Study of biomarkers p53, Ki-67, Bcl-2, and VEGF in pterygium

Abstract

Purpose:

To study the biomarkers present in primary pterygium samples of patients of Indian ethnicity and compare it with the samples obtained from the unaffected conjunctiva of the same eye.

Methods:

A prospective case-control study of 17 eyes in patients above 10 years of age with primary pterygium who underwent pterygium excision using limbal conjunctival autograft technique. The pterygium samples (cases) and conjunctival samples (controls) were sent for immunohistochemical (IHC) staining for the following biomarkers: p53, Bcl-2, Ki-67, and vascular endothelial growth factor (VEGF).

Result:

The immunohistochemistry of the samples and the controls revealed p53 positivity in 47.05% of pterygium samples and 29.4% of controls (P < 0.587). Nine cases each in pterygium and control samples were positive for Ki-67 expression. Differences in the staining pattern between the two groups were not statistically significant (P < 1.000). Bcl-2 positivity was seen in 10 pterygium samples (58.8%) and 12 controls (70.5%), with no statistical difference between the two groups (P < 0.455). VEGF expression was seen in both epithelial and endothelial cells of the samples and controls, with no statistical difference between the two groups, with P = 1.000 for the epithelial staining and P = 0.637 for endothelial staining.

Conclusion:

The expression of biomarkers was comparable in both groups. We conclude that pterygium, against common belief, might not be a localized disease process but a global ocular phenomenon where the apparently healthy tissue also has some ongoing disease process at a molecular level.

Pterygium is a fibrovascular growth of the conjunctiva encroaching onto the cornea and is a common disorder of the ocular surface.[1,2] It not only affects the cosmetic outlook of the patient but also affects refractive astigmatism and is a potentially blinding disease in the advanced stage due to invasion of the visual axis, which can have a significant impact on vision and may require surgery for visual rehabilitation.[3]

The exact etiology and pathogenesis of pterygium remains unclear. It has been classified as a benign proliferative lesion as well as a neoplastic-like growth disorder owing to the presence of tumor-like characteristics such as altered progenitor cells,[4] loss of cell polarity,[5] corneal invasiveness, matrix remodeling,[6,7] epithelial cell motility,[4,8] and a high recurrence rate with aberrant proliferation.[9] Some researchers postulate that ultraviolet (UV) is a trigger for its development through limbal epithelial stem cell damage and the upregulation of multiple pro-inflammatory cytokines, growth factors, and matrix metalloproteinases.

The standard care of pterygium consists of surgical excision of the tissue followed by covering it with a conjunctival autograft or an amniotic membrane.[10,11] Pterygium surgery is complicated by its high postoperative recurrence rate of up to 89%, and its severity may vary according to the adopted approach and preoperative conditions.[12]

Various biomarkers, such as the tumor suppressor gene p53,[13,14] Bcl-2 gene (which has a role in the inhibition of apoptosis),[15] Ki-67 protein (an important marker in determining cell proliferation),[15] and vascular endothelial growth factor (VEGF) acting on the vascular endothelial cells (which stimulates growth of neovascularization),[16] have been studied and thought to play an important role in the genesis and recurrence of pterygium.

The study of these biomarkers not only provides new insights into the pathways and molecular mechanisms of pterygium development but may also pave way for novel treatment modalities and targeted therapy for pterygia and recurrence prevention.

The current study aims to study the biomarkers present in primary pterygium samples of patients of Indian ethnicity and compare them with the samples obtained from the unaffected conjunctiva of the same eye.

Methods

A case-control study was conducted on 17 eyes with primary pterygium in patients above 10 years of age attending the cornea services at the tertiary care ophthalmic service in New Delhi. The following grading system was used:

• Grade 0: Pingeculum, posterior to the limbus

• Grade 1: Tissue involvement to the limbus

• Grade 2: Tissue just onto the limbus

• Grade 3: Tissue between the limbus and the pupillary margin

• Grade 4: Tissue central to the pupillary margin.

Cases of recurrent pterygium were excluded from the study. The study period was 12 months. Approval from the institutional ethics committee was taken before starting the study. The study was registered with the Trial Registry of India (CTRI/2022/01/039609) and followed the tenets of the Declaration of Helsinki. Informed consent was obtained from the subjects after explanation of the nature of the study.

A comprehensive ophthalmological workup of both eyes was done, which included a detailed slit-lamp examination to measure the size of pterygium (length and width), conjunctival, and corneal vascularization. Assessment of visual acuity, measurement of the thickness of pterygium over cornea and conjunctiva by AS-OCT (anterior segment optical coherence tomography, REVO60, OPTOPOL technology), and intraocular pressure was also performed in all the patients preoperatively.

