Abstract
Aim:
This study aimed to evaluate the immunohistochemical co-expression of MUC1 and MUC4 in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and assess their potential as biomarkers for diagnosing and prognosticating oral malignancies.
Materials and Methods:
A retrospective observational study was conducted on formalin-fixed, paraffin-embedded tissue specimens from the archives of the Department of Oral Pathology and Microbiology. A total of 80 histopathologically confirmed cases, consisting of 40 OL and 40 OSCC cases, were selected. Immunohistochemical staining for MUC1 and MUC4 was performed using specific monoclonal antibodies, and the expression was evaluated based on localization, intensity, and extent of staining. Statistical analysis was performed using SPSS, with P < 0.05 considered statistically significant.
Results:
The study found that MUC1 and MUC4 expression were significantly higher in OSCC compared to OL. High MUC1 expression was observed in 82.5% of OSCC cases versus 30% in OL cases (P < 0.001), and high MUC4 expression was found in 70% of OSCC cases versus 30% in OL (P = 0.002). Co-expression of MUC1 and MUC4 was seen in 90% of OSCC cases and 65% of OL cases (P = 0.027). Logistic regression analysis confirmed that OSCC diagnosis was a strong predictor of high MUC1 and MUC4 expression (P < 0.001).
Conclusion:
The findings suggest that MUC1 and MUC4 are significantly overexpressed in OSCC compared to OL, and their co-expression may serve as a valuable biomarker combination for early diagnosis and prognosis. Elevated expression of both mucins is associated with poor prognosis, indicating their potential for use in clinical practice for risk stratification and personalized treatment in OSCC patients.
Keywords: Immunohistochemistry, MUC1, MUC4, oral leukoplakia, oral squamous cell carcinoma
Résumé
Objectif:
Cette étude visait à évaluer la co-expression immunohistochimique de MUC1 et MUC4 dans la leucoplasie orale (LO) et le carcinome épidermoïde oral (CEO) et à évaluer leur potentiel en tant que biomarqueurs pour le diagnostic et le pronostic des cancers de la cavité buccale.
Matériel et Méthodes:
Une étude observationnelle rétrospective a été menée sur des échantillons tissulaires fixés au formol et inclus en paraffine provenant des archives du Département de pathologie et de microbiologie buccales. Au total, 80 cas confirmés histopathologiquement, dont 40 cas de LO et 40 cas de CEO, ont été sélectionnés. La coloration immunohistochimique de MUC1 et MUC4 a été réalisée à l’aide d’anticorps monoclonaux spécifiques, et l’expression a été évaluée en fonction de la localisation, de l’intensité et de l’étendue de la coloration. L’analyse statistique a été réalisée à l’aide du logiciel SPSS, avec un seuil de signification statistique fixé à P < 0,05.
Résultats:
L’étude a révélé que l’expression de MUC1 et MUC4 était significativement plus élevée dans le carcinome épidermoïde de la cavité buccale (OSCC) que dans la leucémie orale (OL). Une forte expression de MUC1 a été observée dans 82,5 % des cas d’OSCC contre 30 % dans les cas d’OL (P < 0,001), et une forte expression de MUC4 a été constatée dans 70 % des cas d’OSCC contre 30 % dans les cas d’OL (P = 0,002). La co-expression de MUC1 et MUC4 a été observée dans 90 % des cas d’OSCC et 65 % des cas d’OL (P = 0,027). L’analyse de régression logistique a confirmé que le diagnostic d’OSCC était un facteur prédictif important d’une forte expression de MUC1 et MUC4 (P < 0,001).
