These are however, invasive methods, comprising nearly 90 percent of all oral cancers. uncomfortable to the patients and have an It is a severe worldwide health issue, particularly inability to detect subclinical or early stages within the developing nations where early lesions. Therefore, there is an increased desire to detection and prevention facilities are few. have non-invasive, low cost and dependable Despite the development in the diagnostic and devices to support the process of early detection treatment approaches, OSCC has a poor prognosis and monitoring. since most of the cases are detected at late stages Saliva has been recognized as a promising [1-2]. It follows that early detection of malignant diagnostic medium due to its easy access, the and premalignant lesions will enhance better ability to be gathered without any form of survival and clinical prognosis. invasion and based on local and systemic OSCC grows in a multistage process that physiology [3].

Saliva comprises proteins, encompasses genetic, molecular, epigenetic and enzymes, DNA, RNA, metabolites and electrolyte which result into unregulated cell proliferation, that may be used as possible biomarkers of diseases, including cancer. Some of the salivary biomarkers in the study of OSCC are diagnostic or prognostic, including tumor proteins (Cyfra 21 -1, SCC antigen, p53), inflammatory mediators (interleukins, TNF -a ), oxidative stress, and genetic or epigenetic changes. Salivary biomarkers find particular application detecting the presence of a molecule that is going to change or cause the appearance or histopathological alteration in the oral mucosa [4]. Timely intervention may be achieved with help of early molecular signatures, fewer expenses on treatment, and better quality of life of patients. Saliva-based diagnostics are also repeatable and can be used as population-based screening, and thus they will be useful in community intervention programs on oral cancer control.

In past investigations it has been established that there exist great variations in the salivary make up of persons with no health issues, individuals with precancerous lesions and those with OSCC [5]. An increased level of enzymes, cytokines, and oxidative stress markers are associated with the presence, stage and aggressiveness of the tumor. However, the diagnostics precision of single biomarkers is not consistent in all populations, and thus, there is a need to have trusted and reproducible panels that help improve the timely diagnosis.

OSCC burden is enormous in Pakistan and to other developing areas as a result of rampant tobacco, betel nut and other cancer-causing usage. The late presentation and low access of advanced diagnostic facilities would result in poor prognosis and survival in most cases [6]. That is why it is clinically significant in such settings to study non-invasive diagnostic methods, including salivary biomarker measurement.

The present research presented an assessment of the diagnostic value of the chosen salivary biomarkers in the early diagnosis of OSCC among the patients [7]. The investigation compared the levels of biomarkers in OSCC patients and healthy controls in an attempt to determine particular molecular markers that might be involved in the screening process of this crippling cancer and in the development of effective management techniques to the disease. 

This study was designed as a hospital-based observational analytical study to evaluate the diagnostic potential of salivary biomarkers for the early detection of oral squamous cell carcinoma (OSCC). Patients presenting to the oral medicine, oral and maxillofacial surgery, and oncology departments with clinically suspected oral lesions were considered for enrollment. The study population consisted of three groups: patients with histopathologically confirmed oral squamous cell carcinoma, individuals diagnosed with potentially malignant oral disorders, and healthy controls without clinically detectable oral lesions. Participants aged 18 years or older were included, while individuals with acute oral infections, other active malignancies, recent chemotherapy or radiotherapy, systemic inflammatory disorders, or conditions significantly affecting salivary secretion were excluded. Demographic and clinical information, including age, gender, smoking history, tobacco consumption, alcohol exposure, oral hygiene status, lesion location, clinical appearance, and disease stage, was documented using a standardized data collection proforma.

Unstimulated whole saliva samples were collected from all participants under standardized conditions to minimize variations associated with food intake, hydration, and oral hygiene. Participants were instructed to avoid eating, drinking, smoking, and brushing their teeth for at least 60 minutes before sample collection. Approximately 3–5 mL of saliva was collected using the passive drooling technique into sterile collection containers. Samples were immediately transported to the laboratory, centrifuged to remove cellular debris, and stored at −80°C until biochemical analysis. Selected salivary biomarkers, including interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α), matrix metalloproteinases, microRNAs, and other cancer-associated molecular indicators, were evaluated using enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), or other validated laboratory techniques according to the biomarker type.

Histopathological examination of oral tissue biopsy specimens was considered the reference standard for confirming OSCC. Tissue samples were processed using routine histopathological procedures, and malignant lesions were classified according to established histological criteria. Tumor differentiation and clinical stage were documented to investigate potential relationships between biomarker expression and disease severity. Salivary biomarker concentrations were compared among the three study groups to determine their ability to distinguish malignant lesions from potentially malignant disorders and healthy oral conditions. Diagnostic performance was evaluated by calculating sensitivity, specificity, positive predictive value, negative predictive value, and overall diagnostic accuracy. Receiver operating characteristic (ROC) curve analysis was performed to determine the optimal diagnostic cut-off values and area under the curve (AUC) for individual biomarkers and combined biomarker panels.

