Evaluation of Salivary Cytokine Profiles as Biomarkers for Early Detection of Oral Potentially Malignant Disorders

BackgroundOral cancer (OC) is usually diagnosed at advanced clinical stages due to its asymptomatic nature and absence of pathognomonic signs in its early development phase. Delayed diagnosis is one of the major causes of OC treatment failure and poor prognosis. Development of alternative diagnostic approaches are imperative for improving early detection and therapeutic success rates. Salivary cytokines (SC) have been studied as potential diagnostic biomarkers for OC and may represent a potential tool for improvement of its early detection.MethodsIn this systematic review and meta-analysis we identified SC studied as OC biomarkers by systematically reviewing the PubMed and Cochrane Library databases using the terms: “oral cancer”, “cytokine”, and “saliva”, and also combined with “interleukin” or “interferon”. Only case-control studies that measured SC by ELISA from treatment naïve patients were included in the qualitative review. For the meta-analysis were included all comparable studies that provided enough data (sample size, mean and standard deviation or standard error of the mean) for SC levels in OC patients, non-cancer controls and patients with oral potentially malignant disorders (OPMD), including leukoplakia. Comparisons with patients with oral lichen planus (OLP) and gingivitis were included in the qualitative analysis.ResultsA total of 28 articles (from 2004 to 2018) were included in the systematic review, describing 10 different SC, being IL-8 and IL-6 the most studied ones. SC levels were consistently higher among OC patients when compared to healthy controls and to patients with OPMD, OLP and gingivitis. Meta-analysis including 23 eligible studies showed that IL-8, IL-6, TNF-α, IL-1β and IL-10 salivary levels were significantly higher in OC patients compared to controls; and that IL-8, IL-6, TNF-α and IL-1β salivary levels were also higher in OC patients compared to individuals with OPMD. When compared to healthy controls, OPMD patients showed significantly higher IL-6 and TNF-α salivary levels.ConclusionsOur analyses showed that the salivary levels of some cytokines are consistently different among OC, OPMD and healthy patients, indicating that these SC may represent potential diagnostic biomarkers for OC and OPMD. Despite of that, SC levels were highly variable among studies, suggesting that further technical improvement and standardization for SC measurement by ELISA is needed in order to successfully translate these biomarkers to the clinical practice.

Background: Oral squamous cell carcinoma (OSCC) is a life-threatening disease. It could be preceded by oral potentially malignant disorders (OPMDs). It was confirmed that chronic inflammation can promote carcinogenesis. Cytokines play a crucial role in this process. The aim of the study was to evaluate interleukin-1alpha (IL-1α), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor alpha (TNF-α) in tissue specimens and saliva of patients with OSCC and OPMDs. Methods: Cytokines were evaluated in 60 tissue specimens of pathological lesions (OSCCs or OPMDs) and in 7 controls (normal oral mucosa, NOM) by immunohistochemistry and in saliva of 45 patients with OSCC or OPMDs and 9 controls (healthy volunteers) by enzyme-linked immunosorbent assays. Results: Immunohistochemical analysis revealed significantly higher expression of IL-8 in OSCC specimens and TNF-α in OSCCs and OPMDs with dysplasia as compared to NOM. Moreover, expression of TNF-α was significantly higher in oral leukoplakia and oral lichen planus without dysplasia, whereas expression of IL-8 only in oral leukoplakia without dysplasia in comparison with NOM. Salivary concentrations of all evaluated cytokines were significantly higher in patients with OSCC than in controls. Moreover, levels of IL-8 were significantly higher in saliva of patients with OPMDs with dysplasia as compared to controls and in OSCC patients as compared to patients with dysplastic lesions. There was also significant increase in salivary concentrations of IL-6, IL-8 and TNF-α in patients with OSCC as compared to patients with OPMDs without dysplasia. Conclusion: The study confirmed that proinflammatory, NF-kappaB dependent cytokines are involved in pathogenesis of OPMDs and OSCC. The most important biomarker of malignant transformation process within oral mucosa among all assessed cytokines seems to be IL-8. Further studies on a larger sample size are needed to corroborate these results.

