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Exploring the significance of EGFR, MMP-2, MMP-9, HIF-1α, and CAIX as molecular biomarkers in ocular surface squamous neoplasia
For correspondence: Dr Subramanian Krishnakumar, Department of L&T Ocular Pathology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai 600 006, Tamil Nadu, India e-mail: drkk@snmail.org
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Received: ,
Accepted: ,
How to cite this article: Ramachandran RG, Parameswaran S, Rajagopal R, Biswas J, Krishnakumar S. Exploring the significance of EGFR, MMP-2, MMP-9, HIF-1α, and CAIX as molecular biomarkers in ocular surface squamous neoplasia. Indian J Med Res. 2026;163:456-60. doi: 10.25259/IJMR_2438_2025
Abstract
Background and objectives
Ocular surface squamous neoplasia (OSSN) encompasses a spectrum of tumours, including conjunctival squamous intraepithelial neoplasia (CSIN), carcinoma in situ (CIS), and invasive squamous cell carcinoma (iSCC). The roles of biomarkers such as epidermal growth factor receptor (EGFR), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), hypoxia-inducible factor-1α (HIF-1α), and carbonic anhydrase IX (CAIX) in OSSN progression are incompletely understood. This study aimed to investigate their expression patterns in Asian Indian OSSN tumours.
Methods
This observational immunohistochemistry (IHC) study was conducted on 21 formalin-fixed, paraffin-embedded tissue specimens from patients with CSIN, CIS, or iSCC. OSSN tumours were categorised into Group A (CSIN and CIS) and Group B (iSCC). Immunohistochemical staining was used to assess the expression of EGFR, MMP-2, MMP-9, HIF-1α, and CAIX. Statistical analysis was performed to determine significant differences in biomarker expression between the groups.
Results
Significantly higher expression of EGFR (P=0.03), MMP-2 (P=0.02), and CAIX (P=0.03) was observed in Group B (iSCC) compared to Group A (CSIN and CIS). MMP-9 expression did not differ significantly between the groups (P=0.14). HIF-1α expression in tumour cells was also not significantly different (P=0.41). HIF-1α expression in inflammatory cells was significantly higher in Group B (iSCC) compared to Group A (P=0.038).
Interpretation and conclusions
This study suggests that EGFR, MMP-2, and CAIX may play a role in OSSN progression from CSIN/CIS to iSCC. Elevated HIF-1α expression in inflammatory cells highlights the potential involvement of inflammation in OSSN development. These biomarkers may serve as potential therapeutic targets and could also be integrated into diagnostic tools for OSSN.
Keywords
Epidermal growth factor receptor
Hypoxia-inducible factor-1α
Matrix metalloproteinase-2
Matrix metalloproteinase-9
Ocular surface squamous neoplasia
Ocular surface squamous neoplasia (OSSN) represents a spectrum of neoplastic lesions that can arise on the cornea and conjunctiva. Risk factors for OSSN include ultraviolet-B radiation, ocular surface injury, cigarette smoking, human papillomavirus (HPV16–18), xeroderma pigmentosum, and human immunodeficiency virus (HIV).1,2 These factors contribute to the development of OSSN by promoting genetic mutations and cellular dysregulation, leading to a range of lesions from conjunctival squamous intraepithelial neoplasia (CSIN) with varying severity to carcinoma in situ (CIS) and, ultimately, invasive squamous cell carcinoma (iSCC).
Various imaging modalities, such as high-resolution optical coherence tomography, in vivo confocal microscopy, and ultrasound biomicroscope, are increasingly employed to support the clinical diagnosis of OSSN and to monitor treatment response.3-6 Surgical excision remains the definitive management for OSSN tumours; however, accurate subtyping through biopsy and histopathological evaluation is critical, particularly for aggressive variants like mucoepidermoid carcinoma and adenosquamous cell carcinoma, which have the potential to invade intraocular structures and metastasize.7 Prior studies have implicated matrix metalloproteinases (MMPs) and epidermal growth factor receptor (EGFR) in the progression of conjunctival squamous cell carcinomas, indicating their potential as targets for therapy.8-13 Notably, EGFR overexpression has been associated with poorer clinical outcomes in several cancers.
In this study, we examined the protein expression of EGFR, MMP-2, MMP-9, hypoxia-inducible factor-1α (HIF-1α), and carbonic anhydrase IX (CAIX) in OSSN, with a focus on Asian Indian tumours. The inclusion of HIF-1α and CAIX was motivated by their established roles in tumour growth and metastasis.14,15
Methods
This observational study was undertaken by the Department of L&T Ocular Pathology, Vision Research Foundation, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Chennai, Tamil Nadu, India. This study adhered to the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Vision Research Foundation.
