Evaluation of the clinical use of MGMT methylation in extracellular vesicle-based liquid biopsy as a tool for glioblastoma patient management.

Glioblastoma (GB) is a devastating tumor of the central nervous system characterized by a poor prognosis. One of the best-established predictive biomarker in IDH-wildtype GB is O6-methylguanine-DNA methyltransferase (MGMT) methylation (mMGMT), which is associated with improved treatment response and survival. However, current efforts to monitor GB patients through mMGMT detection have proven unsuccessful. Small extracellular vesicles (sEVs) hold potential as a key element that could revolutionize clinical practice by offering new possibilities for liquid biopsy. This study aimed to determine the utility of sEV-based liquid biopsy as a predictive biomarker and disease monitoring tool in patients with IDH-wildtype GB. Our findings show consistent results with tissue-based analysis, achieving a remarkable sensitivity of 85.7% for detecting mMGMT in liquid biopsy, the highest reported to date. Moreover, we suggested that liquid biopsy assessment of sEV-DNA could be a powerful tool for monitoring disease progression in IDH-wildtype GB patients. This study highlights the critical significance of overcoming molecular underdetection, which can lead to missed treatment opportunities and misdiagnoses, possibly resulting in ineffective therapies. The outcomes of our research significantly contribute to the field of sEV-DNA-based liquid biopsy, providing valuable insights into tumor tissue heterogeneity and establishing it as a promising tool for detecting GB biomarkers. These results have substantial implications for advancing predictive and therapeutic approaches in the context of GB and warrant further exploration and validation in clinical settings.

Biomarkers for Pre-Treatment Risk Stratification of Prostate Cancer Patients: A Systematic Review.

BACKGROUND: Prostate cancer (PCa) is one of the most frequently occurring malignancies. Although most cases are not life-threatening, approximately 20% endure an unfavorable outcome. PSA-based screening reduced mortality but at the cost of an increased overdiagnosis/overtreatment of low-risk (lrPCa) and favorable intermediate-risk (firPCa) PCa. PCa risk-groups are usually identified based on serum Prostate-Specific Antigen (PSA), the Gleason score, and clinical T stage, which have consistent although variable specificity or subjectivity. Thus, more effective and specific tools for risk assessment are needed, ideally making use of minimally invasive methods such as liquid biopsies. In this systematic review we assessed the clinical potential and analytical performance of liquid biopsy-based biomarkers for pre-treatment risk stratification of PCa patients. METHODS: Studies that assessed PCa pre-treatment risk were retrieved from PubMed, Scopus, and MedLine. PCa risk biomarkers were analyzed, and the studies' quality was assessed using the QUADAS-2 tool. RESULTS: The final analysis comprised 24 full-text articles, in which case-control studies predominated, mostly reporting urine-based biomarkers (54.2%) and biomarker quantification by qPCR (41.7%). Categorization into risk groups was heterogeneous, predominantly making use of the Gleason score. CONCLUSION: This systematic review unveils the substantial clinical promise of using circulating biomarkers in assessing the risk for prostate cancer patients. However, the standardization of groups, categories, and biomarker validation are mandatory before this technique can be implemented. Circulating biomarkers might represent a viable alternative to currently available tools, obviating the need for tissue biopsies, and allowing for faster and more cost-effective testing, with superior analytical performance, specificity, and reproducibility.

Pancreatic cancer biomarkers: A pathway to advance in personalized treatment selection.

Pancreatic cancer is one of the tumors with the worst prognosis, and unlike other cancers, few advances have been made in recent years. The only curative option is surgery, but only 15-20% of patients are candidates, with a high risk of relapse. In advanced pancreatic cancer there are few first-line treatment options and no validated biomarkers for better treatment selection. The development of targeted therapies in pancreatic cancer is increasingly feasible due to tumor-agnostic treatments, such as PARP inhibitors in patients with BRCA1, BRCA2 or PALB2 alterations or immunotherapies in patients with high microsatellite instability/tumor mutational burden. In addition, other therapeutic molecules have been developed for patients with KRAS G12C mutation or fusions in NTRK or NRG1. Consequently, there has been a growing interest in biomarkers that may help guide targeted therapy in pancreatic cancer. Therefore, this review aims to offer an updated perspective on biomarkers with therapeutic potential in pancreatic cancer.