Oral squamous cell carcinoma (OSCC) is a highly prevalent and aggressive malignancy of the oral cavity, frequently preceded by oral potentially malignant disorder (OPMD). Despite therapeutic advances, survival rates remain unsatisfactory, primarily due to late diagnosis, recurrence, and molecular alterations in histologically tumor-free surgical margins. Programmed cell death ligand-1 (PD-L1), an immune checkpoint molecule, contributes to tumor immune evasion and has been implicated in cancer progression. Its expression in OPMDs and OSCC surgical margins may serve as an early indicator of malignant transformation and recurrence risk. This study aims to assess PD-L1 expression in OPMDs and histologically negative surgical margins of OSCC and evaluate their association with 3-year survival outcomes. This retrospective cross-sectional study will be conducted over 12 months at a tertiary care hospital in Sawangi Meghe, Wardha, India. Archived formalin-fixed, paraffin-embedded samples of OPMDs and tumor-free surgical margins from OSCC cases (2018-2020) will be retrieved. Immunohistochemistry for PD-L1 will be performed using the SP263 clone, and expression will be evaluated using the combined positive score. Demographic, clinical, and survival data will be collected from patient records. Statistical analysis will determine correlations among PD-L1 expression, clinicopathological variables, and 3-year survival. The study is expected to provide insights into the role of PD-L1 as a biomarker for early detection, prognostication, and risk stratification in patients with OPMDs and OSCC. Data collection and immunohistochemical analysis have not yet commenced at the time of submission. By evaluating PD-L1 expression in premalignant lesions and histologically negative margins, this study aims to identify molecular predictors of OSCC progression and survival. The findings may help establish PD-L1 as a prognostic biomarker and support its integration into precision oncology and immunotherapy strategies.

Oral squamous cell carcinoma (OSCC) remains one of the most common malignancies in the head and neck region, often preceded by a spectrum of oral potentially malignant disorders (OPMDs). Despite advances in diagnostic methods, reliable and non-invasive biomarkers for early detection and prognostic stratification are still lacking. In recent years, circulating cell-free DNA (cfDNA) has emerged as a promising liquid biopsy tool in several solid tumors, offering insights into tumor burden, heterogeneity, and molecular dynamics. However, its application in oral oncology remains underexplored. This study aims to review and discuss the current evidence on cfDNA quantification and mutation analysis (including TP53, NOTCH1, and EGFR) in patients with OPMDs and OSCC. Particular attention is given to cfDNA fragmentation patterns, methylation signatures, and tumor-specific mutations as prognostic and predictive biomarkers. Moreover, we highlight the challenges in standardizing pre-analytical and analytical workflows in oral cancer patients and explore the potential role of cfDNA in monitoring oral carcinogenesis. Understanding cfDNA dynamics in the oral cavity might offer a novel, minimally invasive strategy to improve early diagnosis, risk assessment, and treatment decision-making in oral oncology.