Study design
This was an observational immunohistochemistry (IHC) study on archived formalin-fixed paraffin-embedded (FFPE) specimens collected between 2011 and 2019. All IHC staining and masked dual-observer scoring were performed in 2024–2025 using a single standardised protocol and unified detection platform.
Sample collection criteria
(i) Inclusion: histopathology-confirmed OSSN; adequate FFPE tumour tissue; complete slides/blocks available; no prior topical/systemic anti-neoplastic therapy before excision. (ii) Exclusion: non-OSSN ocular surface tumours; severely autolysed/insufficient tissue; prior neoadjuvant therapy; duplicate specimens from the same episode. (iii) Selection: consecutive eligible archival cases from 2011–2019 were screened; 21 unique cases met all criteria and had sufficient material for the full IHC panel and scoring. Groups A (CSIN/CIS) and B (iSCC) were defined by histopathological invasion status (as described).
OSSN sample details
The samples were categorised into two groups based on histopathological assessment of stromal invasion, local spread, or metastasis. Group A (n=7) consisted of OSSN cases with varying grades of dysplasia, ranging from mild, moderate, and severe conjunctival intraepithelial dysplasia to carcinoma in situ (CIS). Group B (n=14) comprised OSSN cases with invasive squamous cell carcinoma (iSCC). Within Group B, two cases of adenosquamous cell carcinoma exhibited intraocular extension and were derived from exenterated samples, while one case of mucoepidermoid carcinoma presented with regional lymph node metastasis. Clinicopathological details of patients in each group, including age, sex, and tumour characteristics, Immunoreactive Score (IRS)are summarised in Supplementary Tables I and II.
Immunohistochemistry techniques
Immunohistochemistry was employed to evaluate the expression levels of EGFR, MMP-2, MMP-9, HIF-1α, and CAIX in the FFPE tissue blocks. Sections were deparaffinised, rehydrated, and subjected to antigen retrieval based on established protocols. Commercially available mouse or rabbit monoclonal antibodies (detailed in Supplementary Table III) were used, and IHC staining was performed using a Ventana automated IHC stainer, following the manufacturer’s instructions.
Two senior ocular pathologists, blinded to the clinicopathological data, independently evaluated the immunostaining. The IRS system was used to quantify biomarker expression. The IRS score was determined by multiplying the percentage of positive tumour cells (0: negative,1=<10%,2=10% to 50%, 3=51% to 80%,4=>80%) by the staining intensity (0: negative, 1: weak, 2: moderate, 3: strong).14 The final IRS score ranged from 0 to 12. For HIF-1α, the IRS was assessed separately in tumour cells and in inflammatory cells within the tumour microenvironment. Any discrepancies in scoring between the two pathologists were resolved by consensus review.
Statistical analysis
Statistical analyses were performed using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics version 29.0.1.0 (IBM Corp., Armonk, NY, USA). Results are presented as the mean±standard deviation (SD) of the IRS score. The Mann-Whitney U test was used to compare IRS scores between Group A and Group B. A P value of less than 0.05 was considered statistically significant.
To assess interobserver variability in immunohistochemical scoring, the strength of agreement between the two pathologists was evaluated using weighted Kappa statistics. Kappa values were interpreted as follows: ≤0.20, poor agreement; 0.21–0.40, fair agreement; 0.41–0.60, moderate agreement; 0.61–0.80, good agreement; and 0.81–1.00, excellent agreement.
Results
EGFR expression
EGFR expression was significantly higher in Group B (iSCC) compared to Group A (CSIN and CIS) Supplementary Figure 1A. Immunohistochemical analysis revealed intense EGFR staining in the cytoplasm and plasma membrane of tumour cells in iSCC cases (Supplementary Fig. 1 B and C), with a mean±SD IRS scores of 7.3±3.3 compared to 4.4±2.5 in CSIN and CIS cases (P=0.03).
MMP-2 and MMP-9 expression
MMP-2 expression was significantly elevated in Group B (iSCC), with a mean±SD IRS score of 8.7±3.4 compared to 5±2.7 in Group A (P=0.02) (Supplementary Fig. 2A). MMP-2 staining was observed in both the nucleus and cytoplasm of atypical epithelial cells, as shown in Supplementary Figure 2 B and C. MMP-9 expression did not show a statistically significant difference between the groups (P=0.14). Its staining pattern was faint and heterogeneous, detected primarily in carcinoma cells, endothelial cells, and macrophages (Supplementary Fig. 3 A-C).