Procedure

All cases were primary pterygium cases in the nasal quadrant. The majority of the samples were grades 2–3 as per the grading system mentioned before. Pterygium excision with the limbal conjunctival autograft technique was performed under peribulbar anesthesia by the same surgeon.

Approximately 0.5 mL of saline was injected under the belly of the pterygium by using a 26-G needle mounted on a 2-mL syringe. The head of the pterygium was avulsed from its attachment at the cornea by reverse stripping by using slow and deliberate traction holding its free end parallel to the cornea. The fibrovascular tissue underneath the cut end of the conjunctiva was dissected and excised, leaving bare sclera and the muscle free from the episcleral tissue.

Donor conjunctiva harvested from the superotemporal quadrant of the same eye was 4 mm × 4 mm larger than the size of the bare sclera; 3 mm × 3 mm of this donor conjunctiva was sent for IHC to serve as control. The dissection was continued at the donor site for approximately 1 mm into the peripheral cornea, beyond the vascular arcade to include the limbal tissue. The conjunctival piece was excised using a sharp Vanna’s scissors. The graft was then transferred to the bare sclera, with epithelial side up, preserving the limbal orientation, and secured with 8-0 vicryl interrupted sutures. The donor site was sutured. At the end of the surgery, antibiotic-steroid ointment was applied to the conjunctival sac.

Immunohistochemistry

Samples were fixed in 10% formalin, processed, and embedded in paraffin. All samples were sectioned at 3-μm thickness and mounted on positively charged glass slides labeled with barcodes after sectioning and fixed overnight at 37°C for immunohistochemical (IHC) staining. After deparaffinization in alcohol, samples were washed in distilled water. These sections were stained with hematoxylin and eosin for histopathological evaluation.

IHC staining for p53, Bcl-2, VEGF, and Ki-67 was performed on 17 primary pterygium (cases) and 17 healthy conjunctiva tissue samples (controls) from the same eye. IHC using the streptavidin–biotin peroxidase method was performed on paraffin-embedded tissues, and antigen retrieval was achieved by heat retrieval methods by using Tris Buffer (10 mM Tris base, 0.05% Tween20, pH 10).

Samples were incubated with the following antibodies for 60 min at 25°C. The primary antibodies used were as follows:

1. p53 Mouse Monoclonal Antibody dilution 1:100 (Clone BP-53-12, (PathnSitu, Livermore, CA, USA)

2. Ki-67 (Clone: MIB1) Mouse Monoclonal Antibody dilution 1:100 (PathnSitu, Pleasanton, CA, USA)

3. Bcl-2 alpha AB1; (clone 100/D5, Thermo Scientific, Kalamazoo, USA)

4. VEGF RTU (ready to use),7 mL, (Clone RBT, BIOSB, SantaBarbara, CA, USA).

A light microscope (OLYMPUS BX53) was used, and the immune-positive cells were examined manually. Staining intensity was graded in the following manner for all the biomarkers: 0 –negative, 1+ – weak, 2+ – intermediate, and 3+ – strong staining.

1. p53 positivity: The basal epithelial cells were counted. At least 100 cells of tissue (including immune-positive cells) in three microscopic fields (magnification 100×) from the sample were studied. The expression of the p53 protein was marked positive if it was present in more than 10% of tissue cells and negative if present in less than 10% of cells.

2. Ki-67 positivity: The basal epithelial cells were counted, and positive staining cells of five microscopic fields (400×) from each tissue was performed. The positive staining cells were noted by their labeling index as a percentage in each specimen, and the measurements were averaged.

3. Bcl-2 positivity: The basal and parabasal layer of epithelial cells were observed at 100× for any positive staining. Staining intensity and the percent positive cells were graded on a scale of 0–3 as mentioned before.

4. VEGF expression: VEGF expressivity either in the epithelial or endothelial layer or both in the samples was assessed. Staining intensity and the percent positive cells were graded.

Statistical analysis

The Statistical Package for Social Sciences (SPSS) PC 21version was used for data analysis. Fisher’s exact test was used to calculate the association and its significance between categorical variables, for example, case/control and positivity. Because the sample size was small, the P value was considered significant at the 10% (0.10) level. Wherever enough samples were available, odds ratios were calculated for risk of exposure among cases as compared to controls.

Result

The immunohistochemistry of the samples and controls revealed p53 positivity (The expression of p53 protein was marked positive if it was present in more than 10% of tissue cells and negative if present in less than 10% cells.) in 47.05% (8 out of 17) pterygium samples [Fig. 1a] and 29.4% (5 out of 17) controls [Fig. 1b] (P < 0.587) [Table 1].

Figure 1.

(a) IHC staining of p53-positive pterygium specimen at 200×. (b) IHC staining of control specimen with p53 showing less than 10% positivity

Table 1.