Interleukin-33 (IL-33) and its receptor Suppression of tumorigenicity 2 (ST2), along with IL-10 and IFN-γ, exert contrasting roles in tumor growth, immune evasion, and host defense. IL-33/ST2 signalling can either promote tumor progression or, in some contexts, enhance anti-tumor immunity. However, the role of serum IL-33 and soluble ST2 in Oral squamous cell carcinoma (OSCC) or its precursor, Oral potentially malignant disorders (OPMDs), remains largely unexplored. To evaluate whether IL-33 and ST2, in conjunction with their interactions with IL-10 and IFN-γ, influence tumor dynamics in OSCC, as reflected in their serum levels. Ninety participants were enrolled in this cross-sectional observational study and divided into three groups: Healthy controls (HC) (n = 30), OPMDs (n = 30), and OSCC (n = 30). Clinicopathological data were recorded, and 5 mL of venous blood was collected from each subject prior to treatment. Serum IL-33, soluble ST2, IL-10, and IFN-γ levels were quantified using ELISA. The data were analysed by applying the Kruskal-Wallis/Mann-Whitney U-tests, Receiver operating characteristics (ROC) curve analysis, DeLong's test and binomial logistic regression (BLR), with significance set at p < 0.05. The analysis demonstrated a progressive and significant increase in IL-33, ST2, and IL-10 levels from HC to OPMDs to OSCC (p < 0.001). In contrast, IFN-γ levels exhibited a significant inverse trend, being highest in OPMDs, comparable in HC, and lowest in OSCC (p < 0.001). Immune mediators in OSCC showed significant associations with clinicopathological parameters, including tumor stage, depth of invasion, lymph nodal metastasis (LNM), tumor budding, and surgical margin status (p < 0.05). Serum IL-10 was the strongest positive predictor, effectively discriminating lymph node status (LNS). IL-33 and soluble ST2 showed positive trends toward predicting LNM with high classification accuracy. ROC analysis showed excellent discriminatory ability of all immunomodulatory mediators for distinguishing OSCC from OPMDs. Although soluble ST2 had the highest AUC, DeLong's test (p = 0.592) showed no significant difference, indicating comparable diagnostic potential. BLR confirmed their diagnostic relevance, with elevated IL-33, soluble ST2, and IL-10 increasing the odds of OSCC, while higher IFN-γ reduced the risk. The panel of immunomodulatory mediators analysed here reflects a biologically relevant shift toward pro-tumorigenic inflammation and immune evasion, underscoring their role as biomarkers of malignant progression. Collectively, these immunomodulatory mediators demonstrated strong diagnostic accuracy in differentiating OSCC from OPMDs and showed potential for risk stratification.

Background/Objectives: Oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs) remain major clinical challenges due to late diagnosis and limited prognostic markers. This study evaluated salivary interleukin-6 (IL-6) and cotinine as potential biomarkers for oral epithelial transformation and exposure to risk factors. Methods: From 267 individuals with histopathologically confirmed OPMDs or OSCC, 47 patients met the inclusion criteria and were enrolled (18 OPMDs, 29 OSCC). A control group comprised 40 individuals without oral mucosal pathology. Unstimulated saliva samples were collected and analyzed for IL-6 and cotinine concentrations. Biomarker levels were compared among groups and evaluated in relation to smoking exposure, alcohol use, and clinicopathological parameters. Results: Salivary IL-6 and cotinine levels differed significantly among groups (p < 0.05), with the highest concentrations observed in OSCC patients, intermediate levels in OPMDs, and the lowest levels in controls. Cotinine levels were significantly higher in smokers and individuals with greater tobacco exposure in both study groups, whereas IL-6 concentrations were not significantly associated with smoking or alcohol consumption. No correlation between IL-6 and cotinine was found in OPMDs or OSCC; however, a moderate negative correlation was observed in controls. Conclusions: Salivary IL-6 and cotinine demonstrate potential as complementary, non-invasive biomarkers for assessing oral epithelial transformation and tobacco exposure. Their combined evaluation may support risk stratification and early detection in patients with OPMDs.

Data sources Four electronic databases were searched: Medline (OVID), Web of Science, Embase and Scopus. An initial search was carried out in May 2018, and this was updated in September 2020. There was no time restriction on the studies included, and the final data consisted of studies published from 2004-2020.Study selection The electronic database search yielded 2,764 abstracts, and following de-duplication, 1,873 articles were screened in accordance with the exclusion criteria. In total, 346 articles were selected for full-text screening by four pairs of blinded reviewers and 295 articles were included in the final study. The main objectives of this study were to investigate a suitable biomarker for early detection of oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), and to assess the relationships between salivary biomarkers and risk factors for OSCC and OPMD. The Newcastle-Ottawa Scale was used for quality assessment. Most studies were considered to have a moderate risk of bias. The publications included fulfilled the following criteria: original research, human subjects with oral cavity cancer, OSCC or OPMD, aged 18 years or over, studies analysing biomarkers in saliva or salivary rinse, and studies published in English.Data extraction and synthesis Data extraction followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline process. The following data parameters were included in the studies chosen for assessment: study design, first author, year of publication, country of study population, age, sample size, gender, salivary biomarkers, method used to analyse the biomarkers, relationships between risk factors and salivary biomarkers, and conclusions.Results Following evaluation of 295 articles and selection of suitable salivary biomarkers, 28 articles were chosen to further assess interleukins as potential biomarkers and 33 studies were found to report a relationship between salivary biomarkers and risk factors. From the data reported, IL1β, IL6 and IL8 were identified as being statistically significant and most suitable for early identification of OSCC and OPMDs. In smokers, there were significant differences found in certain biomarkers compared to controls. There were statistically non-significant relationships found between biomarkers and alcohol, as well as other risk factors.Conclusion The authors proposed that a proteomic salivary biomarker panel, including a combination of IL1β, IL6 and IL8, would be suitable for clinical validation for the early detection and screening of OPMDs and OSCC. They have also highlighted the presence of research gaps in the relationship between salivary biomarkers and risk factors for OPMDs and OSCC, and the need for further research to understand the role of biomarkers in disease initiation and progression.

