d e
d
f g
e f
Figure 2. Stromal changes in pterygium. (a) mild perivascular
lymphocytic infiltration, hematoxylin and eosin (h and e)
stain, ×100; (b) diffuse lymphocytic infiltration, h and e, ×100;
(c) prominent stromal solar elastosis, h and e, ×100; (d) stromal
sidrophages, h and e, ×100; (e) sidrophages with positive
iron granules, perl’s stain × 100; (f) positive immunostain
for vascular endothelial growth factor, ×100; (g) positive
g h
immunostain for cd20 at epithelial lymphocytic exocytosis,
Figure 1. Epithelial changes seen in pterygium: (a) goblet ×400.
cell hyperplasia, hematoxylin and eosin (h and e) stain,
×40; (b) epithelial pigmentation, h and e, ×400; (c) epithelial
We noted that an increased duration of pterygium
lymphocytic exocytosis, h and e, ×400; (d) reactive epithelial
atypia, h and e, ×400; (e) immature squamous metaplasia with
is correlated with increased severity of redness, degree
reactive changes, h and e, ×400; (f) mildly dysplastic epithelium, of extension over the cornea, increased width, and
h and e, ×400; (g) p53 positive nuclear stain in basal and some astigmatism. Extension of the pterygium over the cornea
suprabasal keratinocytes in reactive epithelium, ×400; (h) p53 is associated with the degree of severity of redness.
positive nuclear stain in basal and some suprabasal keratinocytes A significant positive association was found between
of reactive immature squamous epithelium, ×400. lesion dimensions over the cornea and vascular density.
The same study showed that pterygium redness
No significant correlation was found between any of and fleshiness were positively correlated with lesion
the examined histopathological features and age, sex, dimensions over the cornea. However, larger lesions over
occupation, or duration of the pterygium. the cornea may be associated with increased thickness
and volume of the pterygium body, resulting in higher
DISCUSSION grades of redness and fleshiness.[8]
We found that astigmatism is correlated with
The present study was designed to determine all pterygium grade III redness, grade 3 corneal extension,
histopathological changes in pterygium in relation width >4 mm, and longer duration. We also found that
to the clinical manifestations. The histopathologic pterygium extension over the cornea had the strongest
changes and clinical features of pterygia may provide correlation to astigmatism. Pterygium extension has
a better understanding of the pathogenesis and a stronger correlation with corneal astigmatism than
reveal additional clues for management strategies[12] with width.
(surgical or non‑surgical) to reduce recurrence, severity Previous work has suggested an association between
of inflammation, tissue invasion, proliferation, and the increasing size of a pterygium and increasing degrees
vascularization.[13] of corneal astigmatism.[14] When a primary pterygium
In our study, 72.5% of pterygium patients were reaches more than 1.0 mm in size from the limbus, it
25‑35 years of age; 27.7% were aged 36‑45. The data induces significant astigmatism. This astigmatism tends
indicate that age is not a contributing factor. Pterygium to increase with increasing lesion size, and is improved
can occur as early as 25 years of age. In this study, by successful removal of the pterygium.[15,16]
59.6% of patients were men and 40.4% were women. This study found many histopathological variables
This finding has been reported in most of the literature, of pterygia. The most important variable is epithelial
indicating a higher incidence of pterygium in males, yet atypia. Many studies discuss this issue.[17] In the present
female predomination has also been reported.[14] study, epithelial atypia was found in 53.2% of cases and
Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018 113
Clinicopathological Study on Pterygia; Reda et al
ranged from reactive atypia to mild dysplasia. The results Epithelial pigmentation was evident in 48.9% of the
concur with Gaton et al,[17] who also found only mild cases in our study. Melanocytic lesions can be related to
dysplasia in a few cases of pterygium (3 of 45, 6.6%). In sun exposure according to Dodd et al[20] Thus, it is not
the cases of this study, P53 immune staining was done strange to find increased melanin pigment in pterygium
on four cases only (with no atypia, with reactive atypia, epithelium that is related to ultraviolet radiation
with immature looking squamous metaplasia, and mild exposure. Moreover, Perra et al[21] studied melanocytic
looking dysplasia). No staining difference was detected pigmented lesions in 80 cases of pterygia and found nine
between the four studied cases, so no more cases were cases of pigmented lesions.
subjected to P53 immune staining. In the present study, both epithelial and stromal
This result concurred with Kang et al,[18] who studied the inflammation were detected, but the inflammatory
effect of P53 on reactive, immature, and dysplastic uterine responses were mild (not prominent) and chronic
cervix squamous epithelium and found limited significant (rather than mixed or acute) in both the epithelium
value in differentiation. Van Der Pols et al,[13] reported and the stroma. This concurred with Nassar et al,[2]
that P53 expression is increased in the basal layer and in who reported an inflammatory response in all studied
the whole epidermis with increased time spent outdoors. pterygium cases; a mild inflammatory response, whether
Chan et al [19] found that pterygium commonly epithelial (84.2%) or stromal (71.8%), was most common.
