A Study on Cancer-Associated Fibroblast Using Alpha-Smooth Muscle Actin Immunohistochemistry in Oral Squamous Cell Carcinoma

Background Oral squamous cell carcinoma (OSCC) represents a significant global health burden with complex pathophysiology involving tumor microenvironment interactions. The tumor stroma, particularly cancer-associated fibroblasts (CAFs), plays a crucial role in tumor advancement, invasion, and metastasis. CAFs, identified by alpha-smooth muscle actin (α-SMA) expression, influence tumor behavior through extracellular matrix remodelling and pro-tumorigenic signalling. Despite emerging evidence of their prognostic significance, the relationship between CAF expression patterns and clinicopathological parameters in OSCC remains inadequately characterized. Objectives This study aimed to detect CAFs using α-SMA immunohistochemistry in OSCC and evaluate their association with LNM and pTNM staging, potentially identifying new prognostic markers for clinical management. Methodology This laboratory-based analytical study was conducted between September 2022 and December 2023. Histopathologically confirmed OSCC cases (n=88) treated by composite resection and cervical lymph node dissection were included, excluding recurrent cases, patients who received neoadjuvant chemotherapy, and second primary cancers. H&E slides were reviewed for LNM and pTNM staging. Immunohistochemical staining for α-SMA was performed on 4 μm formalin-fixed paraffin-embedded tissue sections using heat-induced antigen retrieval, followed by incubation with primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody. CAF expression was quantified using Kellermann et al.'s scoring system (Score 1 is <1%, Score 2 is between 1% and 50%, and Score 3 is >50% stained cells) and categorized by distribution pattern (focal, network, or spindle). Additional parameters assessed included tumor-stroma ratio (TSR), worst pattern of invasion (WPOI), tumor budding (TB), and tumor-infiltrating lymphocytes (TILs). Results The study population comprised 88 patients (69.3% female, 30.7% male) (average age: 56.97 years). The buccal mucosa represented the most frequent site (50%), with well-differentiated tumors predominating (80.7%). Pathological staging revealed Stage IVA as the most prevalent (33%), followed by Stage III (31.8%). Regarding CAF expression, Score 3 (abundant CAFs) was observed in 55.7% of cases, Score 2 in 40.9%, and Score 1 in only 3.4%. Network pattern CAF distribution predominated (38.6%), with equal representation of focal and spindle configurations (30.7% each). Statistical analysis revealed no significant association between CAF scores and LNM (p=0.758); however, CAF distribution patterns demonstrated a statistically significant association with pTNM staging (χ²=26.716; p=0.001), with advanced stages showing a distinct pattern shift toward network and spindle arrangements. Notably, significant correlations were observed between TSR and CAF score (p<0.0001), TB and CAF score (p=0.021), and TB and LNM (p=0.047). More aggressive invasion patterns demonstrated higher CAF scores and increased TB intensity. Conclusion While CAF scores alone did not predict LNM, CAF architectural patterns demonstrated significant associations with pathological staging in OSCC. The correlations between CAF expression, TB, and invasion patterns suggest that CAF distribution may serve as a valuable prognostic indicator. These findings highlight the potential of CAF architectural evaluation as an adjunctive histopathological parameter for risk stratification in OSCC patients.