Trefoil Family Factor 1 Expression in the Indian Cohort of Treatment-Naïve Retinoblastoma

Abstract

Introduction

Retinoblastoma (RB) is the most common intraocular malignancy of childhood. Advanced RB has a poor prognosis and is associated with high-risk histopathological factors (HRPFs), metastasis, and relapse. Trefoil family factor 1 (TFF1), a secreted peptide previously associated with adverse clinicopathological features in RB, has emerged as a potential biomarker of aggressive disease; however, its clinical significance in Indian patients remains poorly understood. This study evaluated TFF1 expression in correlation with HRPFs, metastasis, and survival outcomes.

Methods

This study included 40 treatment-naïve patients with RB who underwent primary enucleation as their initial management at a tertiary ocular oncology center. Immunohistochemistry was performed to assess TFF1 expression, which was quantified using a quick score (QS) ranging from 0 to 300, based on staining intensity and percentage of positive cells. Tumors were classified into low-expression (HS1; QS < 50) groups and high-expression (HS2; QS ≥ 50) groups. Associations between QS and high-risk pathological features, metastasis, and survival outcomes were analyzed.

Results

Among 40 patients, 45% were classified as HS2 and 55% as HS1. HRPFs were identified in 55% (22/40) of cases. HRPF positivity was more frequently observed in HS2 tumors (57.1%) than HS1 tumors (42.9%), although the difference did not reach statistical significance (p = 0.105). Massive choroidal invasion (>3 mm) was more frequently observed in HS2 tumors (38.9%) than HS1 tumors (13.6%) (p = 0.14). Post-laminar optic nerve invasion was higher in HS2 tumors (22.2% vs. 13.6%; p = 0.498). Metastasis, tumor recurrence, and disease-related mortality occurred exclusively in the HS2 subgroup (2/2, 100%; p = 0.196), although these associations were not statistically significant. Poorly differentiated ([5/8], 62.5%) and ([2/2], 100%) (p = 0.136) undifferentiated tumors were more frequently observed in HS2 cases, although no statistically significant association with clinicopathological features was identified.

Conclusion

Higher TFF1 expression was associated with high-risk factors, including massive choroidal invasion, post-laminar optic nerve invasion, metastasis, recurrence, and disease-related mortality. Although these associations did not reach statistical significance, the observed trends suggest that TFF1 may contribute to the biological stratification of RB and warrant further evaluation in larger cohorts.

Introduction

Retinoblastoma (RB) is the most common pediatric ocular malignancy originating from cone precursor cells within the retina. The primary cause of malignant transformation of these cells in the developing retina is biallelic inactivation of the RB1 tumor suppressor gene [1], and in about 2% of cases with intact RB1 genes, MYCN amplification can occur. Although RB is highly curable in high-income settings, nearly 50% of affected children in low-resource regions still succumb to metastatic disease [2]. Lack of awareness, delayed diagnosis, limited access to specialized healthcare, and socioeconomic constraints often result in late-stage presentation, the need for enucleation, and poorer outcomes in such countries. Advanced RB is characterized by high-risk histopathological factors (HRPFs), extraocular tumor extension, metastatic spread, or recurrence. HRPFs include uveal invasion, invasion of the optic nerve into the lamina cribrosa, retro-laminar region, or at transection, scleral infiltration, and extra-scleral extension [3]. Identification of features of choroidal invasion, optic nerve invasion, and extra-scleral extension is clinically important because their presence is associated with a significantly higher risk of metastasis or disease recurrence [4].