Conclusion:
Ces résultats suggèrent que MUC1 et MUC4 sont significativement surexprimés dans l’OSCC par rapport à l’OL, et que leur co-expression pourrait constituer une combinaison de biomarqueurs précieuse pour le diagnostic précoce et le pronostic. L’expression élevée des deux mucines est associée à un mauvais pronostic, ce qui indique leur potentiel d’utilisation en pratique clinique pour la stratification des risques et un traitement personnalisé chez les patients atteints de carcinome épidermoïde de la cavité buccale
Mots-clés: Immunohistochimie, MUC1, MUC4, leucoplasie orale, carcinome épidermoïde oral
INTRODUCTION
Oral squamous cell carcinoma (OSCC) is a major cause of morbidity and mortality globally, especially in regions where tobacco and alcohol consumption are prevalent. Early detection and accurate prediction of malignant transformation from premalignant lesions, such as oral leukoplakia (OL), remain essential to improving patient outcomes. In this context, immunohistochemical analysis, particularly the study of mucin expression, has gained attention as a promising diagnostic and prognostic tool. Mucins are high-molecular-weight glycoproteins that play an essential role in various biological processes, including cell signaling, adhesion, and protection. Their overexpression has been associated with the progression of numerous cancers, including OSCC, highlighting their potential as biomarkers for malignancy.[1,2]
MUC1, a transmembrane glycoprotein, is widely expressed in the epithelial lining of various organs, including the oral cavity. In the context of cancer, MUC1 overexpression has been linked to poor prognosis, invasiveness, and metastasis in several malignancies, including breast and colorectal cancer, as well as OSCC. In premalignant oral lesions, such as OL, MUC1 expression has been shown to correlate with disease progression and invasion, suggesting its utility as a biomarker for predicting malignant transformation. The overexpression of MUC1 has been associated with increased invasion and lymph node metastasis in OSCC, making it a valuable marker for assessing the aggressiveness of the disease.[3]
MUC4, another important transmembrane mucin, is also involved in tumor progression. Like MUC1, MUC4 is overexpressed in several epithelial cancers, including OSCC. Studies have demonstrated that MUC4 overexpression correlates with poor clinical outcomes and metastasis, making it a significant prognostic factor in OSCC. The role of MUC4 in cancer is thought to involve its ability to promote cell adhesion, invasion, and epithelial-mesenchymal transition (EMT), which are key processes in the metastatic spread of tumors. The co-expression of MUC1 and MUC4 has been shown to provide a more accurate prediction of clinical outcomes in OSCC patients, with elevated levels of both mucins correlating with a more aggressive phenotype.[3,4]
The expression of MUC1 and MUC4 in OL has also been investigated, with studies suggesting that their upregulation may indicate an increased risk of malignant transformation. The transition from OL to OSCC is a complex process, and the identification of reliable biomarkers is crucial for early diagnosis and intervention. MUC4 expression has been found to be significantly higher in dysplastic epithelium compared to normal mucosa, supporting its potential role in the early detection of OSCC. Similarly, MUC1 has been shown to be upregulated in both premalignant lesions and OSCC, further emphasizing its relevance as a marker of malignancy.[5,6]
In addition to their diagnostic potential, MUC1 and MUC4 also serve as prognostic indicators. High expression levels of MUC1 have been linked to poor clinical outcomes in various cancers, including OSCC. Elevated MUC1 levels are associated with increased tumor invasion, lymph node metastasis, and a higher likelihood of recurrence following treatment. Likewise, MUC4 overexpression has been shown to correlate with worse prognosis, particularly in cancers such as intrahepatic cholangiocarcinoma and gastric adenocarcinoma. The expression of these mucins is not only relevant in assessing tumor aggressiveness but also in predicting patient survival and response to treatment.[,8,9,10]
Studies have also explored the molecular mechanisms through which MUC1 and MUC4 contribute to tumor progression. MUC1, through its extracellular domain, interacts with several signaling pathways, including those involved in cell adhesion and migration, which are crucial for metastasis. In OSCC, MUC1 has been shown to promote cell adhesion and migration, facilitating the spread of cancer cells to distant sites. Similarly, MUC4 contributes to tumor progression by enhancing cell adhesion and promoting EMT, a process that facilitates the transition of epithelial cells into a more invasive, mesenchymal-like phenotype. These findings suggest that both MUC1 and MUC4 play critical roles in the invasive behavior of OSCC.[,12,13]
Despite the growing body of evidence supporting the role of MUC1 and MUC4 in OSCC, the exact mechanisms through which these mucins influence cancer progression remain poorly understood. Further research is needed to investigate how MUC1 and MUC4 interact with the tumor microenvironment, immune cells, and other molecular factors that contribute to tumor development and metastasis. In addition, while MUC1 and MUC4 show promise as biomarkers, more studies are needed to validate their use in clinical practice and to explore their potential for guiding therapeutic decisions in OSCC patients.[14,15]
MATERIALS AND METHODS
This retrospective observational study was conducted on formalin-fixed, paraffin-embedded (FFPE) tissue specimens obtained from the archives of the department of oral pathology and microbiology. A total of 80 histopathologically confirmed cases were selected, comprising 40 cases of OL and 40 cases of OSCC. Ethical clearance was obtained from the Institutional Ethics Committee prior to the commencement of the study.
Inclusion criteria
Histologically confirmed cases of OL and OSCC
Adequate tissue blocks with well-preserved epithelial components
Cases with complete clinical data.