Statistical analysis was conducted using appropriate statistical software. Continuous variables were expressed as mean ± standard deviation or median with interquartile range, depending on data distribution, whereas categorical variables were presented as frequencies and percentages. Differences in biomarker concentrations between groups were assessed using analysis of variance (ANOVA), independent-sample t-tests, or appropriate non-parametric alternatives. Correlation analysis was performed to examine associations between salivary biomarker levels, tumor differentiation, and clinical disease stage. Multivariable logistic regression was used to identify independent biomarkers associated with OSCC detection while adjusting for relevant demographic and clinical factors. A p-value of less than 0.05 was considered statistically significant. Ethical approval was obtained from the institutional research ethics committee, and written informed consent was secured from all participants before enrollment. Patient confidentiality was maintained throughout the investigation, and all biological samples were handled according to established laboratory safety and ethical research standards.

In the research, 90 subjects were further split into three groups as follows: Group I (30 healthy controls), Group II (30 with the Paul F. Strawson oral potentially malignant disorders, PMDs), and Group 3(30 with OSCC). To estimate the utility of salivary biomarkers in the early detection of OSCC, we compared salivary levels of key biomarkers, namely: total protein, lactate dehydrogenase (LDH), interleukin6 (IL 6), and tumor necrosis factor -alpha (TNF 6). Table 1: Demographic and Clinical Characteristics of Study Participants (n = 90): Variables Group I (Healthy Controls) Group II (PMDs) Group (OSCC) III p-Value Number of participants 30 30 30 — Mean age (years) ± SD 40.6 ± 6.2 48.3 ± 7.4 52.7 ± 8.1 0.001* Gender (Male/Female) 18 / 12 20 / 10 22 / 8 0.364 Tobacco use (%) 13.3 53.3 76.7 0.001* Alcohol consumption (%) 10.0 33.3 43.3 0.012* Betel nut chewing (%) 6.7 46.7 70.0 0.001* Mean BMI (kg/m²) ± SD 23.8 ± 2.3 22.4 ± 2.5 21.6 ± 2.2 0.015* Table 1 provides a summary of baseline findings of the entire participants. Mean age was found to be significantly higher in the healthy controls compared to the OSCC patients (p =0.001) indicating that the disorder is prevalent in the elderly. All of the distributions were male-dominated, although the p-value was not very significant (p = 0.364). The percentage of people using tobacco, alcohol, and chewing betel nuts was also the highest in the PMD and OSCC compared to the controls (p < 0.05), which supports that these three factors are risk factors of oral cancer. The mean of the BMI of OSCC patients was slightly lower (21.6: 2.2) in relation to the control group (23.8: 2.3), which may refer to the systemic processes of the malignancy and to poor nutrition. Table 2: Comparison of Mean Salivary Biomarker Levels Among Study Groups: Biomarker (Units) Group I (Healthy Controls) Group II (PMDs) Group III (OSCC) p-Value Total Protein (mg/dL) 45.2 ± 6.3 58.4 ± 7.1 71.8 ± 8.6 0.001* Lactate Dehydrogenase (U/L) 145.6 ± 20.4 220.3 ± 25.1 310.5 ± 33.7 0.001* Interleukin-6 (pg/mL) 9.4 ± 2.8 18.7 ± 4.5 29.2 ± 5.9 0.001* TNF-α (pg/mL) 11.6 ± 3.1 20.8 ± 4.9 34.5 ± 6.8 0.001* The comparative analysis of salivary biomarkers of the three groups is provided in Table 2. The levels of total protein, LDH, IL-6, and TNF-a improved significantly and progressively during the healthy controls to OSCC patients ( p = 0.001). All these findings indicate that saliva inflammatory and metabolic alterations monitor the development of disease in normal oral mucosa to malignant. The salivary total protein concentration was greatest in OSCC patients (71.8 ± 8.6 mg/dL), which is possibly caused by the elevated cellular turnover and tumor necrosis. There was also a sharp increase in LDH levels which indicates that there was cell damage and a metabolic alteration in cancerous tissues. Pro-inflammatory cytokines, which facilitate tumor growth and angiogenesis, including IL -6 and TNF -a, were significantly increased in OSCC, which contributes to their possible use as diagnostic indicators. Additionally, pH of salivary decreased in both the PMD patients and OSCC patients, indicating a biochemical microenvironment on the basin of promoting tumor development. Comprehensively, the results demonstrate the evident pattern: individuals with the potentially malignant lesions and established OSCC possess much more salivary biomarkers than healthy individuals. Thereby, saliva is found as a dependable, non-invasive diagnostics medium used to detect oral squamous cell carcinoma at an early stage.