Established classification systems are used for clinical risk stratification in RB. The International Classification of Retinoblastoma (ICRB) stratifies tumors based on size, location, vitreous seeding, subretinal fluid, and associated risks, thereby guiding decisions on suitability for chemoreduction and focal therapy [5]. The American Joint Committee on Cancer (AJCC) TNM staging system, applicable to both intraocular and extraocular RB, provides a universal staging framework essential for treatment planning [6]. Despite these advances, the greatest benefit of adjuvant therapy remains uncertain, given the considerable variability in metastatic risk across groups, particularly with varying treatment regimens and dosages. In addition to conventional histopathological risk factors, emerging evidence underscores the importance of underlying molecular and biological determinants in driving advanced disease progression, relapse and metastasis. Among these, trefoil factor family 1 (TFF1), a secreted protein aberrantly expressed in various human cancers, has emerged as a compelling candidate biomarker. This underscores the vital importance of further research to elucidate the role of the molecular marker TFF1 in optimizing personalized therapeutic strategies [7]. Liu et al. [8] identified two molecular RB subtypes – subtypes 1 and 2 – with TFF1 enriched in subtype 2, which refers to RB patients having aggressive features, including patients with high-risk disease-like HRPF and metastatic outcomes. Subsequently, Ashechero et al. [9] demonstrated that high TFF1 expression is associated with poor prognosis in RB. However, the role of TFF1 as a biomarker for advanced disease and the context of Indian RB patients, who represent a population with unique demographic, clinical, and socioeconomic characteristics, remain poorly understood. Also, prior studies have included a mixed cohort of previously treated and germline patients.

This study aims to analyze TFF1 expression in primary (treatment-naïve) enucleated unilateral RB eyeballs of Indian patients and examine its association with histopathological features such as choroidal invasion and optic nerve invasion, along with their anatomical extent/stratification, tumor differentiation, recurrence, metastasis, and survival outcomes, ultimately assessing its clinical relevance in this high-burden, low- to middle-income country (LMIC) setting. It will also serve as validation of the previously published data in the Indian context.

Methods

A total of 40 RB patients who underwent primary enucleation as first-line treatment at a tertiary ocular oncology center were included in the study. Approval was obtained from the Institutional Review Board, and informed consent was obtained. Exclusion criteria included patients who did not undergo primary enucleation as part of their treatment, patients whose parents/guardians did not consent to the study, and patients who were lost to follow-up after enucleation. RB grouping and staging were performed according to the IIRC and IRSS [10]. Standard treatment protocol: post-enucleation, patients with HRPF (massive choroidal invasion of >3 mm or full thickness), optic nerve invasion – post-laminar and beyond, scleral extension, anterior segment invasion, ciliary body, iris, anterior chamber, and combined factors (any optic nerve invasion + choroidal invasion) received 6 cycles of standard-dose adjuvant chemotherapy [11]. External beam radiotherapy was administered in cases with extraocular extension of RB as per the protocol. Patients were categorized by clinical demographics, HRPFs, metastasis, recurrence/relapse status, and survival outcomes.

The ocular pathology department processed enucleated eyeballs from treatment-naïve RB cases. Enucleated eyeballs were processed for histological analysis as per the standard processing and reporting protocol. An ocular pathologist reviewed slides with well-preserved tumor tissue for high-risk features.