Epigenomic analysis reveals a unique DNA methylation program of metastasis-competent circulating tumor cells in colorectal cancer.

Circulating tumor cells (CTCs) and epigenetic alterations are involved in the development of metastasis from solid tumors, such as colorectal cancer (CRC). The aim of this study was to characterize the DNA methylation profile of metastasis-competent CTCs in CRC. The DNA methylome of the human CRC-derived cell line CTC-MCC-41 was analyzed and compared with primary (HT29, Caco2, HCT116, RKO) and metastatic (SW620 and COLO205) CRC cells. The association between methylation and the transcriptional profile of CTC-MCC-41 was also evaluated. Differentially methylated CpGs were validated with pyrosequencing and qMSP. Compared to primary and metastatic CRC cells, the methylation profile of CTC-MCC-41 was globally different and characterized by a slight predominance of hypomethylated CpGs mainly distributed in CpG-poor regions. Promoter CpG islands and shore regions of CTC-MCC-41 displayed a unique methylation profile that was associated with the transcriptional program and relevant cancer pathways, mainly Wnt signaling. The epigenetic regulation of relevant genes in CTC-MCC-41 was validated. This study provides new insights into the epigenomic landscape of metastasis-competent CTCs, revealing biological information for metastasis development, as well as new potential biomarkers and therapeutic targets for CRC patients.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

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.

Epigenomic reprogramming of therapy-resistant circulating tumor cells in colon cancer.

Therapy resistance is a major challenge in colorectal cancer management. Epigenetic changes, such as DNA methylation, in tumor cells are involved in the development of acquired resistance during treatment. Here, we characterized the DNA methylation landscape of colon circulating tumor cells (CTCs) during cancer progression and therapy resistance development. To this aim, we used nine permanent CTC lines that were derived from peripheral blood samples of a patient with metastatic colon cancer collected before treatment initiation (CTC-MCC-41) and during treatment and cancer progression (CTC-MCC-41.4 and CTC-MCC-41.5 [A-G]). We analyzed the DNA methylome of these nine CTC lines using EPIC arrays and also assessed the association between DNA methylation and gene expression profiles. We confirmed DNA methylation and gene expression results by pyrosequencing and RT-qPCR, respectively. The global DNA methylation profiles were different in the pre-treatment CTC line and in CTC lines derived during therapy resistance development. These resistant CTC lines were characterized by a more hypomethylated profile compared with the pre-treatment CTC line. Most of the observed DNA methylation differences were localized at CpG-poor regions and some in CpG islands, shore regions and promoters. We identified a distinctive DNA methylation signature that clearly differentiated the pre-treatment CTC line from the others. Of note, the genes involved in this signature were associated with cancer-relevant pathways, including PI3K/AKT, MAPK, Wnt signaling and metabolism. We identified several epigenetically deregulated genes associated with therapy resistance in CTCs, such as AP2M1. Our results bring new knowledge on the epigenomic landscape of therapy-resistant CTCs, providing novel mechanisms of resistance as well as potential biomarkers and therapeutic targets for advanced CRC management.

Integrating digital pathology with transcriptomic and epigenomic tools for predicting metastatic uterine tumor aggressiveness.