HIF-1α expression
HIF-1α expression in tumour cells did not differ significantly between Group B (mean±SD IRS score: 2.3±1.9) and Group A (mean IRS±SD score: 2.9±1.8; P=0.41). However, HIF-1α expression in inflammatory cells was significantly higher in Group B (mean±SD IRS score: 5.3±3.3) compared to Group A (mean±SD IRS score: 2.3±1.4; P=0.038). Supplementary Figure 4A shows weaker staining in CSIN/CIS cases, consistent with their respective IRS scores. Supplementary Figure 4B and C illustrate this upregulation in inflammatory cells within iSCC cases.
CAIX expression
CAIX expression was confined to the cell membrane of tumour cells and was significantly higher in Group B (mean±SD IRS score: 3.8±2.3) compared to Group A (mean±SD IRS score: 1.6±0.5; P=0.03). No detectable staining was observed in stromal or inflammatory cells. Supplementary Figure 5A shows weaker expression in CSIN/CIS, consistent with their respective IRS scores. Supplementary Figure 5B and C demonstrate intense CAIX staining in iSCC cases.
Interobserver agreement
The weighted kappa analysis demonstrated excellent agreement between observers for all markers assessed: EGFR (0.915), MMP-2 (0.943), MMP-9 (0.901), HIF-1α (1.0), and CAIX (1.0). This consistency underscores the reliability of the immunohistochemical scoring system used.
These findings collectively suggest that EGFR, MMP-2, HIF-1α (inflammatory cells), and CAIX may play critical roles in OSSN progression from CSIN/CIS to iSCC, while MMP-9 appears to have limited relevance as a distinguishing biomarker for these stages.
Discussion
This study highlights the potential roles of EGFR, MMP-2, MMP-9, HIF-1α and CAIX in the progression of ocular surface squamous neoplasia (OSSN) from conjunctival squamous intraepithelial neoplasia (CSIN) to invasive squamous cell carcinoma (iSCC). The higher expression of EGFR, MMP-2 and CAIX in iSCC compared with CSIN/CIS supports their association with tumour invasion, aggressive behaviour and treatment resistance, is in line with observations in other epithelial cancers.8-13,14-22 Although HIF-1α expression in tumour cells did not differ between groups, its stronger signal in inflammatory cells in iSCC suggests a hypoxia–inflammation axis that may contribute to OSSN progression.23
The differential expression of EGFR, MMP-2, MMP-9 and CAIX underscores their potential utility as adjunctive biomarkers for identifying higher-risk OSSN and refining histopathological risk stratification.24-26 EGFR, MMPs and CAIX are already being targeted in systemic malignancies with agents such as erlotinib, gefitinib, marimastat and CAIX-directed antibodies including girentuximab.27-31 Their overexpression in OSSN raises a plausible rationale for carefully exploring drug repurposing in selected, refractory ocular tumours. The predominant HIF-1α signal in inflammatory cells further supports targeting the microenvironment; this pattern may partly align with the clinical effectiveness of topical 5-fluorouracil, which combines antimetabolite and secondary anti-inflammatory effects.32
This study has limitations. The cohort is modest, with few intraepithelial lesions (Group A, n=7), and tissues were accrued between 2011 and 2019. Although all immunohistochemistry was performed recently using a protocol-harmonised approach on archival FFPE samples, changes in OSSN epidemiology and treatment patterns over time cannot be excluded. These findings should therefore be regarded as hypothesis-generating and require validation in larger, series; a 2020–2025 cohort using the same scoring framework is in progress.
In conclusion, the differential expression of EGFR, MMP-2, MMP-9, HIF-1α and CAIX in OSSN supports the use of an IHC-based panel to flag tumours that may warrant wider excision margins, adjuvant topical chemotherapy and closer surveillance, thereby directly informing day-to-day clinical decisions in patients at higher risk of recurrence or progression.
Author contributions
RGR: Immunohistochemistry experiments, data compilation, statistical analysis, interpretation of results, manuscript writing; SP: Immunohistochemistry techniques, data analysis, statistical evaluation, compilation of study data, manuscript writing; RRMS: Clinical evaluation, diagnosis, procurement of ocular surface squamous neoplasia (OSSN) clinical samples; JB: Histopathological review of tissue sections, interpretation of pathology findings, review of immunohistochemistry results; SK: Conceived and designed the study, secured funding support, supervised the project, reviewed the data, manuscript writing. All authors have read and approved the final printed version of the manuscript
Financial support and sponsorship
This study was supported by the Department of Biotechnology, Government of India (BT/PR26757/NNT/28/1429/2017).
Conflicts of Interest
None.
Use of Artificial Intelligence (AI)-Assisted Technology for manuscript preparation
The authors confirm that there was use of AI-assisted technology for language editing.
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