We obtained tissue sections from paraffin-embedded eyeball blocks on positively charged slides after evaluation by an ocular pathologist. To assess TFF1 expression, immunohistochemistry was performed on RB tissues, and healthy gastric mucosal tissue from an adult served as a positive control (Fig. 1). For immunohistochemical analysis, slides were baked at 55–60°C for 45 min, deparaffinized with xylene, and rehydrated through a graded ethanol series. Heat-induced epitope retrieval was performed using sodium citrate buffer preheated in a water bath at 98°C, followed by washing with 1× Tris-buffered saline (TBS). Endogenous peroxidase activity was quenched with the kit’s hydrogen peroxide for 15 min in a humid chamber, and the slides were then washed with 1× TBS. Slides were then blocked with 1% BSA in TBS for 30 min at room temperature in a humid chamber. Slides were incubated overnight at 4°C with anti-TFF1 antibody (Sigma-Aldrich, HPA003425; dilution 1:2,000), 1× TBS, and then incubated with DAB chromogen from the PathnSitu kit (PEH002, PathnSitu Biotechnologies). Next, they were washed with 1× TBS and incubated with DAB chromogen from the PathnSitu kit (PEH002, PathnSitu Biotechnologies), followed by hematoxylin counterstaining and DPX mounting. Staining intensity was scored as follows: 0 = no staining, 1 = mild staining (light brown), 2 = moderate staining (brown), and 3 = strong staining (dark brown). TFF1 exhibits primarily cytoplasmic localization, with diffuse nuclear staining in some tissues, consistent with its secretory function, as indicated by tissue protein expression. Intensity scoring was performed independently by two experienced ocular onco-pathologists using defined reference images (Fig. 2). This helped to identify the true TFF1 score without bias. Final intensity categories were established by consensus after review and approval by the two independent pathologists. Only viable tumor cells were included in the assessment of staining intensity and percentage positivity. The following were excluded from scoring: ghost cells, apoptotic cells, necrotic/nonviable cells, stromal cells, inflammatory cells, and nonneoplastic elements. The percentage (P) of tumor cells stained (0–100%) was recorded, and a quick score (QS = I × P; range 0–300) was calculated by combining TFF1 staining intensity and percentage positivity, which was used to classify tumors into two expression categories: HS1 (low expression): QS < 50, HS2 (high expression): QS ≥ 50 [8, 9], in the viable cells. The histological level of tumor differentiation was recorded separately as well differentiated, moderately differentiated, or poorly differentiated.

Fig. 1.

Representative image of TFF1 localization in RB tissue and positive control. a Negative TFF1 expression in RB tissue. b Positive TFF1 expression in RB tissue. c TFF1 expression in gastric mucosal epithelium. IHC images were taken on the Olyvia whole-slide scanner at ×20 magnification.

Fig. 2.

TFF1 expression in HS1 vs. HS2 in HRPF. a, b TFF1 expression in enucleated eyeball with intraocular tumor in nonmetastatic RB tissue and corresponding histopathology image. c, d TFF1 expression in enucleated eyeball in a patient with metastatic RB with PLONI and corresponding histopathology image. e QS of positive TFF1 expression in nonmetastatic vs. metastatic RB. f, g TFF1 expression in non-HRPF RB tissue and corresponding histopathology image of an intraocular tumor. h, i TFF1 expression in HRPF RB tissue and corresponding histopathology image. j QS of positive TFF1 expression in non-HRPF vs. HRPF RB tissue. Images taken in Olyvia whole-slide scanner at ×20 magnification. Hematoxylin and eosin images taken in Olympus CKX53 microscope with Metzer-M 4k Ultra HD camera at ×10 magnification.

Raw data were entered into MS Excel and analyzed in R after cleaning and deriving clinically meaningful variables, including multiple binary and ordinal indicators of metastasis and HRPF. Descriptive statistics were summarized overall and by HS subtype, with appropriate statistical tests applied using the gtsummary package. Correlation between QS and HRPF positivity, metastasis, and survival outcome was assessed using numeric transformations and correlation matrices. At the same time, the discriminatory performance of QS was evaluated using ROC curve analysis to estimate AUC, optimal cut-offs (Youden index), sensitivity, and specificity, with statistical significance assessed using DeLong’s method.

Results

This study included 40 patients with RB, with a mean age of 39.75 ± 25.22 months. The study involved 40 RB patients, with 22 (55.0%) classified as HS1 and 18 (45.0%) as HS2. The mean age at diagnosis was similar between the groups (40.50 ± 21.83 vs. 38.83 ± 29.47 months; p = 0.529), as was the follow-up duration (16.14 ± 13.85 vs. 22.92 ± 15.82 months; p = 0.161). A near-significant difference in sex distribution was observed (p = 0.053): females comprised 32.5% (13/40), predominantly HS1 (76.92%), while males comprised 67.5% (27/40), with a higher representation in HS2 (55.56%). Histopathological analysis revealed significant differences (p = 0.042): both group D tumors were HS1, whereas among 35 group E tumors, 57.14% were HS1 and 42.86% were HS2. Notably, all 3 cases with extraocular disease were HS2 (100%). Clinical outcomes showed a trend toward worse prognosis with HS2: tumor recurrence occurred exclusively in HS2 (100%), metastasis was confined to HS2, and mortality was observed only in HS2 patients.

