DNA methylation markers for oral cancer detection in non- and minimally invasive samples: a systematic review.

More than 50% of oral cancer (OC) patients are diagnosed with advanced-stage disease associated with poor prognosis and quality of life, supporting an urgent need to improve early OC detection. The identification of effective molecular markers by minimally invasive approaches has emerged as a promising strategy for OC screening. This systematic review summarizes and evaluates the performance of the DNA methylation markers identified in non- or minimally invasive samples for OC detection. PubMed's MEDLINE, Scopus, Embase, and Cochrane Library databases were systematically searched for studies that evaluated DNA methylation markers in non-invasive and/or minimally invasive samples (oral rinse/saliva, oral brush, and blood) from OC patients. Two investigators independently extracted data on study population characteristics, candidate methylation markers, testing samples, DNA methylation assay, and performance diagnostic outcomes. Methodological study quality was assessed with the Quality Assessment for Studies of Diagnostic Accuracy-2 tool. Thirty-one studies met the inclusion criteria for this systematic review. DNA methylation markers were evaluated in oral rinse/saliva (n = 17), oral brush (n = 9), and blood (n = 7) samples. Methylation-specific PCR (MSP) and quantitative-MSP were the most common DNA methylation assays. Regarding diagnostic performance values for salivary, oral brush, and blood DNA methylation markers, sensitivity and specificity ranged between 3.4-100% and 21-100%, 9-100% and 26.8-100%, 22-70% and 45.45-100%, respectively. Different gene methylation panels showed good diagnostic performance for OC detection. This systematic review discloses the promising value of testing DNA methylation markers in non-invasive (saliva or oral rinse) or minimally invasive (oral brush or blood) samples as a novel strategy for OC detection. However, further validation in large, multicenter, and prospective study cohorts must be carried out to confirm the clinical value of specific DNA methylation markers in this setting.

Liquid Biopsies Based on Cell-Free DNA Integrity as a Biomarker for Cancer Diagnosis: A Meta-Analysis.

Liquid biopsies have been identified as a viable source of cancer biomarkers. We aim to evaluate the diagnostic accuracy of cell-free DNA integrity (cfDI) in liquid biopsies for cancer. A comprehensive literature search was conducted through PubMed, Embase, Web of Science, and Cochrane Library up to June 2024. Seventy-two study units from forty-six studies, comprising 4286 cancer patients, were identified and evaluated. The Quality Assessment for Studies of Diagnostic Accuracy-2 (QUADAS-2) was used to assess study quality. Meta-regression analysis was employed to investigate the underlying factors contributing to heterogeneity, alongside an evaluation of publication bias. The bivariate random-effect model was utilized to compute the primary diagnostic outcomes and their corresponding 95% confidence intervals (CIs). The pooled sensitivity, specificity, and positive and negative likelihood ratios of cfDI in cancer diagnosis were 0.70 and 0.77, 3.26 and 0.34, respectively. The overall area under the curve was 0.84, with a diagnostic odds ratio of 10.63. This meta-analysis suggested that the cfDI index has a promising potential as a non-invasive and accurate diagnostic tool for cancer. Study registration: The study was registered at PROSPERO (reference No. CRD42021276290).

Liquid biopsies based on cell-free DNA as a potential biomarker in head and neck cancer.

In the era of 'precision medicine', liquid biopsies based on cell-free DNA (cfDNA) have emerged as a promising tool in the oncology field. cfDNA from cancer patients is a mixture of tumoral (ctDNA) and non-tumoral DNA originated from healthy, cancer and tumor microenvironmental cells. Apoptosis, necrosis, and active secretion from extracellular vesicles represent the main mechanisms of cfDNA release into the physiological body fluids. Focused on HNC, two main types of cfDNA can be identified: the circulating cfDNA (ccfDNA) and the salivary cfDNA (scfDNA). Numerous studies have reported on the potential of cfDNA analysis as potential diagnostic, prognostic, and monitoring biomarker for HNC. Thus, ctDNA has emerged as an attractive strategy to detect cancer specific genetic and epigenetic alterations including DNA somatic mutations and DNA methylation patterns. This review aims to provide an overview of the up-to-date studies evaluating the value of the analysis of total cfDNA, cfDNA fragment length, and ctDNA analysis at DNA mutation and methylation level in HNC patients.

Liquid biopsy-based microRNA models as potential biomarkers of bowel conditions.

