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.

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.

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.

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.

Global Gene Expression Characterization of Circulating Tumor Cells in Metastasic Castration-Resistant Prostate Cancer Patients.

BACKGROUND: Current therapeutic options in the course of metastatic castration-resistant prostate cancers (mCRPC) reinforce the need for reliable tools to characterize the tumor in a dynamic way. Circulating tumor cells (CTCs) have emerged as a viable solution to the problem, whereby patients with a variety of solid tumors, including PC, often do not have recent tumor tissue available for analysis. The biomarker characterization in CTCs could provide insights into the current state of the disease and an overall picture of the intra-tumor heterogeneity. METHODS: in the present study, we applied a global gene expression characterization of the CTC population from mCRPC (n = 9), with the goal to better understand the biology of these cells and identify the relevant molecules favoring this tumor progression. RESULTS: This analysis allowed the identification of 50 genes specifically expressed in CTCs from patients. Six of these markers (HOXB13, QKI, MAOA, MOSPD1, SDK1, and FGD4), were validated in a cohort of 28 mCRPC, showing clinical interest for the management of these patients. Of note, the activity of this CTC signature was related to the regulation of MYC, a gene strongly implicated in the biology of mCRPC. CONCLUSIONS: Overall, our results represent new evidence on the great value of CTCs as a non-invasive biopsy to characterize PC.

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.

Detection of MET Alterations Using Cell Free DNA and Circulating Tumor Cells from Cancer Patients.

MET alterations may provide a potential biomarker to evaluate patients who will benefit from treatment with MET inhibitors. Therefore, the purpose of the present study is to investigate the utility of a liquid biopsy-based strategy to assess MET alterations in cancer patients. We analyzed MET amplification in circulating free DNA (cfDNA) from 174 patients with cancer and 49 healthy controls and demonstrated the accuracy of the analysis to detect its alteration in patients. Importantly, a significant correlation between cfDNA concentration and MET copy number (CN) in cancer patients (r = 0.57, p <10-10) was determined. Furthermore, we evaluated two approaches to detect the presence of MET on circulating tumor cells (CTCs), using the CellSearch® and Parsortix systems and monitored patients under anti-EGFR treatment (n = 30) combining both cfDNA and CTCs analyses. This follow-up provides evidence for the potential of MET CN assessment when patients develop resistance to anti-EGFR therapy and a significant association between the presence of CTCs MET+ and the Overall Survival (OS) in head and neck cancer patients (P = 0.05; HR = 6.66). In conclusion, we develop specific and noninvasive assays to monitor MET status in cfDNA/CTCs and demonstrate the utility of plasma MET CN determination as a biomarker for monitoring the appearance of resistance to anti-EGFR therapy.

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.

A Novel Saliva-Based miRNA Signature for Colorectal Cancer Diagnosis.

Salivary microRNAs (miRNAs) are of high interest as diagnostic biomarkers for non-oral cancer. However, little is known about their value for colorectal cancer (CRC) detection. Our study aims to characterize salivary miRNAs in order to identify non-invasive markers for CRC diagnosis. The screening of 754 miRNAs was performed in saliva samples from 14 CRC and 10 healthy controls. The differential expressed miRNAs were validated by RT-qPCR in 51 CRC, 19 adenomas and 37 healthy controls. Receiver operating characteristic (ROC) curves and logistic regression models were performed to analyze the clinical value of these miRNAs. Twenty-two salivary miRNAs were significantly deregulated in CRC patients vs. healthy individuals (P < 0.05) in the discovery phase. From those, five upregulated miRNAs (miR-186-5p, miR-29a-3p, miR-29c-3p, miR-766-3p, and miR-491-5p) were confirmed to be significantly higher in the CRC vs. healthy group (P < 0.05). This five-miRNA signature showed diagnostic value (72% sensitivity, 66.67% specificity, AUC = 0.754) to detect CRC, which was even higher in combination with carcinoembryonic antigen (CEA) levels. Overall, after the first global characterization of salivary miRNAs in CRC, a five-miRNA panel was identified as a promising tool to diagnose this malignancy, representing a novel approach to detect cancer-associated epigenetic alterations using a non-invasive strategy.

Circulating microRNAs as Promising Biomarkers in Colorectal Cancer.

Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related deaths worldwide. Despite numerous advances in therapeutic approaches, this cancer has a poor prognosis when it is diagnosed at late stages. Therefore, the scientific effort is nowadays directed towards the development of new non-invasive and dynamic biomarkers to improve the survival expectancy of CRC patients. In this sense, deregulated expression of many miRNAs has been shown to play an important role for CRC carcinogenesis and dissemination. Noticeably, an increasing number of studies highlight that circulating miRNAs, including those traveling inside exosomes or those released by tumor cells into circulation, constitute a promising tool for early detection, prognosis and therapy selection of CRC. Therefore, in this review we focus on the clinical potential of blood circulating miRNAs as emerging biomarkers with high value to improve the clinical management of CRC patients, providing a deep and complete perspective of the realities and challenges to translate these biomarkers to the clinical context.

Liquid Biopsy in Oral Cancer.

Oral cancer is one of the most prevalent forms of cancer worldwide. Carcinogenesis is a complex process, in which heterogeneity plays an important role in the development and progression of the disease. This review provides an overview of the current biological and clinical significance of circulating tumour cells (CTCs), circulating tumour DNA (ctDNA), and exosomes for diagnosis and prognosis of oral cancer. We highlight the importance of liquid biopsy­using blood and saliva­which represents a potential alternative to solid biopsy for diagnosis and prognosis. Moreover, liquid biomarkers allow for the real-time monitoring of tumour evolution and therapeutic responses, initiating the era of personalized medicine. However, in oral cancer, the impact of liquid biopsies in clinical settings is still limited, requiring further studies to discover the best scenario for its clinical use.

Human salivary microRNAs in Cancer.

Circulating microRNAs (miRNAs) have emerged as excellent candidates for cancer biomarkers. Several recent studies have highlighted the potential use of saliva for the identification of miRNAs as novel biomarkers, which represents a great opportunity to improve diagnosis and monitor general health and disease. This review summarises the mechanisms of miRNAs deregulation in cancer, the value of targeting them with a therapeutic intention and the evidence of the potential clinical use of miRNAs expressed in saliva for the detection of different cancer types. We also provide a comprehensive review of the different methods for normalising the levels of specific miRNAs present in saliva, as this is a critical step in their analysis, and the challenge to validate salivary miRNAs as a reality to manage cancer patients.