Exclusion criteria
Cases with a prior history of radiotherapy or chemotherapy
Inadequate or poorly preserved tissue samples
Recurrent OSCC cases.
Immunohistochemistry procedure
Four-micron-thick sections were obtained from FFPE tissue blocks using a rotary microtome and mounted on poly-L-lysine-coated glass slides to enhance tissue adherence. The slides were allowed to dry overnight at 37°C, followed by incubation at 60°C for 1 h to improve tissue adhesion and reduce detachment during staining.
Immunohistochemical staining was carried out using the avidin-biotin peroxidase complex (ABC) method with diaminobenzidine (DAB) as the chromogen. The procedure was standardized for the detection of MUC1 and MUC4 glycoproteins using specific monoclonal antibodies.
Primary antibodies used
Anti-MUC1 monoclonal antibody: Clone: Ma695; Dilution: 1:100; Supplier: Abcam, UK
Anti-MUC4 monoclonal antibody: Clone: 8G7; Dilution: 1:100; Supplier: Thermo Fisher Scientific, USA.
Detailed staining protocol
Deparaffinization and rehydration
Tissue sections of four-micron thickness mounted on poly-L-lysine-coated slides were first subjected to deparaffinization. This was achieved by immersing the slides in three changes of xylene, each for 5 min. Rehydration followed through a series of graded alcohols, beginning with two changes of 100% ethanol for 3 min each, then 95% ethanol for 2 min, followed by 70% ethanol for 2 min. The slides were finally rinsed in distilled water for 5 min to complete the rehydration process.
Antigen retrieval
To unmask antigenic sites, heat-induced epitope retrieval was performed. Slides were immersed in 10 mM citrate buffer (pH 6.0) and placed in a microwave-safe container. They were then heated in a microwave oven at 750 watts for 15 min. Following this, the slides were cooled at room temperature for 20 min and rinsed in phosphate-buffered saline (PBS, pH 7.4) to prepare for the next step.
Blocking of endogenous peroxidase
To prevent nonspecific staining due to endogenous peroxidase activity, the slides were treated with 3% hydrogen peroxide in methanol for 10 min at room temperature. After this blocking step, the slides were washed in PBS for 5 min to remove excess reagent.
Blocking nonspecific binding
In order to reduce nonspecific antibody binding, the sections were incubated with 5% normal goat serum for 20 min. The serum used was matched to the host species of the secondary antibody, and this step helped improve the specificity of the immunoreaction.
Primary antibody incubation
The slides were then incubated with primary antibodies against MUC1 and MUC4, each prepared in PBS containing 1% bovine serum albumin. The incubation was carried out for 1 h at room temperature in a humidified chamber to maintain optimal conditions. Negative control slides were treated in the same way, except PBS was used in place of the primary antibody to confirm the specificity of staining.
Secondary antibody application
After the primary antibody step, the slides were washed in PBS and incubated with a biotinylated secondary antibody for 30 min at room temperature. This was followed by the application of an avidin-biotin complex (ABC reagent) for another 30 min, enhancing the sensitivity of the staining reaction.
Visualization
Visualization of the antigen-antibody reaction was carried out using DAB chromogen. Slides were incubated with DAB for 3-5 min until a brown precipitate, indicating positive staining, appeared. The reaction was stopped by washing the slides in distilled water.
Counterstaining and mounting
Finally, the slides were counterstained with Harris hematoxylin for 30 s to 1 min to highlight cell nuclei. Following this, the sections were dehydrated through graded alcohols, cleared in xylene, and mounted with DPX mounting medium to preserve the stained slides for microscopic examination.
Controls
Positive controls
Breast carcinoma sections were used as positive controls for MUC1. Pancreatic carcinoma sections were used as positive controls for MUC4.
Negative controls
Parallel sections were processed without primary antibodies to serve as negative controls, confirming the specificity of staining.
Evaluation of immunohistochemical staining
All stained slides were independently evaluated by two experienced oral pathologists who were blinded to the clinical data to ensure unbiased and consistent interpretation. The immunohistochemical staining was assessed based on three main parameters: Localization, intensity, and extent of staining. The localization of the immunopositivity was categorized as membranous, cytoplasmic, or a combination of both.
Staining intensity was scored on a scale from 0 to 3, where a score of 0 indicated no staining, 1 indicated weak staining, 2 represented moderate staining, and 3 denoted strong staining. The extent of staining was evaluated based on the percentage of positively stained cells, with a score of 0 assigned for <5% positivity, 1 for 5%–25%, 2 for 26%–50%, 3 for 51%–75%, and 4 for more than 75% of the cells showing positive staining.