Immunohistochemical analysis demonstrated a difference in staining scores (p < 0.001): HS2 tumors exhibited a higher burden (167.72 ± 70.22) compared to HS1 (11.95 ± 13.55), underscoring a strong association between molecular marker expression and high-risk histopathological phenotype (see Table 1). Of 40 patients, 32 were TFF1-positive and categorized as HS1 or HS2 based on the QS.

Table 1.

RB patient demographics and clinicopathological information

Characteristic	Overall (N = 40)	HS1 (N = 22)	HS2 (N = 18)	p value
Gender, n (%)	​	​	​	0.053
F	13	76.92 (10)	23.08 (3)	​
M	27	44.44 (12)	55.56 (15)	​
Age, months	39.75±25.22	40.50±21.83	38.83±29.47	0.529
Follow-up, months	19.19±14.97	16.14±13.85	22.92±15.82	0.161
Disease severity group/Stage, n (%)	​	​	​	0.042
Group D	2	100.00 (2)	0.00 (0)	​
Group E	35	57.14 (20)	42.86 (15)	​
Extra ocular	3	0.00 (0)	100.00 (3)	​
Tumor recurrence, n (%)	​	​	​	0.196
No	38	57.89 (22)	42.11 (16)	​
Yes	2	0.00 (0)	100.00 (2)	​
Metastasis, n (%)	​	​	​	0.196
No	38	57.89 (22)	42.11 (16)	​
Yes	2	0.00 (0)	100.00 (2)	​
Final outcome, n (%)	​	​	​	0.196
Alive	38	57.89 (22)	42.11 (16)	​
Expired	2	0.00 (0)	100.00 (2)	​
QS	82.05±91.69	11.95±13.55	167.72±70.22	<0.001

High-Risk Stratification Based on TFF1 QS

Based on the QS, 55% (n = 22) cases were classified as HS1 and 45% (n = 18) as HS2. The mean QS was significantly higher in HS2 compared to HS1 (167.72 ± 70.22 vs. 11.95 ± 13.55, p < 0.001).

Clinicopathological stratification by TFF1-associated histological subgroups showed that HS2 tumors exhibited more adverse pathological and clinical features than HS1 tumors. HRPF positivity was higher in HS2 than HS1 tumors (57.1% vs. 42.9%; p = 0.105). Choroidal invasion was more frequent in HS2 (70.0% vs. 30.0%; p = 0.14), including massive choroidal invasion (>3 mm) (38.9% vs. 13.6%; p = 0.14). Similarly, optic nerve invasion was more prevalent in HS2 (33.3% vs. 22.7%; p = 0.498), with post-laminar optic nerve invasion (PLONI) detected in 22.2% and 13.6% of cases, respectively (p = 0.498). Multiple HRPF site involvement (C+ON+S) was observed in 80.0% of HS2 tumors compared with 20.0% of HS1 tumors (p = 0.155). Histopathologically, poorly differentiated tumors were more common in HS2 (62.5% vs. 37.5%; p = 0.136), while all undifferentiated tumors occurred exclusively in HS2 (100%; p = 0.136). HS2 tumors showed numerically higher rates of adverse pathological features; however, these differences were not statistically significant and should be interpreted as hypothesis-generating (Table 2; Fig. 2).

Table 2.