The incidence of new cancer cases is expected to increase significantly in the future, posing a worldwide problem. In this regard, precision oncology and its diagnostic tools are essential for developing personalized cancer treatments. Digital pathology (DP) is a particularly key strategy to study the interactions of tumor cells and the tumor microenvironment (TME), which play a crucial role in tumor initiation, progression and metastasis. The purpose of this study was to integrate data on the digital patterns of reticulin fiber scaffolding and the immune cell infiltrate, transcriptomic and epigenetic profiles in aggressive uterine adenocarcinoma (uADC), uterine leiomyosarcoma (uLMS) and their respective lung metastases, with the aim of obtaining key TME biomarkers that can help improve metastatic prediction and shed light on potential therapeutic targets. Automatized algorithms were used to analyze reticulin fiber architecture and immune infiltration in colocalized regions of interest (ROIs) of 133 invasive tumor front (ITF), 89 tumor niches and 70 target tissues in a total of six paired samples of uADC and nine of uLMS. Microdissected tissue from the ITF was employed for transcriptomic and epigenetic studies in primary and metastatic tumors. Reticulin fiber scaffolding was characterized by a large and loose reticular fiber network in uADC, while dense bundles were found in uLMS. Notably, more similarities between reticulin fibers were observed in paired uLMS then paired uADCs. Transcriptomic and multiplex immunofluorescence-based immune profiling showed a higher abundance of T and B cells in primary tumor and in metastatic uADC than uLMS. Moreover, the epigenetic signature of paired samples in uADCs showed more differences than paired samples in uLMS. Some epigenetic variation was also found between the ITF of metastatic uADC and uLMS. Altogether, our data suggest a correlation between morphological and molecular changes at the ITF and the degree of aggressiveness. The use of DP tools for characterizing reticulin scaffolding and immune cell infiltration at the ITF in paired samples together with information provided by omics analyses in a large cohort will hopefully help validate novel biomarkers of tumor aggressiveness, develop new drugs and improve patient quality of life in a much more efficient way.

A genome-wide cell-free DNA methylation analysis identifies an episignature associated with metastatic luminal B breast cancer.

Breast cancers of the luminal B subtype are frequent tumors with high proliferation and poor prognosis. Epigenetic alterations have been found in breast tumors and in biological fluids. We aimed to profile the cell-free DNA (cfDNA) methylome of metastatic luminal B breast cancer (LBBC) patients using an epigenomic approach to discover potential noninvasive biomarkers. Plasma cfDNA was analyzed using the Infinium MethylationEpic array in a cohort of 14 women, including metastatic LBBC patients and nontumor controls. The methylation levels of cfDNA and tissue samples were validated with droplet digital PCR. The methylation and gene expression data of 582 primary luminal breast tumors and 79 nontumor tissues were obtained from The Cancer Genome Atlas (TCGA). We found an episignature of 1,467 differentially methylated CpGs that clearly identified patients with LBBC. Among the genes identified, the promoter hypermethylation of WNT1 was validated in cfDNA, showing an area under the ROC curve (AUC) of 0.86 for the noninvasive detection of metastatic LBBC. Both paired cfDNA and primary/metastatic breast tumor samples showed hypermethylation of WNT1. TCGA analysis revealed significant WNT1 hypermethylation in the primary tumors of luminal breast cancer patients, with a negative association between WNT1 methylation and gene expression. In this proof-of-principle study, we discovered an episignature associated with metastatic LBBC using a genome-wide cfDNA methylation approach. We also identified the promoter hypermethylation of WNT1 in cfDNA as a potential noninvasive biomarker for luminal breast cancer. Our results support the use of EPIC arrays to identify new epigenetic noninvasive biomarkers in breast cancer.

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.

Noninvasive early detection of colorectal cancer by hypermethylation of the LINC00473 promoter in plasma cell-free DNA.