BackgroundDyslipidaemia is associated with cancers. However, the specific expression of serum lipids in oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) remains unclear, and it remains unknown whether serum lipids are associated with the development of OPMD and OSCC. This study investigated the serum lipid profiles of OPMD and OSCC patients, and the association of serum lipids with the occurrence of OPMD and OSCC.MethodsA total of 532 patients were recruited from the Affiliated Hospital of Stomatology, Nanjing Medical University. Serum lipid parameters including total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (Apo-A), apolipoprotein B (Apo-B), and lipoprotein (a) (Lpa) were analysed, and clinicopathological data were collected for further analysis. Furthermore, a regression model was used to evaluate the relationship between serum lipids and the occurrence of OSCC and OPMD.ResultsAfter adjusting for age and sex, no significant differences were observed in serum lipid or body mass index (BMI) between OSCC patients and controls (P > 0.05). HDL-C, Apo-A, and Apo-B levels were lower in OSCC patients than in OPMD patients (P < 0.05); HDL-C and Apo-A levels were higher in OPMD patients than in controls (P < 0.05). Furthermore, female OSCC patients had higher Apo-A and BMI values than males. The HDL-C level was lower in patients under 60 years of age than in elders (P < 0.05); and age was related to a higher risk of developing OSCC. Female patients with OPMD had higher TC, HDL-C, and Apo-A levels than males (P < 0.05); OPMD patients over 60 years of age had higher HDL-C than youngers (P < 0.05), whereas the LDL-C level was lower in elders (P < 0.05). The HDL-C and BMI values of the patients with oral leukoplakia (OLK) with dysplasia were more elevated than those of the oral lichen planus group, and the LDL-C, and Apo-A levels in patients with OLK with dysplasia were decreased (P < 0.05). Sex, high HDL-C and Apo-A values were associated with the development of OPMD.ConclusionSerum lipids exhibited certain differences according to the occurrence and development of OSCC; high levels of HDL-C and Apo-A might be markers for predicting OPMD.

Biopsy and histopathological examination continue to be the gold standards for diagnosing oral cancer and oral potentially malignant disorders (OPMD). The prognosis of these conditions is significantly influenced by established factors such as the degree of dysplasia, tumour size, grade, and patient age. While these traditional diagnostic approaches are crucial, there is a growing need for innovative methods to improve diagnosis and monitoring of these conditions. We hypothesize that identifying specific diagnostic biomarkers in human saliva, alongside traditional methods, could enhance clinical outcomes in managing both malignant and non-malignant oral pathologies. In this study, salivary levels of previously described biomarkers BDNF, IL-6, IL-8, IL-10, IL-15, IL-1RA, LIF, and TNF-α were compared among oral cancer patients, patients with OPMD, periodontal disease patients, and healthy volunteers. The results indicated that these cytokines have the potential to distinguish cancer cases, though they are less effective in differentiating between OPMD and periodontitis. Using pairs or groups of biomolecules could improve the ability to differentiate between different pathological entities. We found the following panel IL-6, IL-15, and IL-1RA to be particularly significant as diagnostic biomarkers for oral squamous cell carcinoma and OPMD.

