Salivary gland tumors (SGTs) are a rare and histologically heterogeneous group of tumors with a wide range of clinical presentation, biological behaviour, and prognosis.[1] These tumors account for approximately 3–10% of all head and neck neoplasms and nearly 0.3% of human neoplasms.[2] About 64- 80% occur in the parotid gland and the submandibular and minor salivary glands.[3] Benign tumors make up about 54-79% of cases, with pleomorphic adenoma being the most common benign lesion, while mucoepidermoid carcinoma and adenoid cystic carcinoma are common malignant lesions. [4]

Proper differentiation between benign and malignant salivary gland tumors is crucial for the correct treatment planning and prognosis.[5] Malignant salivary gland tumors can present with infiltrative growth, perineural invasion, local recurrence, and distant metastasis, which involve complex molecular mechanisms involved in the progression of tumors.[6] Immunohistochemistry and molecular pathology have come a long way in facilitating the characterization of these neoplasms in recent years, especially in diagnostically challenging cases.[7] The enzymes that participate in the extracellular matrix remodeling process have been of interest due to their role in the invasion and malignant transformation of tumors.[8] Out of these, two different types of proteases, Cathepsin D and matrix metalloproteinase-9 (MMP-9), have been identified that could offer complementary information on the invasive potential of salivary gland neoplasms.[9, 10]

Cathepsin D is an aspartic protease localized to the lysosomes that plays a role in intracellular protein degradation, but is also related to tumor growth and the degradation of extracellular matrix and malignant progression.[11] Increased expression and abnormal secretion of Cathepsin D have been associated with aggressive biological behavior in various malignancies.[12] Mohan et al. (2022) investigated its immunohistochemical expression in 40 salivary gland tumors, including 20 benign and 20 malignant, and found that the expression was significantly higher in the malignant tumors.[10] The mean immunoreactive score was 6.284 ± 4.377 in the malignant neoplasms as opposed to 2.281 ± 2.459 in the benign neoplasms (p = 0.004).[10] Moreover, 95% of malignant tumors were reported to be Cathepsin D immunopositive, while 80% of benign tumors were positive in the same staining, and mucoepidermoid carcinoma showed the highest expression.[10]

Gelatinase B (MMP9) is a zinc-dependent protease involved in extracellular matrix degradation, basement membrane remodeling, angiogenesis, and tumor cell invasion.[13] Recent studies have highlighted its role beyond the degradation of the extracellular matrix, in regulating adhesion and invasive cell behavior.[14, 15] Biological relevance of MMP-9 in cancer progression is known, but the connection between the immunohistochemical expression of MMP-9 and the benign or malignant nature of salivary gland tumors remains unclear.[16]

Proteolytic pathways with Cathepsin D and MMP-9 have been linked to tumor progression, but their relative immunohistochemical expression in benign and malignant salivary gland tumors is under-studied. It is particularly significant that histologically indistinguishable salivary gland neoplasms can have very different clinical characteristics, and it is important to be able to identify markers to reflect the differences in tumor biology. Examining these two proteases together may provide valuable insights into the relationship between lysosomal proteolytic activity, extracellular matrix remodeling, and malignant potential. Furthermore, this staining intensity and distribution may be useful for determining their expression patterns that are linked to benign and malignant histological features. By exploring these molecular characteristics, the study seeks to contribute to a better understanding of salivary gland tumor biology and establish a foundation for further research into their diagnostic and prognostic applications. The present study is therefore designed to assess and compare the expression of Cathepsin D and MMP-9 in both benign and malignant salivary gland tumors and to explore their possible value as markers of malignant biological behaviour in combination.