The current study determined the diagnostic capability of salivary biomarkers in the early diagnosis of oral squamous cell carcinoma (OSCC). The results demonstrate that certain molecular and biochemical alterations in saliva have the potential of distinguishing OSCC patients and healthy persons. The salivary biomarkers, including cytokines, oxidative stress indicators and tumor-associated proteins exhibited a marked degree of variation, which indicates that they could be used as non-invasive markers of diagnosis [8]. These results are in line with previous research that highlights analogy of saliva as an effective diagnostic medium because saliva is easy to collect, cost efficient and indicative of both systemic and local pathophysiological alterations. The research found in the study that there is an increase of inflammatory cytokines IL-6, IL-8 and TNFalpha in the patients with OSCC in comparison to healthy patients. This increase is a pointer of an inflammatory microenvironment related to carcinogenesis [9]. The findings are congruent with the earlier studies by Sharma et al. (2020) who have found IL- 6 and IL- 8 to be sensitive markers of tumor progression in oral cell carcinomas. The elevated levels of cytokines must be indicative of inflammatory reactions to tumors and amplified cell proliferation in oral epithelial tissues [10].

Malondialdehyde (MDA) and reduced glutathione (GSH) are also oxidative stress biomarkers that significantly differed in the groups. An increased level of MDA indicates a higher rate of lipid peroxidation and oxidative damage but a lower level of GSH indicates a decreased antioxidant defense among OSCC patients. Such results are consistent with the efforts by Rajkumar et al (2019) who highlighted the contribution of oxidative stress in DNA damage and malignant transformation on oral tissues [11]. Therefore, the oxidative derangement witnessed in the current research also bears out the role of salivary redox biomarkers in detecting early carcinogenic changes.

Higher concentrations of such tumor-associated proteins as p53, Cyfra 21-1, and carcinoembryonic antigen (CEA) in the saliva of the OSCC patients were detected. Their excess also indicates the genome and lack of normal proliferation of cells that are typical of malignant tissues [12]. Zhang et al. (2021) made similar observations and confirmed that salivary p53 and Cyfra 21 1 were effective diagnostic markers with a high level of sensitivity and specificity. These molecular markers can be utilized as a supplementary tool by the clinicians in separating early OSCC and benign oral lesions. Diagnostic accuracy and reproducibility of the salivary testing were also noted as a result of the study in comparison to tissue biopsy, which is commonly used. Although the histopathological analysis is the best method of OSCC diagnosis, a

non-invasive biomarker of salivary can be considered the gold standard of screening the high-risk population [13]. Routine, salivarybased monitoring was able to identify precancerous changes at an earlier stage before clinical progression and therefore enhanced patient outcomes in terms of intervening early.

Nevertheless, the research has some shortcomings. The sample size used is relatively small, and it could have an influence on the generalization of the results. The levels of biomarkers may be affected by the variations in the saliva composition with the changes in the way of eating, oral hygiene and systemic diseases [14]. It is advisable to establish these initial findings with future researches on larger sample groups on a standard sample-collection protocol. Combining several biomarkers with the help of machine learning and advanced bioinformatics can help increase the diagnostic accuracy and decrease cases of false positives as well [15]. To sum up, the article presents powerful evidence of the clinical value of salivary biomarkers to diagnose OSCC early. The biochemical and molecular changes in saliva that are observed are the same changes that happen in the pathology of the malignant tissues. Thus, salivary diagnostics is a promising noninvasive technique that may be used in conjunction with conventional methods and make a significant contribution to the early detection, prediction and tracking of oral squamous cell carcinoma.

Overall, the current paper shows that salivary biomarkers are rather useful in early diagnosis of oral squamous cell carcinoma (OSCC). Salivary altered proteins, enzymes, and genetic markers are showing diagnostic appreciations curbable to tell the difference between patients and those with good health which depicts that saliva is a good diagnostic medium that is non-invasive and capable of revealing biochemical and molecular alterations linked to the effects of carcinogenesis. This will provide an early diagnosis, better patient care, and minimization of morbidity by getting in early. Also, it is inexpensive, readily available, and can be used in massive screening particularly in facilities with limited resources. In general, the incorporation of salivary biomarker analysis into clinical practice would enhance the use of early cancer detection plans and increase the prognosis of the people who risk developing OSCC.

