Oral squamous cell carcinoma (OSCC), accounting for over 90% of oral malignancies, is a significant health concern globally, with India reporting a particularly high burden due to prevalent risk factors such as tobacco, alcohol, and betel nut use. These exposures, along with poor oral hygiene and HPV infections, contribute to OSCC initiation and progression. Commonly affected sites—the tongue, the floor of the mouth, and buccal mucosa—impact vital functions, leading to considerable morbidity.1

Despite diagnostic and therapeutic advances, OSCC outcomes remain poor, with a low five-year survival rate due to its invasive nature, early lymphatic spread, and frequent recurrences. The tumour's ability to invade surrounding tissues is driven by epithelial-mesenchymal transition (EMT), a biological process where epithelial cells acquire mesenchymal properties, increasing their migratory and invasive potential—especially at the invasive front of the tumour.2

E-cadherin and vimentin are key EMT markers. Loss of E-cadherin, which maintains epithelial integrity, facilitates tumour cell detachment, while vimentin upregulation enhances cell motility and invasiveness. This "cadherin switch" at the invasive front reflects tumour aggressiveness and represents a critical interface for tumour-stroma interaction and EMT modulation.3,4

Although EMT is known to influence tumour progression, its exact role in OSCC, particularly at the invasive front, remains underexplored. The association between EMT marker expression and clinicopathological parameters such as tumour grade and nodal metastasis is yet to be established, limiting its clinical utility in routine prognostication.5,6

This study investigates the expression of E-cadherin and vimentin at the invasive front of OSCC and their correlation with histological grade and lymph node metastasis. By elucidating these patterns, the research aims to improve understanding of OSCC biology, support the identification of prognostic biomarkers, and guide targeted therapeutic interventions.

This observational study, comprising both retrospective and prospective components, was conducted in the Department of Pathology at Bharati Vidyapeeth (Deemed to Be University) Medical College, Pune, in association with Bharati Hospital and Research Centre.

Study Population and Duration: The population included patients histologically diagnosed with oral squamous cell carcinoma (OSCC). The study's retrospective arm covered 24 months, while the prospective arm spanned 18 months. A total of 57 OSCC cases were included based on the availability of adequate histopathological material.

Sample Size and Sampling Methodology: The estimated sample size was 57 cases, calculated using the standard formula: n = [Z2 × P(1−P))/d²]. The cases were selected by purposive sampling from incisional biopsy and surgical resection specimens diagnosed as OSCC. Cases were included if adequate stroma at the tumour-invasive front was present. Specimens with inadequate or superficial tissue lacking invasive front and retrospective cases where paraffin blocks were unavailable were excluded from the study.

Data Collection Tools and Ethical Consideration: Clinical data and histopathological findings were obtained from test requisition forms, printed pathology reports, and biopsy registers. The study involved no direct patient intervention, so there were no anticipated risks.

Tissue Processing and Sectioning: Tissue specimens were received in 10% buffered formalin. Small biopsies were processed the same day, while resected specimens were grossed after overnight fixation. Tumour sections were selected per the College of American Pathologists (CAP) protocol. The tissues underwent automated processing in a closed retort-based system. Fixation was followed by dehydration using a graded series of 100% 2-propanol, clearing with multiple xylene steps, and wax impregnation using molten paraffin wax. The total processing time was approximately 14 hours. Paraffin blocks were embedded using a dedicated tissue embedding station, and 3–4 µm thick sections were cut using a microtome. Continuous ribbons of sections were floated on warm water and mounted on Poly-L-Lysine-coated glass slides.

Hematoxylin and Eosin Staining: The mounted slides underwent manual H&E staining. Sections were initially deparaffinized using xylene and rehydrated through descending grades of alcohol. Nuclear staining was done with Harris hematoxylin, followed by differentiation in acid alcohol and bluing under tap water. Counterstaining was performed with eosin. Dehydration and clearing were completed using absolute alcohol and xylene, respectively, and slides were mounted using DPX. The nuclei appeared violet, and the cytoplasm pink under light microscopy.