BACKGROUND: Current noninvasive assays have limitations in the early detection of colorectal cancer. We evaluated the clinical utility of promoter methylation of the long noncoding RNA LINC00473 as a noninvasive biomarker to detect colorectal cancer and associated precancerous lesions. METHODS: We evaluated the epigenetic regulation of LINC00473 through promoter hypermethylation in colorectal cancer cell lines using bisulfite genomic sequencing and expression analyses. DNA methylation of LINC00473 was analyzed in primary colorectal tumors using 450K arrays and RNA-seq from The Cancer Genome Atlas (TCGA). Tissue-based findings were validated in several independent cohorts of colorectal cancer and advanced colorectal polyp patients by pyrosequencing. We explored the clinical utility of LINC00473 methylation for the early detection of colorectal cancer in plasma cell-free DNA by quantitative methylation-specific PCR and droplet digital PCR. RESULTS: LINC00473 showed transcriptionally silencing due to promoter hypermethylation in colorectal cancer cell lines and primary tumors. Methylation of the LINC00473 promoter accurately detected primary colorectal tumors in two independent clinical cohorts, with areas under the receiver operating characteristic curves (AUCs) of 0.94 and 0.89. This biomarker also identified advanced colorectal polyps from two other tissue-based clinical cohorts with high diagnostic accuracy (AUCs of 0.99 and 0.78). Finally, methylation analysis of the LINC00473 promoter in plasma cell-free DNA accurately identified patients with colorectal cancer and advanced colorectal polyps (AUCs of 0.88 and 0.84, respectively), which was confirmed in an independent cohort of patients. CONCLUSIONS: Hypermethylation of the LINC00473 promoter is a new promising biomarker for noninvasive early detection of colorectal cancer and related precancerous lesions.

DNA methylome in visceral adipose tissue can discriminate patients with and without colorectal cancer.

Adipose tissue dysfunction, particularly the visceral (VAT) compartment, has been proposed to play a relevant role in colorectal cancer (CRC) development and progression. Epigenetic mechanisms could be involved in this association. The current study aimed to evaluate if specific epigenetic marks in VAT are associated with colorectal cancer (CRC) to identify epigenetic hallmarks of adipose tissue-related CRC. Epigenome-wide DNA methylation was evaluated in VAT from 25 healthy participants and 29 CRC patients, using the Infinium HumanMethylation450K BeadChip. The epigenome-wide methylation analysis identified 170,184 sites able to perfectly separate the CRC and healthy samples. The differentially methylated CpG sites (DMCpGs) showed a global trend for increased methylated levels in CRC with respect to healthy group. Most of the genes encoded by the DMCpGs belonged to metabolic pathways and cell cycle, insulin resistance, and adipocytokine signalling, as well as tumoural transformation processes. In gene-specific analyses, involved genes biologically relevant for the development of CRC include PTPRN2, MAD1L1, TNXB, DIP2C, INPP5A, HDCA4, PRDM16, RPTOR, ATP11A, TBCD, PABPC3, and IER2. The methylation level of some of them showed a discriminatory capacity for detecting CRC higher than 90%, showing IER2 to have the highest capacity. This study reveals that a specific methylation pattern of VAT is associated with CRC. Some of the epigenetic marks identified could provide useful tools for the prediction and personalized treatment of CRC connected to excess adiposity.

Aberrant integration of Hepatitis B virus DNA promotes major restructuring of human hepatocellular carcinoma genome architecture.

Most cancers are characterized by the somatic acquisition of genomic rearrangements during tumour evolution that eventually drive the oncogenesis. Here, using multiplatform sequencing technologies, we identify and characterize a remarkable mutational mechanism in human hepatocellular carcinoma caused by Hepatitis B virus, by which DNA molecules from the virus are inserted into the tumour genome causing dramatic changes in its configuration, including non-homologous chromosomal fusions, dicentric chromosomes and megabase-size telomeric deletions. This aberrant mutational mechanism, present in at least 8% of all HCC tumours, can provide the driver rearrangements that a cancer clone requires to survive and grow, including loss of relevant tumour suppressor genes. Most of these events are clonal and occur early during liver cancer evolution. Real-time timing estimation reveals some HBV-mediated rearrangements occur as early as two decades before cancer diagnosis. Overall, these data underscore the importance of characterising liver cancer genomes for patterns of HBV integration.

Evaluation of a Targeted Next-Generation Sequencing Panel for the Non-Invasive Detection of Variants in Circulating DNA of Colorectal Cancer.