To quantify the overall expression level, an Immunoreactivity Score (IRS) was calculated by multiplying the intensity score by the extent score. Based on the resulting IRS, the expression was classified as low when the score ranged from 0 to 3, and high when the score was 4 or more. Any discrepancies between the two observers were discussed and resolved through consensus to ensure accuracy and reliability in the evaluation.
Statistical analysis
Data were analyzed using SPSS software version 25.0 was used (The software used is IBM SPSS Statistics for Windows, developed and maintained by IBM Corp. of Armonk, NY, USA). Chi-square test was used to compare expression patterns between OL and OSCC groups. P < 0.05 was considered statistically significant.
RESULTS
The study included 80 patients, evenly divided into two groups: 40 with OL and 40 with OSCC. The mean age of patients with OL was 49.2 ± 10.1 years, whereas OSCC patients had a significantly higher mean age of 56.8 ± 9.4 years. The age difference between the two groups was statistically significant (P = 0.001), indicating that OSCC tends to occur in an older population. In terms of gender distribution, 70% of OL cases and 65% of OSCC cases were male, while females accounted for 30% and 35%, respectively. There was no statistically significant difference in gender distribution between the two groups (P = 0.617), suggesting that both conditions affect males more commonly, but gender alone may not be a distinguishing factor [Table 1].
Table 1.
Demographic distribution of study participants
| Parameter | OL (n=40) | OSCC (n=40) | Total (n=80) | P |
|---|---|---|---|---|
| Age (years), mean | 30–70 (49.2±10.1) | 35–75 (56.8±9.4) | - | 0.001* |
| Gender, n (%) | ||||
| Male | 28 (70) | 26 (65) | 54 (67.5) | 0.617 |
| Female | 12 (30) | 14 (35) | 26 (32.5) |
*P<0.05 statistically significant. OL=Oral leukoplakia, OSCC=Oral squamous cell carcinoma
Lesions in both OL and OSCC groups were most frequently located on the buccal mucosa, accounting for 45% and 40% of cases, respectively. The tongue was the second most common site, followed by the gingiva/alveolar ridge, floor of the mouth, and other or unspecified areas. The distribution of lesion sites between OL and OSCC was not statistically significant (P = 0.901), indicating that while site predilection exists, it is not a major differentiating factor between premalignant and malignant lesions in this study [Table 2].
Table 2.
Site-wise distribution of lesions
| Site | OL (n=40), n (%) | OSCC (n=40), n (%) | Total (n=80), n (%) | P |
|---|---|---|---|---|
| Buccal mucosa | 18 (45) | 16 (40) | 34 (42.5) | 0.901 |
| Tongue | 10 (25) | 12 (30) | 22 (27.5) | |
| Floor of mouth | 4 (10) | 3 (7.5) | 7 (8.75) | |
| Gingiva/alveolar ridge | 5 (12.5) | 6 (15) | 11 (13.75) | |
| Other/unspecified | 3 (7.5) | 3 (7.5) | 6 (7.5) |
OL=Oral leukoplakia, OSCC=Oral squamous cell carcinoma
MUC1 expression showed a marked difference between OL and OSCC. In OL, 20% of cases were negative for MUC1 expression, while 50% exhibited low expression and only 30% showed high expression (IRS ≥4). In contrast, OSCC cases demonstrated significantly higher MUC1 expression, with only 2.5% being negative, 15% showing low expression, and a substantial 82.5% showing high expression. The total positivity (IRS ≥1) was 80% in OL and 97.5% in OSCC. The difference in high MUC1 expression between the two groups was statistically significant (P < 0.001), indicating a strong association between elevated MUC1 levels and malignant transformation [Table 3, Figures 1 and 2].
Table 3.
Mucins 1 expression pattern in oral leukoplakia and oral squamous cell carcinoma
| MUC1 expression (IRS) | OL (n=40), n (%) | OSCC (n=40), n (%) | Total (n=80), n (%) | P |
|---|---|---|---|---|
| Negative (0) | 8 (20) | 1 (2.5) | 9 (11.25) | <0.001* |
| Low (1–3) | 20 (50) | 6 (15) | 26 (32.5) | |
| High (≥4) | 12 (30) | 33 (82.5) | 45 (56.25) | |
| Total positive | 32 (80) | 39 (97.5) | 71 (88.75) |
*P-value is less than 0.05. OL=Oral leukoplakia, OSCC=Oral squamous cell carcinoma, IRS=Immunoreactivity score, MUC1=Mucins 1
Figure 1.