Immunohistochemical Staining Protocol: For immunohistochemical evaluation, specific primary antibodies were used: E-cadherin (clone HECDE-1, Vitro, Batch No. 076100395) and vimentin (clone V9, Vitro, Batch No. 41379699). Paraffin sections were taken on Poly-L-Lysine-coated slides and incubated overnight in a hot air oven. Deparaffinization was carried out using two xylene baths, each for five minutes. Slides were rehydrated in descending grades of alcohol. Antigen retrieval was performed in a microwave oven at 97°C for two cycles of ten minutes each. After cooling, slides were washed with distilled water and then rinsed three times in Tris-buffered saline (TBS). Endogenous peroxidase activity was blocked using hydrogen peroxide for 30 minutes, and sections were demarcated using a PAP pen to restrict reagent spread. Primary antibodies were applied and incubated for two hours, followed by washing with phosphate-buffered saline (PBS). A secondary antibody conjugated with horseradish peroxidase (HRP) was applied for one hour then washed off with tris buffer. Visualization was achieved using 3,3'-diaminobenzidine (DAB) chromogen, and the brown reaction product was monitored under a microscope. Counterstaining was performed using Harris hematoxylin, followed by bluing under tap water. Slides were dehydrated in ascending alcohol grades, cleared in xylene, and mounted with DPX.

Evaluation and Data Management: Staining intensity and distribution were examined under a light microscope to assess the immunoreactivity of E-cadherin (membranous) and vimentin (cytoplasmic). All clinical data, histopathological grading, and IHC results were documented and entered into Microsoft Excel for further statistical analysis. The correlation between marker expression and histological grade or lymph node metastasis was explored using appropriate statistical tools.

Table 1 summarizes the clinicopathological characteristics of 57 OSCC patients, with the majority aged between 41–50 years (31.6%) and predominantly male (86.0%). The most common tumour site was the buccal mucosa (35.1%), followed by the tongue (24.5%). Incisional biopsies constituted the most significant proportion of specimen types (50.9%). Most tumours were moderately differentiated (68.4%), and the predominant pattern of invasion was type 5 (26.3%). Immunohistochemical analysis revealed that E-cadherin expression was lost in 77.2% of cases, while vimentin was expressed in 64.9% of the tumours.

Table 1: Characteristics of study participants with OSCC (n=57)

A progressive loss of E-cadherin expression was observed, increasing from 40.0% in WPOI 1 to 93.3% in WPOI 5, while vimentin expression showed a corresponding rise from 30.0% in WPOI 1 to 86.6% in WPOI 5. Preserved E-cadherin was more frequent in lower WPOI categories (60.0% in WPOI 1), whereas its loss predominated in higher grades. Conversely, vimentin was not expressed in the majority of WPOI 1 cases (70.0%) but was markedly expressed in WPOI 4 (83.3%) and WPOI 5 (86.6%). Both markers demonstrated statistically significant associations with the pattern of invasion, with p-values of 0.015 for E-cadherin and <0.05 for vimentin. (Table 2)

Table 2: Expression of IHC markers concerning different patterns of invasion (n=57)

Table 3: Case distribution in view of E-cadherin and Vimentin expression within the central area of tumour and invasive front

As shown in Table 3, E-cadherin expression was preserved in 63.15% of cases and lost in 36.84% within the central tumour area, whereas at the invasive front, preservation decreased to 43.85% and loss increased to 56.15%. Conversely, vimentin was expressed in 29.82% of central areas and not expressed in 70.18%, while its expression rose to 52.63% at the invasive front, with non-expression declining to 47.36%. These differences were statistically significant, with p-values of 0.038 for E-cadherin and 0.013 for vimentin.

Table 4 shows that in cases with lymph node metastasis, E-cadherin loss (42.85%) exceeded its preservation (28.57%), while in non-metastatic cases, both preserved and lost expressions were equally represented at 12.28%. Vimentin expression was markedly higher in metastatic cases (57.14%) than non-metastatic ones (12.28%), with non-expression being predominant in the absence of metastasis. Despite these patterns, no statistically significant association was found, as indicated by p-values of 0.80 for E-cadherin and 0.42 for vimentin.