Molecular profiling of circulating cell-free DNA (cfDNA) has shown utility for the management of colorectal cancer (CRC). TruSight Tumor 170 (TST170) is a next-generation sequencing (NGS) panel that covers 170 cancer-related genes, including KRAS, which is a key driver gene in CRC. We evaluated the capacity of TST170 to detect gene variants in cfDNA from a retrospective cohort of 20 metastatic CRC patients with known KRAS variants in tumor tissue and in cfDNA previously analyzed by pyrosequencing and BEAMing, respectively. The cfDNA of most of the patients (95%) was successfully sequenced. We frequently detected variants with clinical significance in KRAS (79%, 15/19) and PIK3CA (26%, 5/19) genes. Variants with potential clinical significance were also identified in another 27 cancer genes, such as APC. The type of KRAS variant detected in cfDNA by TST170 showed high concordance with those detected in tumor tissue (77%), and very high concordance with cfDNA analyzed by BEAMing (94%). The variant allele fractions for KRAS obtained in cfDNA by TST170 and BEAMing correlated strongly. This proof-of-principle study indicates that targeted NGS analysis of cfDNA with TST170 could be useful for non-invasive detection of gene variants in metastatic CRC patients, providing an assay that could be easily implemented for detecting somatic alterations in the clinic.

Modulation of Colorectal Tumor Behavior via lncRNA TP53TG1-Lipidic Nanosystem.

Long non-coding RNAs (lncRNAs) are an emerging group of RNAs with a crucial role in cancer pathogenesis. In gastrointestinal cancers, TP53 target 1 (TP53TG1) is an epigenetically regulated lncRNA that represents a promising therapeutic target due to its tumor suppressor properties regulating the p53-mediated DNA damage and the intracellular localization of the oncogenic YBX1 protein. However, to translate this finding into the clinic as a gene therapy, it is important to develop effective carriers able to deliver exogenous lncRNAs to the targeted cancer cells. Here, we propose the use of biocompatible sphingomyelin nanosystems comprising DOTAP (DSNs) to carry and deliver a plasmid vector encoding for TP53TG1 (pc(TP53TG1)-DSNs) to a colorectal cancer cell line (HCT-116). DSNs presented a high association capacity and convenient physicochemical properties. In addition, pc(TP53TG1)-DSNs showed anti-tumor activities in vitro, specifically a decrease in the proliferation rate, a diminished colony-forming capacity, and hampered migration and invasiveness of the treated cancer cells. Consequently, the proposed strategy displays a high potential as a therapeutic approach for colorectal cancer.

Clinical potential of circulating free DNA and circulating tumour cells in patients with metastatic non-small-cell lung cancer treated with pembrolizumab.

Immune checkpoint inhibitors, such as pembrolizumab, are revolutionizing therapeutic strategies for different cancer types, including non-small-cell lung cancer (NSCLC). However, only a subset of patients benefits from this therapy, and new biomarkers are needed to select better candidates. In this study, we explored the value of liquid biopsy analyses, including circulating free DNA (cfDNA) and circulating tumour cells (CTCs), as a prognostic or predictive tool to guide pembrolizumab therapy. For this purpose, a total of 109 blood samples were collected from 50 patients with advanced NSCLC prior to treatment onset and at 6 and 12 weeks after the initiation of pembrolizumab. Plasma cfDNA was measured using hTERT quantitative PCR assay. The CTC levels at baseline were also analysed using two enrichment technologies (CellSearch® and Parsortix systems) to evaluate the efficacy of both approaches at detecting the presence of programmed cell death ligand 1 on CTCs. Notably, patients with high baseline hTERT cfDNA levels had significantly shorter progression-free survival (PFS) and overall survival (OS) than those with low baseline levels. Moreover, patients with unfavourable changes in the hTERT cfDNA levels from baseline to 12 weeks showed a higher risk of disease progression. Additionally, patients in whom CTCs were detected using the CellSearch® system had significantly shorter PFS and OS than patients who had no CTCs. Finally, multivariate regression analyses confirmed the value of the combination of CTCs and cfDNA levels as an early independent predictor of disease progression, identifying a subgroup of patients who were negative for CTCs, who presented low levels of cfDNA and who particularly benefited from the treatment.

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.

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.