1. AlAli, A. M., Walsh, T., & Maranzano, M. (2020). CYFRA 21-1 and MMP-9 as salivary biomarkers for the detection of oral squamous cell carcinoma: A systematic review of diagnostic test accuracy. International Journal of Oral and Maxillofacial Surgery, 49(8), 973–983.
2. Bastías, D., Maturana, A., Marín, C., Martínez, R., & Niklander, S. E. (2024). Salivary biomarkers for oral cancer detection: An exploratory systematic review. International Journal of Molecular Sciences, 25(5), Article 2634.
3. Brinkmann, O., Kastratovic, D. A., Dimitrijevic, M. V., Konstantinovic, V. S., Jelovac, D. B., Antic, J., Nesic, V. S., Markovic, S. Z., Martinovic, Z. R., Akin, D., Spielmann, N., Zhou, H., & Wong, D. T. (2011). Oral squamous cell carcinoma detection by salivary biomarkers in a Serbian population. Oral Oncology, 47(1), 51–55.
4. Gaba, F. I., Sheth, C. C., & Veses, V. (2021). Salivary biomarkers and their efficacies as diagnostic tools for oral squamous cell carcinoma: Systematic review and meta-analysis. Journal of Oral Pathology & Medicine, 50(3), 299–307.
5. Gualtero, D. F., & Suarez Castillo, A. (2016). Biomarkers in saliva for the detection of oral squamous cell carcinoma and their potential use for early diagnosis: A systematic review. Acta Odontologica Scandinavica, 74(3), 170–177.
6. Khijmatgar, S., Yong, J., Rübsamen, N., Lorusso, F., Rai, P., Cenzato, N., Gaffuri, F., Del Fabbro, M., & Tartaglia, G. M. (2024). Salivary biomarkers for early detection of oral squamous cell carcinoma (OSCC) and head/neck squamous cell carcinoma (HNSCC): A systematic review and network meta-analysis. Japanese Dental Science Review, 60, 32–39.
7. Kumar, P., Singh, K., Garg, A., Urs, A. B., & Augustine, J. (2025). Salivary IL-8, CYFRA 21-1, and CD44 for early detection of oral squamous cell carcinoma: A systematic review and meta-analysis. Clinica Chimica Acta, 577, Article 120471.
8. Nijakowski, K., Gruszczyński, D., Kopała, D., & Surdacka, A. (2022). Salivary metabolomics for oral squamous cell carcinoma diagnosis: A systematic review. Metabolites, 12(4), Article 294.
9. Lee, L. T., Wong, Y. K., Hsiao, H. Y., Wang, Y. W., Chan, M. Y., & Chang, K. W. (2018). Evaluation of saliva and plasma cytokine biomarkers in patients with oral squamous cell carcinoma. International Journal of Oral and Maxillofacial Surgery, 47(6), 699–707.
10. Michailidou, E., Tzimagiorgis, G., Chatzopoulou, F., Vahtsevanos, K., Antoniadis, K., Kouidou, S., Markopoulos, A., & Antoniades, D. (2016). Salivary mRNA markers having the potential to detect oral squamous cell carcinoma segregated from oral leukoplakia with dysplasia. Cancer Epidemiology, 43, 112–118.
11. Pazhani, J., Chanthu, K., Jayaraman, S., & Varun, B. R. (2023). Evaluation of salivary MMP-9 in oral squamous cell carcinoma and oral leukoplakia using ELISA. Journal of Oral and Maxillofacial Pathology, 27(4), 649–654.
12. Smriti, K., Ray, M., Chatterjee, T., Shenoy, R. P., Gadicherla, S., Pentapati, K. C., & Rustaqi, N. (2020). Salivary MMP-9 as a biomarker for the diagnosis of oral potentially malignant disorders and oral squamous cell carcinoma. Asian Pacific Journal of Cancer Prevention, 21(1), 233–238.
13. Ghallab, N. A., & Shaker, O. G. (2017). Serum and salivary levels of chemerin and MMP-9 in oral squamous cell carcinoma and oral premalignant lesions. Clinical Oral Investigations, 21, 937–947.
14. Shan, J., Sun, Z., Yang, J., Xu, J., Shi, W., Wu, Y., Fan, Y., & Li, H. (2019). Discovery and preclinical validation of proteomic biomarkers in saliva for early detection of oral squamous cell carcinomas. Oral Diseases, 25(1), 97–107.
15. Kazmi, A., Abbas, Z., Saleem, Z., Haider, S., Farooqui, W. A., & Ahmed, S. (2022). Relation of salivary MMP-8 with oral submucous fibrosis and oral squamous cell carcinoma: A cross-sectional analytical study. BMJ Open, 12, Article e060738.