Table 4: Expression of IHC markers E-cadherin and Vimentin with respect to the lymph node status

Oral squamous cell carcinoma (OSCC) is a malignancy known for its aggressive nature, often emerging from mucosal surfaces exposed to carcinogens such as tobacco, alcohol, and areca nut. Its biological behaviour varies widely, necessitating a combination of histological and molecular assessments for accurate prognostication. EMT markers, particularly E-cadherin and Vimentin, have been extensively studied for their role in modulating tumour invasiveness and progression. The present research examined these markers in relation to demographic trends, the worst pattern of invasion (WPOI), spatial tumour regions, and lymph node metastasis, providing comprehensive insight into their clinicopathological significance in OSCC.6,7

In our study, most OSCC cases were male (86%) and within the 41–60 age range, consistent with earlier reports, all highlighted a middle-aged predominance of the disease. 8,9,10 The buccal mucosa emerged as the most frequently involved site, corresponding with studies from Indian cohorts, where chewing tobacco is commonly retained in this region. 8,11 Histologically, most tumours were moderately differentiated (68.4%), a pattern also seen in the previous studies9,10 and often associated with transitional EMT features. Immunohistochemical evaluation revealed a high prevalence of E-cadherin loss (77.2%) and Vimentin expression (64.9%), indicating active EMT processes in a substantial portion of cases.

The present research found a strong correlation between EMT marker expression and WPOI. E-cadherin loss increased markedly from WPOI 1 to WPOI 5, while Vimentin expression followed an inverse trend. These associations were statistically significant and closely mirror findings by Mishra et al., who linked higher WPOI patterns (IV–V) with poor differentiation and increased perineural and lymphovascular invasion8. Similarly, Chaw SY et al.12 and Liu LK et al.13 demonstrated that as invasion patterns become more infiltrative, E-cadherin downregulation and Vimentin upregulation become more prominent, supporting the notion of EMT-driven aggressiveness in OSCC. These findings underline the prognostic utility of combining morphological invasion patterns with EMT markers in routine diagnostics.

Our analysis also demonstrated a significant regional difference in EMT marker distribution, with E-cadherin being more frequently preserved in the tumour centre while Vimentin expression predominated at the invasive front. This spatial EMT shift has been well-documented by Kim KH et al.,14 who reported E-cadherin loss in 84% and Vimentin overexpression in 53% of recurrent OSCC cases, especially at the invasive front. Costa et al.15 and Vanini et al.16 echoed similar observations, describing the invasive front as a biologically active zone where epithelial cells acquire mesenchymal traits, promoting local invasion and potential metastasis. These findings reinforce the importance of regional sampling and marker analysis to capture the dynamic tumour microenvironment accurately.

However, when EMT marker expression was evaluated in relation to lymph node metastasis, no statistically significant association was observed in the present study. Although E-cadherin loss and Vimentin expression were more common in node-positive cases, the sample size limited the power to detect meaningful correlations. This contrasts with the findings of Akhtar et al.,17 who reported strong inverse expression patterns in metastatic cases, and Zhou et al.,18 who also linked EMT features to nodal involvement. Conversely, Zidar et al.19 did not find consistent EMT expression across nodal status in conventional OSCC, suggesting that EMT marker utility may vary depending on histological subtypes or sample selection criteria. Therefore, while trends toward EMT activation in metastasizing tumours exist, further investigation with larger cohorts is warranted.

In conclusion, the current study highlights the relevance of EMT markers in assessing OSCC aggressiveness. E-cadherin loss and Vimentin expression were significantly associated with higher WPOI and were more prominent at the invasive front, signifying active epithelial-to-mesenchymal transition and tumour invasiveness. Although a clear link with lymph node metastasis could not be established, these markers still offer valuable insights when combined with histological features. Integrating EMT marker analysis into routine OSCC evaluation may enhance risk stratification and guide individualized therapeutic strategies.

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