Objectives:to determine salivary electrolyte concentration of oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) patients. A related systematic review was performed. Methods:Observational study. Unstimulated saliva from 18 patients with OSCC, 18 with OPMD, and 18 without oral lesions was collected. A biochemical analysis was performed to evaluate the salivary concentrations of potassium (K), phosphorus (P), sodium (Na), calcium (Ca), magnesium (Mg), zinc (Zn), copper (Cu), and iron (Fe). Kruskal–Wallis test was performed, and p < 0.05 was interpreted as statistically significant. The literature search for the systematic review retrieved 9 studies that associated salivary electrolyte levels with presence and progression of OSCC. Results:A highly significant increase was found in the salivary Mg levels in the OPMD group (5.41 µg/mL) in comparison with the OSCC (3.71µg/mL) and control group (3.51 µg/mL) (p = 0.041). No differences were observed in other salivary levels elements. The results of the systematic review revealed that one article indicated a decrease, and three papers reported an increase in salivary Na levels in patients with OPMD and OSCC. Two articles indicated a decrease in salivary K levels in OSCC, and the other two reported high Mg levels in OPMD and OSCC. Conclusion:High salivary Mg levels can be a potential biomarker indicating the presence of OPMD, however, the evidence is still contradictory and more studies are required.

Micronuclei (MN) genotoxicity, linked to chromosomal anomalies, is a key biomarker for carcinogen exposure and cancer susceptibility, with higher frequencies observed in cancer patients. The micronuclei assay, using exfoliated buccal cells, offers a non-invasive method for diagnosing oral lesions caused by tobacco, betel nut, and alcohol. This review aims to systematically review micronuclei frequencies in buccal mucosal cells and assess their potential as genotoxicity markers in oral potentially malignant disorders (OPMD). A systematic search updated to 2024 was conducted using PubMed, Scopus, ProQuest, Cochrane, and Google Scholar for original studies analyzing micronuclei frequencies in buccal cells as genotoxicity markers for OPMD. Studies including leukoplakia, lichen planus, and OSMF, were selected. The assessment of risk bias was done using modified Newcastle-Ottawa Scale followed by meta- analysis. The review was registered in PROSPERO (Registration No. CRD42024536661). Results: Twenty-six articles encompassing a pooled sample of 1,078 healthy controls and 1,489 OPMD cases (417 leukoplakia, 180 oral lichen planus, 401 OSMF, and 491 unsegregated OPMDs) were included. A significant increase in micronuclei frequency was observed in OPMD patients compared to controls (meta-Cohen's d = 3.08, 95% CI: 1.80-4.35). Subgroup analysis revealed a gradual rise in MN frequency from healthy controls to leukoplakia (d = 2.75), oral lichen planus (d = 1.47), and the highest in oral submucous fibrosis (d = 5.55). Considerable heterogeneity was detected among studies (overall I² = 99.23%, OSMF I² = 99.85%, lichen planus I² = 49.59%). This variability highlights methodological and population differences across studies. Micronuclei genotoxicity is emerging as a valuable biomarker for the early detection of OPMD's. Due to its non-invasive and cost-efficient characteristics, examining micronuclei in exfoliated buccal cells could be incorporated into regular screenings for groups at high risk of OPMD and oral cancer. However, the considerable variability among studies necessitates careful interpretation and highlights the importance of establishing standardized protocols in future research.

Introduction:Oral squamous cell carcinoma (OSCC) accounts for 95% among all head and neck cancers. Literature reveals saliva as a predictive, diagnostic and prognostic tool in carcinoma, inflammatory and genetic disorders. Expression of salivary interleukin-6 (IL-6) has been reported in patients with OSCC and in oral potentially malignant disorders (OPMDs). This study aims at the following objectives: • To evaluate the changes in the salivary levels of IL-6 in healthy individuals and those with chronic periodontitis (CP), OPMD and OSCC. • To compare the estimated levels of salivary IL-6 individually in healthy individuals and those with CP, OPMD and OSCC. • To assess the estimated levels of salivary IL-6 individually within histopathologically differentiated OSCC. • To analyse salivary IL-6 as a reliable biomarker in the diagnosis of OSCC.Materials and Methods:Totally, 60 patients were divided into four groups consisting of 15 patients in each group. Salivary samples were collected by simple drooling method. The concentration of IL-6 is to be determined by using Quantitative sandwich ELISA technique. All analyses were carried out using Statistical Package for Social Sciences (SPSS).Results:The concentration values of IL-6 were found to be more in OSCC group in comparison with the other three groups and the concentration values of OPMD were found to be more than in the CP and control group and was statistically significant.Discussion:We attempted a study to evaluate the salivary IL-6 in patients with OSCC, OPMDs and CP in comparison with the healthy controls. We achieved a pragmatic result showed that salivary IL-6 can be a reliable biomarker in the detection of OSCC. Saliva, due its wide array of functional characteristics, is an upcoming diagnostic fluid in the field of medicine and salivary IL-6 can be one such biomarker in the diagnosis of OSCC.