MUC1 Expression pattern in OL AND OSCC
Figure 2.
Heatmap of MUC1 expression pattern in OL ans OSCC
Similar to MUC1, MUC4 expression was significantly higher in OSCC compared to OL. In OL cases, 25% were negative, 45% had low expression, and 30% had high expression. Among OSCC cases, only 7.5% were negative, 22.5% had low expression, and 70% demonstrated high MUC4 expression. The total positivity (IRS ≥1) was 75% in OL and 92.5% in OSCC. This difference in high MUC4 expression was also statistically significant (P = 0.002). These findings suggest that MUC4, like MUC1, may play a role in the progression of premalignant lesions toward malignancy [Table 4 and Figure 3].
Table 4.
Mucins 4 expression pattern in oral leukoplakia and oral squamous cell carcinoma
| MUC4 expression (IRS) | OL (n=40), n (%) | OSCC (n=40), n (%) | Total (n=80), n (%) | P |
|---|---|---|---|---|
| Negative (0) | 10 (25) | 3 (7.5) | 13 (16.25) | 0.002* |
| Low (1–3) | 18 (45) | 9 (22.5) | 27 (33.75) | |
| High (≥4) | 12 (30) | 28 (70) | 40 (50) | |
| Total positive | 30 (75) | 37 (92.5) | 67 (83.75) |
*P-value is less than 0.05. OL=Oral leukoplakia, OSCC=Oral squamous cell carcinoma, IRS=Immunoreactivity score, MUC4=Mucins 4
Figure 3.
MUC4 Expression pattern in OL and OSCC
Co-expression analysis of MUC1 and MUC4 revealed that dual positivity (MUC1+/MUC4+) was present in 65% of OL cases and 90% of OSCC cases. The MUC1+/MUC4− and MUC1−/MUC4+ patterns were seen in fewer cases, while MUC1−/MUC4− expression was seen in only 10% of OL cases and none of the OSCC cases. The difference in co-expression patterns between the groups was statistically significant (P = 0.027). This finding supports the hypothesis that co-overexpression of MUC1 and MUC4 may be indicative of malignant potential and could serve as a valuable biomarker combination in distinguishing OL from OSCC [Table 5].
Table 5.
Co-expression pattern of mucins 1 and mucins 4
| Co-expression pattern | OL (n=40), n (%) | OSCC (n=40), n (%) | Total (n=80), n (%) | P |
|---|---|---|---|---|
| MUC1+/MUC4+ | 26 (65) | 36 (90) | 62 (77.5) | |
| MUC1+/MUC4– | 6 (15) | 3 (7.5) | 9 (11.25) | |
| MUC1–/MUC4+ | 4 (10) | 1 (2.5) | 5 (6.25) | |
| MUC1–/MUC4– | 4 (10) | 0 | 4 (5) | 0.027* |
*P-value is less than 0.05. OL=Oral leukoplakia, OSCC=Oral squamous cell carcinoma, MUC=Mucins
Multivariate logistic regression was conducted to evaluate independent predictors of high MUC1 and MUC4 expression (IRS ≥4). OSCC diagnosis emerged as a strong and statistically significant predictor for both high MUC1 (odds ratio [OR] = 7.19, 95% confidence interval [CI]: 2.57-20.08, P < 0.001) and MUC4 expression (OR = 5.75, 95% CI: 2.12-15.61, P = 0.001). Age over 50 years was also a significant predictor for high MUC1 expression (OR = 2.28, P = 0.048) and showed a borderline association for MUC4 (OR = 2.15, P = 0.066). Gender and lesion site (buccal mucosa) did not show statistically significant associations with either marker. These results reinforce the observation that OSCC is independently associated with elevated MUC1 and MUC4 expression, regardless of age, gender, or lesion location [Table 6].
Table 6.