MicroRNAs detected in liquid biopsies have demonstrated to have potential as biomarkers of benign and malignant bowel conditions. Numerous studies have reported on the combination of different microRNAs to improve the diagnostic capabilities in the detection of bowel conditions. This editorial will be reviewing the best scientific evidence available to this date on studies showing microRNA models with sensitivity and specificity values. Also, the current challenges on how to implement microRNAs in the routine clinical practice are exposed.

Somatic mutations in tumor and plasma of locoregional recurrent and/or metastatic head and neck cancer using a next-generation sequencing panel: A preliminary study.

BACKGROUND: We explore the utility of TruSight Tumor 170 panel (TST170) for detecting somatic mutations in tumor and cfDNA from locoregional recurrent and/or metastatic head and neck squamous cell carcinoma (HNSCC). METHODS: Targeted NGS of tumor DNA and plasma cfDNA was performed using TST170 panel. In addition, a set of somatic mutations previously described in HNSCC were selected for validating in tumor, plasma, and saliva by digital droplet PCR. RESULTS: The TST170 panel identified 13 non-synonymous somatic mutations, of which five were detected in tumoral tissue, other five in plasma cfDNA, and three in both tissue and plasma cfDNA. Of the eight somatic mutations identified in tissue, three were also identified in plasma cfDNA, showing an overall concordance rate of 37.5%. CONCLUSIONS: This preliminary study shows the possibility to detect somatic mutations in tumor and plasma of HNSCC patients using a single assay that would facilitate the clinical implementation of personalized medicine in the clinic.

Modelos de microARNs basados en biopsia líquida como potenciales biomarcadores en patología intestinal / Liquid biopsy-based microRNA models as potential biomarkers of bowel conditions

Los microARNs detectados en biopsias líquidas han demostrado tener potencial como biomarcadores en la patología intestinal benigna y maligna. Numerosos estudios han reportado la combinación de diferentes microARNs con el fin de mejorar la capacidad diagnóstica en patología intestinal. En esta editorial se pretende revisar la mejor evidencia disponible sobre estudios que muestran modelos de microARNs, con valores de sensibilidad y especificidad. También se exponen los desafíos que existen actualmente hasta poder llegar a implementar los microARNs en la clínica (AU)

Genome-wide DNA methylation profiling in tongue squamous cell carcinoma.

OBJECTIVES: To provide a comprehensive characterization of DNA methylome of oral tongue squamous cell carcinoma (OTSCC) and identify novel tumor-specific DNA methylation markers for early detection using saliva. MATERIAL AND METHODS: Genome-wide DNA methylation analysis including six OTSCC matched adjacent non-tumoral tissue and saliva was performed using Infinium MethylationEPIC array. Differentially methylated levels of selected genes in our OTSCC cohort were further validated using OTSCC methylation data from The Cancer Genome Atlas database (TCGA). The methylation levels of a set of tumor-specific hypermethylated genes associated with a downregulated expression were evaluated in saliva. Receiver operating characteristic (ROC) curves were performed to assess the diagnostic value of DNA methylation markers. RESULTS: A total of 25,890 CpGs (20,505 hypomethylated and 5385 hypermethylated) were differentially methylated (DMCpGs) between OTSCC and adjacent non-tumoral tissue. Hypermethylation of 11 tumor-specific genes was validated in OTSCC TCGA cohort. Of these 11 genes, A2BP1, ANK1, ALDH1A2, GFRA1, TTYH1, and PDE4B were also hypermethylated in saliva. These six salivary methylated genes showed high diagnostic accuracy (≥0.800) for discriminating patients from controls. CONCLUSIONS: This is the first largest genome-wide DNA methylation study on OTSCC that identifies a group of novel tumor-specific DNA methylation markers with diagnostic potential in saliva.

Age Estimation Using Maxillary Central Incisor Analysis on Cone Beam Computed Tomography Human Images.

Forensic dentistry plays an important role in human identification, and dental age estimation is an important part of the process. Secondary dentin deposition throughout an individual's lifetime and consequent modification in teeth anatomy is an important parameter for age estimation procedures. The aim of the present study was to develop regression equations to determine age in adults by means of linear measurements and ratios on sagittal, coronal and axial slices of maxillary central incisors using cone bean computed tomography (CBCT). Multiplanar measurements of upper central incisors were taken for a sample of 373 CBCTs. Subsequently, one-way analysis of variance (ANOVA) and multivariate linear regressions were performed for age estimation. The equations obtained from axial linear measurements and ratios presented a standard error of the estimate (SEE) of ±10.9 years (R2 = 0.49), and a SEE of ±10.8 years (R2 = 0.50), respectively. The equation obtained for multiplanar linear measurements presented a SEE of ±10.9 years (R2 = 0.52), while the equation for multiplanar ratios presented a SEE of ±10.7 years (R2 = 0.51). Thus, CBCT measurements on upper central incisors were found to be an acceptable method for age estimation. Horizontal measurements, especially pulp measurements, improve the accuracy of age estimate equations.