showed squamous metaplasia (73.2%); hyperplasia Our results were also in agreement with Safi et al,[8] who
was associated with goblet cell hyperplasia (87.5%). detected a significant stromal inflammatory response
This concurred with our results that found epithelial in only 38.6% of their studied cases. No correlation
hyperplasia in 83% of the cases, but our study showed was found between clinical pterygium redness and
less evidence of goblet cell hyperplasia: just 31.9% of the the epithelial or stromal inflammation, even if it was
cases had prominent goblet cells. pathologically very evident.
114 Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018
Clinicopathological Study on Pterygia; Reda et al
Contd...
Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018 115
Clinicopathological Study on Pterygia; Reda et al
Table 3. Contd...
Astigmatism P
>2.5 D (n=8) <2.5 D (n=39)
n % n %
Stromal vascularity
Vascularity overwhelm fibrosis 7 87.5 29 74.4 0.659*
No significant difference 1 12.5 10 25.6
Stromal inflammation
Perivascular 5 62.5 30 76.9 0.403*
Diffuse 3 37.5 9 23.1
Type of inflammatory cells
Chronic (lymphocytes) 3 37.5 31 79.5 0.028†
Mixed acute and chronic (lymphocytes and neutrophils) 5 62.5 8 20.5
Hemorrhage and sidrophages
Present 8 100 28 71.8 0.170*
Absent 0 0 11 28.2
*Indicates the difference is not significant; †indicates the difference is significant
a b
a b
Figure 4. (a) pterygium extension grade 3, clinical case;
(b) the significant histopathological feature was mixed
chronic (lymphocytes and plasma cells) and acute (neutrophils)
c d inflammatory cells, hematoxylin and eosin stain, ×400.
Figure 3. (a) pterygium redness grade III, clinical case;
(b) significant histopathological features including prominent to use anti‑VEGF as target therapy for pterygium. [2,23,24]
vascularity overwhelming fibrosis, hematoxylin and Hoshino et al[25] reported that VEGF has a narrow target
eosin (h and e) stain, ×100; (c) prominent squamous epithelial cell range and is specific for endothelial cells, because
cell hyperplasia, h and e, ×100; and (d) lymphocytic (chronic)
VEGF binds to the high‑affinity cell surface receptors
stromal inflammatory response, h and e, ×400.
KDR/Flk and Flt, which are predominantly expressed
by endothelial cells. This concurred with our results,
Sankar et al[22] found that all pterygium cases showed
which showed positive staining for VEGF in the wall of
elastoid basophilic degeneration. This was in agreement the blood vessels of all studied lesions (17 cases). These
with our results, as solar elasosis was evident in all of our selected cases presented grade 1 pterygium (9 cases) and
studied cases. They also found that this solar elastosis is grade 3 pterygium (8 cases). There was no significant
an important diagnostic histopathological clue for any difference between VEGF expression and the pterygium
pterygium case. grade.
Stromal vascularity of a pterygium is an important Hemorrhage with/without sidrophages in the
and promising therapeutic target. In the present study, pterygium was observed in 76.6% of cases. Recurrent
prominent vascularity that overcame fibrosis was hemorrhage in a pterygium may elicit a chronic
evident in most of the studied cases (76.6%), and the inflammatory response. This may explain the dominance
remaining cases showed both vascularity and fibrosis of the chronic inflammatory response in most of our
with significant clinico‑pathological correlation. studied cases (72.3%).
Thus, pterygium vascularity can be used as a target Clinical redness was significantly correlated with
for treatment, especially for patients with cosmetic histopathological vascular content, as vascularity
complaints. A few years ago, many studies attempted overwhelmed fibrosis in grade II (79.3%) and in
116 Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018
Clinicopathological Study on Pterygia; Reda et al
Journal of Ophthalmic and Vision Research Volume 13, Issue 2, April-June 2018 117
Clinicopathological Study on Pterygia; Reda et al
23. Besharati MR, Manaviat MR, Souzani A. Subconjunctival Pterygium. Korean J Physiol Pharmacol 2015;19:357‑363.
bevacizumab injection in treatment of pterygium. Acta Med Iran 25. Hoshino M, Takahashi M, Aoike N. Expression of vascular
2011;49:179‑183. endothelial growth factor, basic fibroblast growth factor, and
24. Park YM, Kim CD, Lee JS. Effect of Bevacizumab on Human angiogenin immunoreactivity in asthmatic airways and its
Tenon’s Fibroblasts Cultured from Primary and Recurrent relationship to angiogenesis. J Allergy Clin Immunol 2001;107:295‑300.
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