A comparative cross-sectional study was conducted in the Department of Pathology, Rawal Institute of Health Sciences, Islamabad, Pakistan, over a period of six months, from 1st December 2025 to 31st May 2026. The sample size was estimated using the OpenEpi sample size calculator based on an earlier study by Mohan et al. (2022) that had shown a 95% immunohistochemical positivity of Cathepsin D in the malignant salivary gland tumors and 80% in the benign salivary gland tumors.[10] A two-sided confidence level of 95%, statistical power of 80%, and an equal allocation ratio of 1:1 were considered. The minimum sample size was determined to be 176 cases, which included 88 benign and 88 malignant salivary gland tumors. A non-probability consecutive sampling technique was employed. The study included patients of either gender and all age groups with histopathologically confirmed benign or malignant salivary gland tumors arising from the major or minor salivary glands. Cases that had sufficient blocks of formalin-fixed, paraffin-embedded (FFPE) specimens with representative viable tumor tissue were selected. Specimens were only included if there was enough preserved tissue for immunohistochemical staining of both Cathepsin D and MMP-9, and a complete set of essential histopathological data. The cases with poorly preserved, insufficient, or heavily necrotic tissue samples were ruled out. Specimens in which the tumor components were inadequate for immunohistochemical evaluation, full histopathological records were not available, or there was some doubt regarding the histopathological diagnosis were also excluded. Patients who had undergone neoadjuvant chemotherapy or radiotherapy prior to tissue sampling were excluded, as treatment-related changes may affect protein expression. To minimize potential confounding factors, recurrent tumors, metastases from other primary lesions involving the salivary glands, and non-neoplastic lesions of the salivary glands were excluded. Following approval from the institutional ethics review committee, histopathologically confirmed cases of benign and malignant salivary gland tumors were identified from the records of the Department of Pathology, Rawal Institute of Health Sciences, Islamabad. The relevant demographic and clinicopathological data such as age, sex, site, histological diagnosis, and grade of the tumor (when applicable) were collected and recorded on a structured data collection proforma. Histopathological slides were re-evaluated for confirmation of diagnosis and to determine representative areas of viable tumor tissue. Formalin-fixed paraffin-embedded tissue blocks were retrieved, and sections around 3–4 µm thick were cut for immunohistochemical staining. Cathepsin D and MMP-9 were stained with the primary antibodies following the manufacturer's protocol and a standardized laboratory protocol. The tissue sections were deparaffinised, rehydrated, and then treated with the appropriate antigen retrieval; endogenous peroxidase activity was blocked, and the tissue was then incubated with the appropriate primary antibody. Immunoreactivity was visualized using a suitable secondary detection system and chromogen, followed by hematoxylin counterstaining. The staining was done with appropriate positive and negative controls. Light microscopy was used to view the stained slides, and the intensity of the stain and the number of tumour cells stained were used to determine the immunohistochemical expression. The percentage of positive tumor cells was scored as 0 (no staining), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (more than 75%), while staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The proportion score was multiplied by the intensity score to get a final immunoreactive score of 0-12. The expression scores of Cathepsin D and MMP-9 were recorded separately and compared between benign and malignant salivary gland tumors to assess differences in their immunohistochemical expression patterns. The collected data were entered and analyzed using IBM SPSS Statistics. Quantitative variables, including age and immunohistochemical expression scores, were summarized using means and standard deviations or medians and interquartile ranges, depending on data distribution. Qualitative variables, including gender, tumor location, histopathological diagnosis, and immunohistochemical positivity, were presented as frequencies and percentages. The Shapiro–Wilk test was applied to assess the normality of quantitative data. The independent-samples t-test was used to compare Cathepsin D and MMP-9 expression scores between benign and malignant salivary gland tumors. The chi-square test was applied to examine associations between categorical variables. Spearman's rank correlation coefficient was calculated to evaluate the relationship between Cathepsin D and MMP-9 immunohistochemical expression scores. A p-value of less than 0.05 was considered statistically significant.

A total of 176 patients were enrolled, divided into equal numbers between benign and malignant salivary gland tumors. Patients with malignant tumors were significantly older than those with benign tumors (p < 0.001). The distribution by sex was also significantly different between groups (p=0.049), while the anatomic site of tumor was not significantly associated with tumor type (p=0.085). The parotid gland was the predominant site in both groups (Table 1).

The most frequent benign neoplasm was pleomorphic adenoma, and the most frequent malignant ones were mucoepidermoid carcinoma followed by adenoid cystic carcinoma. In the malignant cases, there was a fairly equal distribution between low, intermediate, and high histopathological grades, with some tumors not being graded (Table 2).

Immunohistochemical data revealed that Cathepsin D and MMP-9 had significantly higher levels of positivity in malignant tumors as compared to benign tumors (p=0.001 and p<0.001, respectively). In the same way, intense staining was more common in malignant neoplasms and showed a statistically significant difference for both markers (p < 0.001). Cathepsin D demonstrated greater overall positivity than MMP-9 in both tumor groups (Table 3).

Malignant tumors exhibited a greater proportion of positively stained tumor cells and higher median immunoreactive scores for both Cathepsin D and MMP-9. In contrast, benign tumors showed mainly low staining distribution and low immunoreactive scores. (Table 4).

The proportion of tumors positive for Cathepsin D and MMP-9 increased progressively from low-grade to high-grade malignant neoplasms. The positivity rate for cathepsin D did not change at each grade, while MMP-9 showed a greater degree of positivity with increasing histopathological grade. However, statistical significance for these grade-related trends was not established (Table 5).