Methods for the Detection of Circulating Biomarkers in Cancer Patients.

Liquid biopsy has emerged as one of the main pillars for personalized oncology. The term englobes body-fluid samples which contain tumor-derived material such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), and circulating extracellular vesicles (cEVs). Potential clinical application of liquid biopsy analyses includes cancer screening, detection of minimal residual disease and recurrence, therapy selection, and evaluation of acquired resistance. Despite the great developments of technology focused on circulating biomarkers characterization only cfDNA testing is nowadays implemented for the therapy selection in some advanced tumors. This can be partially explained by the fact that there is still a lack of global standardization of procedures both in the pre-analytical and analytical steps. In the present chapter, we summarize the different strategies for addressing the study of liquid biopsy taking into account their pros and cons to be applied in a clinical context and we also discuss the main technical and clinical challenges in the field of circulating biomarkers and personalized oncology.

Integrity and quantity of salivary cell-free DNA as a potential molecular biomarker in oral cancer: A preliminary study.

BACKGROUND: Differences in cell-free DNA (cfDNA) fragments have been described as a valuable tool to distinguish cancer patients from healthy individuals. We aim to investigate the concentration and integrity of cfDNA fragments in saliva from oral squamous cell carcinoma (OSCC) patients and healthy individuals in order to explore their value as diagnostic biomarkers. METHODS: Saliva samples were collected from a total of 34 subjects (19 OSCC patients and 15 healthy controls). The total concentration of salivary cfDNA (scfDNA) was determined using a fluorometry method and quantitative real-time polymerase chain reaction (qPCR). To evaluate the scfDNA quantity and integrity, qPCR targeting Arthobacter luteus (ALU) sequences at three amplicons of different lengths (60, 115, and 247 bp, respectively) was carried out. ScfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) were calculated as the ratio between the absolute concentration of the longer amplicons 115 bp and 247 bp and the total scfDNA amount (amplicon 60 bp). RESULTS: The total scfDNA concentration (ALU60) was higher in OSCC than in healthy donors, but this trend was not statistically significant. The medians of scfDNA integrity indexes, ALU115/ALU60 and ALU247/ALU60, were significantly higher in OSCC, showing area under the curve values of 0.8211 and 0.7018, respectively. CONCLUSION: Our preliminary results suggest that scfDNA integrity indexes (ALU115/ALU60 and ALU247/ALU60) have potential as noninvasive diagnostic biomarkers for OSCC.

The Dental Aesthetic Index and Its Association with Dental Caries, Dental Plaque and Socio-Demographic Variables in Schoolchildren Aged 12 and 15 Years.

The Dental Aesthetic Index (DAI) was determined in 12- and 15-year-old schoolchildren to ascertain the prevalence of malocclusion and to assess its association with dental caries experience, dental plaque accumulation, and socio-demographic variables. We performed a cross-sectional study with a stratified two-stage sampling design. An oral health survey and oral examination were conducted, and socio-demographic data were recorded. The sample comprised 1453 schoolchildren aged 12 (868) and 15 (585). These two samples were analyzed separately because statistically significant differences were found: the 12-year-old age group displayed a higher frequency of schoolchildren who attended state-run public schools (p = 0.004) and belonged to a lower social class (p = 0.001); the 15-year-old age group registered higher levels of caries (p = 0.001) and lower levels of dental plaque (p < 0.001). The malocclusion was 9.5% higher (p = 0.001), and the global mean DAI score was likewise higher among the 12-year-olds (p < 0.001). The multivariate regression analysis not only showed that caries and dental plaque were the variables that were the most strongly associated with malocclusion, but that caries (OR = 1.5) and dental plaque (OR > 2) were also risk factors for malocclusion in both groups. In conclusion, this study revealed a higher prevalence of malocclusion and dental plaque at age 12. A higher risk of caries and dental plaque was found to be related to the presence of malocclusion in both age groups.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part II: A Cancer Risk Meta-Analysis.