Binary logistic regression analysis for predictors of high mucins 1 and mucins 4 expression
| Predictor variable | B (MUC1) | SE (MUC1) | OR (95% CI) – MUC1 | P (MUC1) | B (MUC4) | SE (MUC4) | OR (95% CI) – MUC4 | P (MUC4) |
|---|---|---|---|---|---|---|---|---|
| Age (>50 years) | 0.822 | 0.415 | 2.28 (1.01–5.14) | 0.048* | 0.764 | 0.421 | 2.15 (0.95–4.86) | 0.066 |
| Male gender | 0.210 | 0.432 | 1.23 (0.52–2.91) | 0.629 | 0.165 | 0.446 | 1.18 (0.48–2.91) | 0.701 |
| OSCC diagnosis | 1.973 | 0.523 | 7.19 (2.57–20.08) | <0.001** | 1.749 | 0.506 | 5.75 (2.12–15.61) | 0.001* |
| Buccal mucosa site | 0.413 | 0.482 | 1.51 (0.58–3.92) | 0.393 | 0.358 | 0.472 | 1.43 (0.56–3.65) | 0.458 |
*P<0.05 statistically significant, **P<0.01 highly significant. B=Regression coefficient, SE=Standard error, OR=Odds ratio, CI=Confidence interval, MUC=Mucins, OSCC=Oral squamous cell carcinoma
These four images illustrate the immunohistochemical analysis of MUC1 and MUC4 expression in OL and OSCC. The brown staining indicates MUC1 and MUC4 positivity, with varying intensity observed in different regions. In the upper left image, MUC1 expression is observed predominantly in the epithelial layers of OL, showing weaker staining at the basal level. The other images show more intense and widespread MUC1 and MUC4 expression in OSCC tissue, especially in the more invasive regions of the carcinoma, suggesting a significant increase in MUC1 and MUC4 as the disease progresses from premalignant to malignant stages. These findings highlight the potential of MUC1 and MUC4 as biomarkers for diagnosing and predicting the prognosis of oral lesions [Figure 4].
Figure 4.
Immunohistochemical staining of MUC1 and MUC4 expression in oral leukoplakia and oral squamous cell carcinoma. These four images illustrate the immunohistochemical analysis of MUC1 and MUC4 expression in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC). The brown staining indicates MUC1 and MUC4 positivity, with varying intensity observed in different regions. In the upper left image, MUC1 expression is observed predominantly in the epithelial layers of OL, showing weaker staining at the basal level. The other images show more intense and widespread MUC1 and MUC4 expression in OSCC tissue, especially in the more invasive regions of the carcinoma, suggesting a significant increase in MUC1 and MUC4 as the disease progresses from premalignant to malignant stages. These findings highlight the potential of MUC1 and MUC4 as a biomarker for diagnosing and predicting the prognosis of oral lesions
DISCUSSION
The study of mucin expression in oral lesions has emerged as a significant area of research, particularly in the context of distinguishing premalignant lesions like OL from OSCC. The findings from this study highlight the differential expression of MUC1 and MUC4, both of which play crucial roles in the malignant transformation of oral tissues. The results suggest that MUC1 and MUC4 could serve as valuable biomarkers for diagnosing and prognosticating OSCC, particularly when analyzed together.
Our study found that MUC1 expression was significantly higher in OSCC compared to OL, with 82.5% of OSCC cases exhibiting high MUC1 expression, compared to only 30% in OL cases. This difference in expression patterns between OL and OSCC was statistically significant (P < 0.001), underscoring the potential of MUC1 as a marker for malignant transformation. These findings are consistent with those reported by Nitta et al.,[4] who also found that MUC1 expression is significantly associated with the progression of premalignant oral lesions to carcinoma. MUC1’s role in promoting tumor progression through various mechanisms, including cell adhesion, invasion, and metastasis, has been well-documented in other malignancies as well.[3] Its upregulation in OSCC likely contributes to the increased invasiveness and metastatic potential of these tumors.
Similarly, MUC4 expression was significantly higher in OSCC compared to OL [Table 4], with 70% of OSCC cases showing high MUC4 expression versus 30% in OL cases (P = 0.002). These results align with previous studies that have suggested MUC4’s critical role in oral cancer. Kamikawa et al.[16] showed that MUC4 overexpression is associated with poor prognosis in OSCC, further supporting its potential utility as a prognostic marker. MUC4, like MUC1, is involved in processes such as EMT, which is a key mechanism in the metastasis of OSCC.[16] The high expression of both MUC1 and MUC4 in OSCC could reflect their collaborative role in promoting tumor invasiveness and metastasis.
The co-expression of MUC1 and MUC4 was also a key finding of this study. We observed that 90% of OSCC cases exhibited dual positivity (MUC1+/MUC4+), compared to 65% in OL [Table 5]. The statistical significance of this difference (P = 0.027) supports the idea that the simultaneous overexpression of both mucins may be indicative of a more aggressive form of oral carcinoma. Our findings are consistent with those of Kamikawa et al.,[16] who reported that the combination of MUC1 and MUC4 expression in OSCC could predict clinical outcomes. The co-expression of these two mucins might be a reflection of their synergistic role in enhancing tumor progression, possibly through their involvement in key signaling pathways that regulate cell growth, adhesion, and invasion.