Spearman's correlation analysis indicated a positive association between Cathepsin D and MMP-9 immunoreactive scores in both benign and malignant tumors. The correlation was found to be stronger for malignant neoplasms (rₛ = 0.58) than for benign tumors (rₛ = 0.39), and both were reported statistically significant (p < 0.001). (Table 6).

Table 1. Demographic and anatomical characteristics (n = 176)

Table 2. Histopathological diagnoses and malignant grade (n = 176)

Table 3. Immunohistochemical positivity and staining intensity (n = 176)

Table 4. Proportion of positive cells and immunoreactive scores (n = 176)

Table 5. Marker positivity by histopathological grade in malignant tumors (n = 88)

Table 6. Correlation of Cathepsin D and MMP-9 immunoreactive scores

Figure 1. Pleomorphic adenoma of the salivary gland showing weak focal

immunohistochemical staining

Figure 2. Pleomorphic adenoma of the salivary gland showing weak to

moderate cytoplasmic immunohistochemical staining

Figure 3. Mucoepidermoid carcinoma of the salivary gland showing

strong diffuse cytoplasmic immunohistochemical staining.

The present study compared the immunohistochemical expression of Cathepsin D and MMP-9 in benign and malignant salivary gland tumors. The results demonstrated that there were significantly higher levels of expression of both markers in malignant tumors, with higher staining intensity and immunoreactive score. These differences indicated that there was a correlation between the expression of the proteolytic markers and the malignant tumor status, but the diagnostic value and prognostic value of these differences should be further investigated. Patients with malignant tumors were statistically significantly older than those with benign tumors, reinforcing the findings of Khan et al. (2025), who found a significant relationship between salivary gland malignancy and age.[17] However, unlike the present findings, Khan et al. observed no significant gender association.[17] Ammar et al. (2021) and Panda et al. (2021) also reported variations in demographic and anatomical distribution of salivary gland tumors, highlighting variations across study populations.[18, 19] The parotid gland predominated in the present study, with no significant difference between the benign and malignant group distributions. Pleomorphic adenoma was the most frequent benign tumor, whereas mucoepidermoid carcinoma was the predominant malignant neoplasm. Mahmood et al. (2022) and Lee et al. (2023), also found pleomorphic adenoma and mucoepidermoid carcinoma as the most common benign and malignant diagnoses, respectively.[20] The relative frequencies of individual tumor types may reflect geographical variation, sample size, and case selection. The distribution of low-, intermediate-, and high-grade malignant tumors in the present study, on the other hand, showed a similar distribution pattern, as the salivary gland carcinomas were found to be histologically heterogeneous. The expression level of Cathepsin D was significantly higher in malignant tumors than in benign tumors (p = 0.001), and the staining intensity and immunoreactive scores were higher. These findings were consistent with Mohan et al. (2022), who reported significantly greater Cathepsin D expression in malignant salivary gland tumors (p = 0.004).[10] Higher immunoreactive scores were also found in malignant lesions in their study, but no significant association was found with the parameters measured as indicators of prognosis.[12] The results of the immunohistochemical studies were also correlated with other studies of molecular markers in salivary gland neoplasms. In malignant salivary gland tumors, associations between PROX1 and MTA1 expression were reported by Soliman et al. (2025).[21] In a similar study, genetic changes linked to poor prognosis in salivary gland cancers were found in a 2025 molecular study.[21] This research examined various molecular pathways, but all emphasized the need for the study of molecular markers associated with tumors. In the present study, Cathepsin D and MMP-9 demonstrated higher expression in malignant lesions, but their association with clinical outcomes was not evaluated. A positive correlation was observed between Cathepsin D and MMP-9 immunoreactive scores in both tumor groups, with a stronger correlation among malignant neoplasms. This result indicated a possible correlation between the expressions of the two markers but did not imply a direct interaction between them. The results, therefore, should be considered evidence that these immunohistochemical patterns of expression are associated with each other, but not proof of coordinated proteolytic activity. The present study was limited in a number of aspects, such as having a single-center design, non-probability sampling, and relatively heterogeneous histopathological composition. Comparisons may also be affected by differences in grading between the various kinds of malignant tumors. In addition, there were no follow-up data or survival outcomes to evaluate the independent prognostic value of either marker. More extensive studies with subtype-specific analysis and clinical outcomes would be valuable in defining their potential diagnostic and prognostic applications.

Cathepsin D and MMP-9 showed higher immunohistochemical expression in malignant salivary gland tumors than in the benign ones, with higher staining intensity and immunoreactive score. The positive association of both markers, especially in the case of malignant tumors, further emphasized the potential association with malignant biological behavior. These observations suggested that Cathepsin D and MMP-9 may serve as complementary markers in the histopathological assessment of salivary gland neoplasms.