Aberrant methylation of tumor suppressor genes has been reported as an important epigenetic silencer in head and neck cancer (HNC) pathogenesis. Here, we performed a comprehensive meta-analysis to evaluate the overall and specific impact of salivary gene promoter methylation on HNC risk. The methodological quality was assessed using the Newcastle-Ottawa scale (NOS). Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association and Egger's and Begg's tests were applied to detect publication bias. The frequency of salivary DNA promoter methylation was significantly higher in HNC patients than in healthy controls (OR: 8.34 (95% CI = 6.10-11.39; p < 0.01). The pooled ORs showed a significant association between specific tumor-related genes and HNC risk: p16 (3.75; 95% CI = 2.51-5.60), MGMT (5.72; 95% CI = 3.00-10.91), DAPK (5.34; 95% CI = 2.18-13.10), TIMP3 (3.42; 95% CI = 1.99-5.88), and RASSF1A (7.69; 95% CI = 3.88-15.23). Overall, our meta-analysis provides precise evidence on the association between salivary DNA promoter hypermethylation and HNC risk. Thus, detection of promoter DNA methylation in saliva is a potential biomarker for predicting HNC risk.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part I: A Diagnostic Accuracy Meta-Analysis.

DNA hypermethylation is an important epigenetic mechanism for gene expression inactivation in head and neck cancer (HNC). Saliva has emerged as a novel liquid biopsy representing a potential source of biomarkers. We performed a comprehensive meta-analysis to evaluate the overall diagnostic accuracy of salivary DNA methylation for detecting HNC. PubMed EMBASE, Web of Science, LILACS, and the Cochrane Library were searched. Study quality was assessed by the Quality Assessment for Studies of Diagnostic Accuracy-2, and sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (dOR), and their corresponding 95% confidence intervals (CIs) were calculated using a bivariate random-effect meta-analysis model. Meta-regression and subgroup analyses were performed to assess heterogeneity. Eighty-four study units from 18 articles with 8368 subjects were included. The pooled sensitivity and specificity of salivary DNA methylation were 0.39 and 0.87, respectively, while PLR and NLR were 3.68 and 0.63, respectively. The overall area under the curve (AUC) was 0.81 and the dOR was 8.34. The combination of methylated genes showed higher diagnostic accuracy (AUC, 0.92 and dOR, 36.97) than individual gene analysis (AUC, 0.77 and dOR, 6.02). These findings provide evidence regarding the potential clinical application of salivary DNA methylation for HNC diagnosis.

Saliva Gene Promoter Hypermethylation as a Biomarker in Oral Cancer.

Oral carcinogenesis is a multistep process characterized by a summation of multiple genetic and epigenetic alterations in key regulatory genes. The silencing of genes by aberrant promoter hypermethylation is thought to be an important epigenetic event in cancer development and progression which has great potential as a biomarker for early diagnosis, tumor molecular subtyping, prognosis, monitoring, and therapy. Aberrant DNA methylation has been detected in different liquid biopsies, which may represent a potential alternative to solid biopsies. The detection of methylated genes in saliva may have clinical application for noninvasive oral cancer screening and early diagnosis. Here, we review the current evidence on gene promoter hypermethylation in saliva.

Detection of the Accessory Mental Foramina on Human Mandibles Using Cone-beam Computed Tomography: A Systematic Review and Meta-analysis.

INTRODUCTION: This study aimed to perform a systematic review and meta-analysis on accessory mental foramen (AMF) research using cone-beam computed tomographic (CBCT) imaging. METHODS: A systematic review was performed in PubMed, Embase, Thomas Reuter's Web of Science, Scopus, and ScienceDirect databases according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis statement. Articles focusing on AMF prevalence and location using CBCT imaging were selected without language restrictions. Studies reporting pooled results only or presenting any pathology in the area surrounding the mental foramen (MF) were excluded. A meta-analysis using random effects was performed. RESULTS: The present meta-analysis included a total of 46 articles involving 21,761 subjects. The overall pooled AMF prevalence was 7.87% (95% confidence interval [CI], 6.69-9.24) in subjects and 4.75% (95% CI, 3.79-5.95) in hemimandibles (n = 31,158). AMF presence was most commonly unilateral, reaching 90.15% (95% CI, 82.98-94.49). AMFs were significantly more frequent in right hemimandibles (χ2 = 5.20, P < .05) and were most commonly located posterior and inferior to the MF. However, AMFs superior to the MF were also observed in 47.43% (95% CI, 38.45-56.58) of cases. The studies conducted over the last 3 years showed significantly higher AMF prevalence levels (χ2 = 5.12, P < .05). CONCLUSIONS: Our meta-analysis demonstrates that AMF prevalence is considerable and should not be underestimated. AMFs are most frequently located in right hemimandibles. The presence of AMFs superior to the MF is frequent. Around 3% of people present superior AMFs. This fact puts those patients at greater risk for injury when performing periapical surgery in this area.