BackgroundOral squamous cell carcinoma (OSCC) is a common type of head and neck cancer, which is often preceded by oral potentially malignant disorders (OPMDs). Though comprehensive examination forms the backbone of oral cancer screening, the combination of salivary proteomic and transcriptomic markers may identify molecular markers specific to OSCC and assess their ability to predict malignant transformation in OPMD. Hence, this study aimed to determine the diagnostic utility of salivary interleukin-8 (IL-8) mRNA and protein in the differential diagnosis of OSCC, OPMD, and healthy controls.MethodologyUnstimulated whole saliva was collected from 90 subjects, 30 in each group (i.e., OSCC, OPMD, and controls). This comparative cross-sectional, multicenter study was conducted after obtaining institutional ethical clearance. The supernatant was treated with RNase inhibitor and separated into the following two fractions: one for RNA extraction and the other for enzyme-linked immunosorbent assay (ELISA). The results were statistically analyzed using SPSS version 21 software (IBM Corp., Armonk, NY, USA), and p-values <0.05 were considered statistically significant.ResultsSalivary IL-8 showed statistically significant differences when all three groups were compared at the protein (p < 0.001) and transcriptomic (p = 0.001) levels. Salivary IL-8 protein showed the best discriminatory ability in distinguishing OSCC from controls, with the highest area under the curve (AUC) of 0.915. It yielded a sensitivity of 80% and a specificity of 76% at a cutoff value of 239.55 pg/mL (p < 0.001) with strong predictive power.ConclusionsSalivary IL-8 protein showed strong discriminatory ability in the present study compared to salivary mRNA in distinguishing OSCC from controls, with the highest AUC of 0.915. The sensitivity and specificity, as well as the predictive value of polymerase chain reaction measurements of salivary IL-8 mRNA, were not as strong as the sensitivity and specificity for salivary IL-8 protein using ELISA. Alterations in OSCC-associated IL-8 expression levels, both at the protein and mRNA levels, could aid in early oral cancer detection during population mass screening to enable identification of high-risk groups with strong discriminatory ability.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with a poor prognosis, primarily due to a late diagnosis. Recent studies have focused on identifying non-invasive biomarkers for early detection, with microRNAs (miRNAs) emerging as promising candidates. This systematic review aims to evaluate the potential of circulating miRNAs as biomarkers for the early detection of PDAC, analyzing their diagnostic accuracy, specificity, and sensitivity. Following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, a comprehensive search across PubMed, Embase, and the Cochrane Library was conducted. Studies published from January 2013 to October 2023 focusing on miRNA biomarkers for early PDAC detection were included. Data synthesis was performed through a narrative approach due to the heterogeneity of the studies. Nine studies met the inclusion criteria. Key findings include the elevated levels of specific miRNAs, such as miR-18a, miR-106a, and miR-25, in early-stage PDAC patients compared to controls. The integration of miRNA profiles with traditional biomarkers like CA19-9 showed improved diagnostic performance. However, challenges in the standardization of miRNA evaluation methodologies were noted. Circulating miRNAs demonstrate significant potential as non-invasive biomarkers for early PDAC detection. Despite promising results, further research and standardization are necessary for clinical application.

CLINICAL AND EPIDEMIOLOGICAL PROFILE OF PATIENTS WITH ORAL POTENT IALLY MALIGNANT DISORDERS IN A ORAL MEDICINE SERVICE IN