The multivariate logistic regression analysis further reinforced the association between OSCC diagnosis and high MUC1 and MUC4 expression. Both MUC1 (OR = 7.19, P < 0.001) and MUC4 (OR = 5.75, P = 0.001) expression were strong predictors of OSCC, independent of age, gender, and lesion site [Table 6]. This finding aligns with the results of Rathee et al.,[15] who also found that elevated MUC1 and MUC4 expression was significantly associated with OSCC. The association between MUC1/MUC4 expression and OSCC could help distinguish malignant lesions from benign or premalignant ones, providing a more reliable marker for early detection and risk stratification. Moreover, the association between MUC1 and MUC4 expression and poor prognosis supports their potential use as prognostic biomarkers in clinical settings.
The study’s findings also corroborate previous studies that have reported a strong association between MUC1/MUC4 expression and metastatic potential in various cancers. For instance, Saitou et al.[12] found that MUC4 was a novel prognostic factor in invasive ductal carcinoma of the pancreas, and Yamashita et al.[11] reported similar findings in cholangiocarcinoma. The ability of MUC4 to promote cell adhesion and migration in both OSCC and other cancers suggests its role in facilitating metastasis. The overexpression of these mucins may contribute to the formation of a more invasive phenotype, which could explain the poor prognosis associated with high MUC1 and MUC4 expression.
The differential expression of MUC1 and MUC4 in OL and OSCC highlights the importance of these mucins as markers of malignant transformation. As indicated in previous studies,[17] the presence of high MUC4 expression in dysplastic epithelium suggests its potential as an early marker of OSCC progression. Similarly, the significant upregulation of MUC1 in OSCC compared to OL provides further evidence that these mucins could be used to predict the likelihood of malignant transformation in oral premalignant lesions. Furthermore, the combination of MUC1 and MUC4 expression might be a useful tool in clinical practice for assessing the severity and aggressiveness of OSCC, as it may reflect the molecular changes that occur during the malignant progression of oral lesions.
While the study demonstrates the potential of MUC1 and MUC4 as diagnostic and prognostic biomarkers for OSCC, there are some limitations. First, the sample size, although adequate for preliminary analysis, may limit the generalizability of the findings. In addition, further studies are needed to explore the underlying molecular mechanisms through which MUC1 and MUC4 contribute to OSCC progression and metastasis. The functional role of these mucins in the tumor microenvironment, as well as their potential interaction with other biomarkers, needs to be elucidated to better understand their role in OSCC pathogenesis.
CONCLUSION
This study demonstrates that MUC1 and MUC4 are significantly overexpressed in OSCC compared to OL, with a strong correlation between high mucin expression and malignancy. The co-expression of MUC1 and MUC4 further highlights their potential as diagnostic and prognostic biomarkers for OSCC. Elevated levels of both mucins were found to predict poor prognosis, emphasizing their role in tumor progression and metastasis. These findings suggest that MUC1 and MUC4 could be valuable tools for early detection, risk stratification, and personalized treatment strategies for OSCC patients. Further studies are needed to explore their mechanistic roles in oral cancer progression and their potential therapeutic applications.
Conflicts of interest
There are no conflicts of interest.
Funding Statement
Nil.