Clinico-pathological features in HS1 vs. HS2 RB patients

Characteristic	HS1 (N = 22)	HS2 (N = 18)	p value
QS	11.95±13.55	167.72±70.22	<0.001
HRPF (any), n (%)	9 (42.9)	12 (57.1)	0.105
Massive choroidal invasion (CI)	3 (30.0)	7 (70.0)	0.14
No CI	19 (86.4)	11 (61.1)	​
Focal (<3 mm)	1 (4.5)	2 (11.1)	​
Massive (>3 mm)	3 (13.6)	7 (38.9)	​
Optic nerve invasion (any), n (%)	5 (22.7)	6 (33.3)	0.498
No ONI	17 (77.3)	12 (66.7)	​
Prelaminar	3 (13.6)	2 (11.1)	​
Laminar	2 (9.1)	2 (11.1)	​
Postlaminar	3 (13.6)	4 (22.2)	​
≥2 HRPF sites (C+ON+S)	1 (20.0)	4 (80.0)	0.155
Tumor differentiation, n (%)	​	​	0.136
Well differentiated	19 (63.3)	11 (36.7)	​
Poorly differentiated	3 (37.5)	5 (62.5)	​
Undifferentiated	0 (0)	2 (100)	​
Metastasis	0 (0)	2 (100)	0.196
Tumor recurrence	0 (0)	2 (100)	0.196
Outcome (DDD)	0 (0)	2 (100)	0.196

HRPF choroidal features in HS1 vs. HS2 RB patients. HRPF optic nerve features in HS1 vs. HS2 RB patients.

C, choroid; ON, optic nerve; S, sclera; DDD, died due to disease.

ROC analysis demonstrated that elevated QS was associated with increased detection of high-risk RB pathology, including choroidal invasion (AUC – 0.603), optic nerve invasion (AUC – 606), composite HRPF involvement (AUC – 0.617), and metastasis (AUC – 0.788) (Fig. 3). The strongest predictive performance was observed for metastasis, showing near-complete discrimination. This aligns with clinicopathological findings, where metastasis, recurrence, and disease-related death occurred exclusively in the HS2 subgroup (2/2 [100%]; p = 0.196). The 2 patients who succumbed to the disease both had optic nerve involvement, and 1 had additional choroidal involvement on histopathology.

Fig. 3.

RoC curve based on TFF1 QS. RoC curve of QS for the detection of HRPF choroid invasion (a), HRPF optic nerve invasion (b), HRPF choroid invasion, optic nerve invasion, and sclera invasion (c), and metastasis (d).

The same patient was also cerebrospinal fluid cytology positive, with CNS metastasis detected on metastatic workup and died within 1 month of enucleation. The other patient received adjuvant chemotherapy (3 cycles of vincristine, etoposide, and carboplatin) post-enucleation and was planned for orbital radiation; was lost to follow-up for 6 months and eventually developed symptoms suggestive of CNS metastasis and passed away after 8 months due to CNS disease.

Consistent with this aggressive phenotype, HS2 tumors also showed higher frequencies of massive choroidal invasion (38.9% vs. 13.6%; p = 0.14), PLONI (22.2% vs. 13.6%; p = 0.498), and multisite HRPF involvement (80.0% vs. 20.0%; p = 0.155). All metastatic cases (2/2 [100%]; p = 0.196), recurrent tumors (2/2 [100%]; p = 0.196), and DDD events (2/2 [100%]; p = 0.196) occurred in HS2 patients, whereas no such events were identified in HS1 tumors (0%; p = 0.196).

TFF1 expression was higher in metastatic than nonmetastatic tumors and in HRPF-positive than HRPF-negative tumors. Among the evaluated outcomes, the highest discriminatory performance was observed for metastasis. In this cohort, HS2 tumors showed higher frequencies of massive choroidal invasion, PLONI, and multisite HRPF involvement compared with HS1 tumors. Metastasis, recurrence, and disease-related mortality were observed only in the HS2 subgroup.