Comparison between p53 positivity in pterygium samples and controls

Mean positive cells (p53)
Staining pattern		Case				Control
		n		%		n		%
1+		2		25		0		0
2+		2		25		2		40
3+		4		50		3		60
Total		8		47.05		5		29.4

Nine out of 17 (52.9%) cases [Fig. 2a] and control samples [Fig. 2b] were positive for Ki-67 expression. The difference in the staining pattern between the two groups was not statistically significant (P < 1.000) [Table 2].

Figure 2.

(a) IHC staining with Ki‐67 labeling antibody of a section of Ki‐67-positive pterygium at 200×. (b) IHC staining of Ki‐67-negative control sample

Table 2.

Comparison between Ki-67 positivity in pterygium samples and controls

Mean positive cells (Ki-67)
Case				Control
n		%		n		%
9		52.9%		9		52.9%

Bcl-2 positivity was seen in 10 out of 17 pterygium samples (58.8%) [Fig. 3a] and 12 out of 17 controls (70.5%) [Fig. 3b], with no statistical difference between the two groups (P < 0.455) [Table 3].

Figure 3.

(a) IHC staining of Bcl-2-positive epithelial cells of pterygium specimen at 200×. (b) IHC staining with Bcl-2 antibody showing negative staining

Table 3.

Comparison between Bcl-2 positivity in pterygium samples and controls

Mean positive cells (Bcl-2)
Staining pattern		Case				Control
		n		%		n		%
1+		1		10		0		0
2+		0		0		0		0
3+		9		90		12		100
Total		10		58.8		12		70.5%

VEGF expression was seen in both epithelial [Table 4a] and endothelial cells [Table 4b] of the samples [Fig. 4a] and controls [Fig. 4b], with no statistical difference between the two groups, with P = 1.000 for the epithelial staining and P = 0.637 for endothelial staining.

Table 4a.

Comparison of VEGF positivity in epithelial cells. Cells showing both epithelial and endothelial positivity have been excluded from the table

Epithelial cell positivity		Case				Control
		n		%		n		%
≤2+		2		25		2		50
>2+		6		75		2		50
Total		8		47.0		4		23.5

Table 4b.

Comparison of VEGF positivity in endothelial cells. Cells showing both epithelial and endothelial positivity have been excluded from the table

Endothelial Cell positivity		Case				Control
		n		%		n		%
≤2+		3		60		4		57.1
>2+		2		40		3		42.8
Total		5		29.4%		7		41

Figure 4.

(a) IHC analysis of VEGF in both epithelial (green arrow) and endothelial cells (black arrow) at 200×. (b) IHC staining showing weak staining in epithelial and endothelial cells

Discussion

Pterygium is regarded as a degenerative condition with multifaceted pathology that displays apparent conflicting characteristics. It is considered benign because of its self-limiting and superficial nature; however, because of its highly proliferative and potentially recurrent nature, it has been said to possess signs of preneoplastic transformation.

Various opinions regarding the mechanism of pterygium development have been discussed in the literature. However, the exact pathogenesis is still a dilemma. Chronic inflammation occurring in the limbal conjunctival vessels or conjunctival epithelium is thought to be one of the factors in the pathogenesis.[17]

Pathophysiology of pterygium due to exposure to UV rays is perhaps the most accepted risk factor for the occurrence of pterygium.[2,18] There is a void in the existing literature regarding the pathophysiology of pterygium and its overlap with other malignant conditions (ocular surface squamous neoplasia and skin cancer). The initial insult with UV rays ensues a cycle of indirect DNA damage as a part of the repair mechanism. There also occurs downstreaming of apoptotic regulators along with resultant cellular adaptations.[19]

Various inflammatory and apoptotic biomarkers have been studied in the literature to help understand the molecular level of the occurrence and recurrence of pterygium. A study of these biomarkers can help with targeted therapy in pterygium patients and prevent recurrence.[20,21]

The purpose of the current study was to ascertain p53, Ki-67, Bcl-2, and VEGF expression in pterygium and healthy conjunctiva of the same eye and if this could further help in the development of targeted therapy. The ethical clearance for the study of biomarkers from the conjunctiva of the normal/healthy eye was refused by the institutional ethics committee.

Spandidos et al.[22] in their study concluded that there is a role for tumor suppressor genes and decreased fidelity in DNA replication and repair in the development of pterygium.

p53 is a tumor suppressor gene that controls the cell cycle and is involved in DNA repair and synthesis, cell differentiation, and apoptosis. The p53 protein can be identified by IHC staining by using a monoclonal antibody against it. Normal cells are negative for the stain as the protein concentration is very low due to the short half-life of the protein (6–20 min). In many types of neoplastic cells, its concentration is higher and IHC staining for the protein is positive.[23,24]

Tan et al.[25] performed IHC staining on eight pterygia specimens and found three of them (37.5%) to be positive for abnormal expression of p53. Dushku and Reid[13] found increased nuclear p53 in the limbal epithelium of pterygia, limbal tumors, and pinguecula and suggested that the existence of p53 mutation in the cells is an early event in the development of pterygium, probably as a result of UV radiation exposure. Onur et al.[14] found that only 7.9% out of the 38 patients had a few p53-stained cells. Chowers et al.[16] in their study on primary, recurrent pterygia and controls concluded that mutation in the p53 gene is not crucial for pterygium formation and recurrence.