REFERENCES
- 1.Angelin D, Nair BJ. Comparative evaluation of survivin expression in leukoplakia, lichen planus, and oral squamous cell carcinoma: An immunohistochemical study. J Cancer Res Ther. 2020;16:569–74. doi: 10.4103/jcrt.JCRT_421_19. [DOI] [PubMed] [Google Scholar]
- 2.Abidullah M, Nahar P, Ahmed SA. Expression of MUC4 in oral dysplastic epithelium. Int J Pharm Investig. 2019;9:85–8. [Google Scholar]
- 3.Nakamori S, Kameyama M, Imaoka S, Furukawa H, Ishikawa O, Ohigashi H, et al. MUC1 mucin expression as a marker of progression and metastasis of human colorectal carcinoma. Gastroenterology. 1994;106:353–61. doi: 10.1016/0016-5085(94)90592-4. [DOI] [PubMed] [Google Scholar]
- 4.Nitta T, Sugihara K, Tsuyama S, Murata F. Immunohistochemical study of MUC1 mucin in premalignant oral lesions and oral squamous cell carcinoma: Association with disease progression, mode of invasion, and lymph node metastasis. Cancer. 2000;88:245–54. doi: 10.1002/(sici)1097-0142(20000115)88:2<245::aid-cncr1>3.0.co;2-t. [DOI] [PubMed] [Google Scholar]
- 5.Matsumura N, Yamamoto M, Aruga A, Takasaki K, Nakano M. Correlation between expression of MUC1 core protein and outcome after surgery in mass-forming intrahepatic cholangiocarcinoma. Cancer. 2002;94:1770–6. doi: 10.1002/cncr.10398. [DOI] [PubMed] [Google Scholar]
- 6.Shibahara H, Tamada S, Higashi M, Goto M, Batra SK, Hollingsworth MA, et al. MUC4 is a novel prognostic factor of intrahepatic cholangiocarcinoma-mass forming type. Hepatology. 2004;39:220–9. doi: 10.1002/hep.20031. [DOI] [PubMed] [Google Scholar]
- 7.Tamura Y, Higashi M, Kitamoto S, Yokoyama S, Osako M, Horinouchi M, et al. MUC4 and MUC1 expression in adenocarcinoma of the stomach correlates with vessel invasion and lymph node metastasis: An immunohistochemical study of early gastric cancer. PLoS One. 2012;7:e49251. doi: 10.1371/journal.pone.0049251. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Shobita K, Lakshmi BR, Kumar GR. Immunohistochemical expression of MUC1 in different grades of oral squamous cell carcinoma. Asian Pac J Health Sci. 2018;5:165–9. [Google Scholar]
- 9.Tashiro Y, Yonezawa S, Maruyama I, Sato E, Kim YS. Immunohistochemical study of mucin carbohydrates and core proteins in human ovarian tumors. Hum Pathol. 1994;25:364–72. doi: 10.1016/0046-8177(94)90144-9. [DOI] [PubMed] [Google Scholar]
- 10.Osako M, Yonezawa S, Siddiki B, Yamada N, Sato E. Immunohistochemical study of mucin carbohydrates and core proteins in human pancreatic tumors. Cancer. 1993;71:2191–9. doi: 10.1002/1097-0142(19930401)71:7<2191::aid-cncr2820710705>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
- 11.Yamashita K, Shibahara H, Higashi M, Yonezawa S, Sato E. Immunohistochemical study of mucin carbohydrates and core proteins in hepatolithiasis and cholangiocarcinoma. Int J Cancer. 1993;55:82–91. doi: 10.1002/ijc.2910550116. [DOI] [PubMed] [Google Scholar]
- 12.Saitou M, Goto M, Horinouchi M, Tamada S, Nagata K, Hamada T, et al. MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas. J Clin Pathol. 2005;58:845–52. doi: 10.1136/jcp.2004.023572. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Kohli M, Shivam AK, Ahuja P, Dutta J. Mucin-4: A novel marker for oral cancer. J Oral Maxillofac Pathol. 2019;23:49–53. doi: 10.4103/jomfp.JOMFP_175_16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Kumar MH, Sanjai K, Kumarswamy J, Keshavaiah R, Papaiah L, Divya S. Expression of MUC1 mucin in potentially malignant disorders, oral squamous cell carcinoma and normal oral mucosa: An immunohistochemical study. J Oral Maxillofac Pathol. 2016;20:214–8. doi: 10.4103/0973-029X.185916. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Rathee R, Devi A, Narwal A, Kamboj M, Singh S. Immunohistochemical coexpression of MUC1 and MUC4 in oral leukoplakia and oral squamous cell carcinoma. Head Neck Pathol. 2021;15:831–42. doi: 10.1007/s12105-021-01291-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Kamikawa Y, Kanmura Y, Hamada T, Yamada N, Macha MA, Batra SK, et al. Combination of MUC1 and MUC4 expression predicts clinical outcome in patients with oral squamous cell carcinoma. Int J Clin Oncol. 2015;20:298–307. doi: 10.1007/s10147-014-0710-6. [DOI] [PubMed] [Google Scholar]
- 17.Narashiman S, Narasimhan M, Venkatraman G. Expression of Mucin 4 in leukoplakia and oral squamous cell carcinoma: An immunohistochemical study. J Oral Maxillofac Pathol. 2014;18:25–31. doi: 10.4103/0973-029X.131887. [DOI] [PMC free article] [PubMed] [Google Scholar]