Discussion

In this study, we evaluated TFF1 expression in treatment-naïve RB in an Indian cohort and examined its association with histopathological risk factors, metastasis, and clinical outcomes. Prognostication in RB continues to rely primarily on established HRPFs identified after enucleation, which are strongly associated with metastatic potential [3, 4]. More recently, molecular markers such as TFF1 have been investigated as adjuncts to risk stratification and to identify patients at increased risk of relapse and mortality [7–9]. Accordingly, we assessed TFF1 expression, quantified using the quantitative score (QS), in relation to HRPFs, metastasis, recurrence, relapse, and clinical outcomes to better characterize adverse disease behavior.

Within our cohort, TFF1 expression demonstrated clear quantitative stratification. HS2 tumors exhibited significantly higher QS values than HS1 tumors (167.7 ± 70.2 vs. 11.9 ± 13.5; p < 0.001). Furthermore, tumors harboring ≥2 high-risk sites were predominantly observed in the HS2 group (80% vs. 20%), suggesting an association between elevated QS and a greater burden of high-risk pathological features.

Although optic nerve invasion (33.3% HS2 vs. 22.7% HS1; p = 0.498) and choroidal invasion (70.0% HS2 vs. 30.0% HS1; p = 0.14) were observed across QS subgroups, further subclassification revealed a distinct gradient of disease severity. More advanced forms of invasion, including PLONI (22.2% vs. 13.6%) and massive choroidal invasion (38.9% vs. 13.6%), were more frequent in HS2 tumors. While these differences did not reach statistical significance, the enrichment of both PLONI and massive choroidal invasion within the HS2 subgroup suggests that higher TFF1 expression may be associated with the anatomical extent and severity of tumor invasion rather than simply the presence or absence of HRPFs. Given that PLONI and massive choroidal invasion are established clinicopathological predictors of metastatic dissemination [3, 4], this trend warrants further investigation in larger cohorts. Metastatic spread is also influenced by anatomical routes of dissemination, particularly through the optic nerve and its proximity to the central nervous system, which may affect outcomes independently of TFF1 expression. In contrast, tumors with no invasion or only focal involvement were largely confined to the HS1 group.

Liu et al. [8] demonstrated universal TFF1 positivity in evaluable metastatic tumors and reported that 18 of 19 HS2 tumors subsequently developed metastasis. Similarly, Aschero et al. [9] observed a higher frequency of HRPFs in TFF1-high tumors and identified TFF1 expression as a marker of poor prognosis. Although our study was limited to a single-center cohort of primary enucleated RB eyes, we observed a similar trend toward enrichment of high-risk pathological features and adverse clinical outcomes in the HS2 subgroup, although this did not reach statistical significance.

Collectively, these findings suggest that QS may reflect not only the presence of HRPFs but also their anatomical depth and extent highlighting the molecular changes associated with disease progression. The increased frequency of PLONI, massive choroidal invasion, and involvement of multiple HRPF sites within the HS2 subgroup support an association between elevated TFF1 expression and more extensive tumor spread. Whether this relationship directly contributes to metastatic potential or instead reflects aggressive underlying tumor biology remains to be determined.

Importantly, despite optic nerve invasion in HS1 tumors, no adverse events were observed in this subgroup. In contrast, all adverse outcomes, including metastasis, recurrence, and mortality, occurred exclusively in HS2 tumors with high QS values. Nevertheless, not all HS2 tumors developed adverse events, indicating that TFF1 expression alone is insufficient to confer aggressive clinical behavior. These observations should be interpreted cautiously, given the limited number of adverse events in the cohort.

In addition, previous studies have not fully clarified the independent prognostic contributions of scleral and choroidal involvement. Although event-free survival and overall survival have been reported [9, 11], the lack of outcome stratification by individual risk factors limits a detailed understanding of their relative prognostic significance, particularly in real-world and LMIC settings.