Our study observed a higher p53 expression in primary pterygium at 47.05% as compared to 29.4% positivity in normal conjunctiva [Table 1], with the majority of cells in both groups showing 3+ positivity; however, it was not statistically significant, which is in concurrence with the above review of the literature.

Ki-67 plays a role in all of the active phases (G1, S, G2, and M) of the cell cycle; it is not related to the resting cells (G0). Thus, the proliferative cellular activity can be evaluated by the detection of Ki-67 nuclear protein.[26,27]

Mahesh et al.[28] studied the correlation between p53 and Ki-67 expression with the severity and duration of the pterygium on 43 patients and found that the expression of cell proliferation marker Ki-67 increases with the duration of pterygium and p53 expression increases with the duration and severity of pterygium. A study by Liang K et al.[29] showed the presence of Ki-67 expressivity in pterygium as well as normal samples; however, no grading of staining intensity was done.

In our study, both pterygium and control in our study showed weak staining on Ki-67 in equal numbers (52.9%) in each group. A greater number of cases with >5% positivity of Ki-67 index in pterygium was observed compared to controls. The correlation between p53 and Ki-67 was not studied by us.

Bcl-2 belongs to the family of apoptosis regulatory proteins, which can induce or inhibit apoptosis. Bcl-2 is primarily expressed in the basal epithelial layer of all pterygium epithelial cells and normal conjunctiva as well. However, the ratio of Bcl-2 to Bcl-associated X protein (BAX, an apoptosis regulatory protein) is believed to determine the fate of the cell.[29]

There was higher expression of Bcl-2 in normal conjunctiva (70.5%) compared to pterygium (58.8%), which was not statistically significant (P < 0.4). However, BAX, which is the apoptosis regulatory protein, has not been evaluated in our study, and hence the ratio of Bcl-2 to BAX cannot be commented upon.

Turan M and Turan[30] showed significantly increased p53, Bcl-2, and Ki-67 expression in primary and recurrent pterygiums when compared to normal conjunctiva of the eye, with no disease process in play (P < 0.001). Suren E et al.[19] demonstrated an increased expression of p53, Bcl-2, and Ki-67 levels in pterygium samples compared to normal conjunctiva obtained from patients posted for strabismus surgery.

Our study, however, was not granted ethical clearance for the use of conjunctiva of the healthy eye without any disease process for control samples.

VEGF plays an important role in the angiogenesis and neovascularization in the pathogenesis of pterygium. It has been demonstrated on IHC in the epithelial and endothelial cells of pterygium samples by Bianchi et al.[31] They reported a statistically significant difference in staining pattern among pterygium and normal conjunctiva, with increased VEGF expression in the epithelial (72.9%), stromal (87.80%), and vascular endothelium (72.9%) of pterygium samples. However, controls comprised only two cadaveric specimens that they believed to have an altered physiology.

The study had assumed that it was impossible to use control specimens from normal conjunctiva adjacent to pterygium as it may also have potential abnormalities, which was evident in our study where VEGF expression was observed in all of the pterygium (100%) and 82.3% of control samples.

Aspiotis M et al.[32] concluded that normal conjunctiva shows intense expression of VEGF in epithelial cells. However, this is not capable of inducing angiogenesis because of low corresponding mean vascular density (MVD) values. Our study showed VEGF expression exclusively in the epithelial cells in 23.5% of the control samples, however these cells were not capable of any neovascularization on their own.

Conclusion

There is a lot of variability in biomarkers expression of pterygium in various studies as controls used were variable in different studies. Most of the reviewed literature either did not have a control group or it was too small when compared to the pterygium group or was taken from the healthy eyes of other or the same patient. Hence, a definite conclusion regarding the expression of biomarkers in pterygium is difficult to draw.

Our study differs in that the control group was taken from the apparently healthy quadrant of the affected eye, with the expression of biomarkers from pterygium and healthy unaffected conjunctiva being comparable. This leads us to conclude that pterygium, against common belief, might not be a localized disease process but a global ocular phenomenon where the apparently healthy tissue also has some ongoing disease process at a molecular level as apparent from no significant difference in the staining patterns from pterygium or apparently normal conjunctiva of the same eye.

Financial support and sponsorship

Self funded.

Conflicts of interest

There are no conflicts of interest.