Association of Salivary Human Papillomavirus Infection and Oral and Oropharyngeal Cancer: A Meta-Analysis.

BACKGROUND: Human papillomavirus (HPV) infection has been recognized as an important risk factor in cancer. The purpose of this systematic review and meta-analysis was to determine the prevalence and effect size of association between salivary HPV DNA and the risk of developing oral and oropharyngeal cancer. METHODS: A systematic literature search of PubMed, EMBASE, Web of Science, LILACS, Scopus and the Cochrane Library was performed, without language restrictions or specified start date. Pooled data were analyzed by calculating odds ratios (ORs) and 95% confidence intervals (CIs). Quality assessment was performed using the Newcastle-Ottawa Scale (NOS). RESULTS: A total of 1672 studies were screened and 14 met inclusion criteria for the meta-analysis. The overall prevalence of salivary HPV DNA for oral and oropharyngeal carcinoma was 43.2%, and the prevalence of salivary HPV16 genotype was 27.5%. Pooled results showed a significant association between salivary HPV and oral and oropharyngeal cancer (OR = 4.94; 2.82-8.67), oral cancer (OR = 2.58; 1.67-3.99) and oropharyngeal cancer (OR = 17.71; 6.42-48.84). Significant associations were also found between salivary HPV16 and oral and oropharyngeal cancer (OR = 10.07; 3.65-27.82), oral cancer (OR = 2.95; 1.23-7.08) and oropharyngeal cancer (OR = 38.50; 22.43-66.07). CONCLUSIONS: Our meta-analysis demonstrated the association between salivary HPV infection and the incidence of oral and oropharyngeal cancer indicating its value as a predictive indicator.

Salivary biomarkers for cancer diagnosis: a meta-analysis.

Background: Saliva represents a promising non-invasive source of novel biomarkers for diagnosis and prognosis cancer. This meta-analysis evaluates the diagnostic value of salivary biomarkers for detection of malignant non-oral tumours to better define the value of saliva as an alternative liquid biopsy.Materials and methods: We performed a systematic review and meta-analysis. PubMed, Embase, LILACS and the Cochrane Library were searched to identify articles that examined the potential of salivary biomarkers for the diagnosis of malignant non-oral tumours. To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR), area under hierarchical summary receiver operating characteristic (AUC) curve, sensitivity, specificity, positive likelihood ratio (PLR) and negative likelihood ratio (NLR) using a random- or fixed-effects model. Heterogeneity and publication bias were assessed. Statistical tests were two-sided.Results: One hundred fifty-five study units from 29 articles with 11,153 subjects were included. The pooled sensitivity, specificity, PLR, NLR, DOR and AUC were 0.76 (95% confidence intervals (CI), 0.74-0.77), 0.76 (95% CI, 0.75-0.77), 3.22 (95% CI, 2.92-3.55), 0.31 (95% CI, 0.28-0.34), 13.42 (95% CI, 12.28-15.96) and 0.85 (95% CI, 0.84-0.87), respectively.Conclusion: Salivary biomarkers may be potentially used for non-invasive diagnosis of malignant non-oral tumours.Key messagesThis meta-analysis evaluates the diagnostic value of salivary biomarkers for detection of malignant non-oral tumours to better define the role of saliva as an alternative liquid biopsy.Salivary biomarkers showed 85% accuracy for cancer distant to the oral cavity.Saliva represents a promising non-invasive source of novel biomarkers in cancer.

Cell-Free microRNAs as Potential Oral Cancer Biomarkers: From Diagnosis to Therapy.

Oral cavity cancer is the most frequent malignancy of the head and neck. Unfortunately, despite educational interventions for prevention and early diagnosis, oral cancer patients are often diagnosed in advanced stages associated with poor prognosis and life expectancy. Therefore, there is an urgent need to find noninvasive biomarkers to improve early detection of this tumor. Liquid biopsy has emerged as a valuable tool in medical oncology which provides new horizons for improving clinical decision making. Notably, cell-free microRNAs (miRNAs), a class of short non-coding RNAs, are emerging as novel noninvasive cancer biomarkers. Here, we provide an overview of the potential clinical application of cell-free miRNAs as diagnostic, prognostic, and therapeutic biomarkers in oral cancer.