Another important consideration is the anatomical extent of tumor involvement, particularly with respect to the optic nerve. The 3 extraocular patients in this cohort, of which 2 patients died, all fall under the HS2 category. The proximity of tumor cells to the surgical transection margin may play a critical role in metastatic dissemination. Post-laminar invasion and optic nerve margin involvement may independently drive metastasis, potentially confounding the prognostic value of molecular markers such as TFF1. Consequently, the clinical relevance of TFF1 expression may be influenced by these anatomical factors and should be evaluated in detail in future studies.

In LMIC settings with constrained healthcare resources and limited access to intensive surveillance or treatment escalation, risk stratification beyond conventional histopathological assessment is especially vital [2]. Although current protocols recommend adjuvant chemotherapy for HRPFs [4, 11], not all HRPF-positive patients develop metastasis. In our cohort, adverse outcomes were confined to the HS2 subgroup despite standard management, including 1 patient with positive surgical cut end and another with optic nerve involvement up to the post-laminar region. While based on few events and requiring validation, these findings suggest TFF1 expression may help identify HRPF-positive patients at higher risk of progression. Rather than replacing existing histopathological criteria, TFF1 could serve as an adjunct biomarker and complement histopathology to refine risk assessment. Future studies should explore whether combining TFF1 expression with high-risk features such as choroidal invasion and post-laminar optic nerve involvement [3, 4] enhances identification of patients who might benefit from intensified surveillance, altered chemotherapy, or targeted therapies. This stratification is especially relevant in LMICs, where optimizing limited healthcare resources remains a key challenge.

From a translational perspective, these findings are particularly important in resource-limited settings where histopathology is essential for diagnosis and risk assessment. Incorporating QS-based molecular markers into standard histopathology may enable more precise, actionable risk stratification. To our knowledge, this is the first study to evaluate TFF1 expression in an Indian RB cohort and correlate it with tumor invasion severity, offering a more comprehensive understanding of tumor behavior in a treatment-naïve cohort. However, the study lacked sufficient power to detect modest associations between TFF1 and adverse outcomes. Larger, multicenter studies and functional research are needed to clarify TFF1’s role, including its potential as a noninvasive biomarker in liquid biopsies [7].

Conclusion

In this cohort, higher TFF1 expression was associated with increased frequencies of adverse histopathological and clinical features, including massive choroidal invasion, PLONI, metastasis, recurrence, and disease-related mortality. However, these associations were not statistically significant. Larger multicenter studies are required to determine whether TFF1 provides independent prognostic value beyond established histopathological risk factors. Combining QS with histopathological risk factors may enhance risk stratification, particularly in resource-limited settings.

Statement of Ethics

This study protocol was reviewed and approved by the Institutional Review Board and Ethics Committee of Dr Shroff’s Charity Eye Hospital with IRB approval no. IRB/2025/Mar/08. Research was conducted ethically in accordance with the World Medical Association Declaration of Helsinki. Written informed consent was obtained from the parent/legal guardian of the patient to participate in this study and for publication of the details of their medical case and any accompanying images.

Conflict of Interest Statement

No conflict of interest to declare.

Funding Sources

This study was funded by ICMR (EMDR/IG/11/2024-01907), Velux Stiftung (1973), and Eicher Shroff Centre for Stem Cell Research.

Author Contributions

Aman Verma, Chhavi Gupta, and Sima Das were involved in the design of the study, data collection, analysis, and manuscript preparation and review. Anureet Kaur, Ria Sachdeva, Akhil Varshney, and Ravindra Kumar Saran were involved in the collection of data and analysis. Sudhir Verma, Anil Tiwari, and Virender S. Sangwan were involved in manuscript review. Arpan Gandhi was involved in data collection. All the authors contributed to the preparation, review, and approval of the manuscript. All authors attest that they meet the current ICMJE criteria for Authorship.

Funding Statement

This study was funded by ICMR (EMDR/IG/11/2024-01907), Velux Stiftung (1973), and Eicher Shroff Centre for Stem Cell Research.

Data Availability Statement

The data that support the findings of this study are not publicly available due to patient confidentiality reasons but are available from the corresponding author, Sima Das, upon request.