Droplet digital PCR of tumor suppressor gene methylation in serial oral rinses of patients with head and neck squamous cell carcinoma.
Ontology highlight
ABSTRACT: Background
Head and neck squamous cell carcinoma (HNSCC) currently lacks sensitive approaches to detect cancer-related traits in body fluid.Methods
Methylation of tumor suppressor genes (TSGs) (PAX5, EDNRB, and DCC) were measured in the oral rinses from 50 HNSCC and 58 control subjects using droplet digital PCR (ddPCR). Diagnostic accuracies in detecting HNSCC and the detection rate of recurrence in the post-treatment monitoring were analyzed.Results
ddPCR TSG methylation detection in oral rinses for diagnosis of HNSCC had an AUC of 0.892 for PAX5, 0.753 for EDNRB, and 0.729 for DCC. Significant drop of TSG methylation was observed after completion of surgery (p < 0.01). 76.9% of the relapse cases had a pre-emptive rebound of methylation above presurgery levels in at least one of the tested markers before confirmed recurrence.Conclusions
Utilizing ddPCR for TSG methylation detection in oral rinses shows potential for detection and monitoring of HNSCC.
SUBMITTER: Fung SYH
PROVIDER: S-EPMC8248028 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Molecular deep surgical margin analysis has been shown to predict locoregional recurrences of head and neck squamous cell carcinoma (HNSCC). To improve the accuracy and versatility of the analysis, we used a highly tumor-specific methylation marker and highly sensitive detection technology to test DNA from surgical margins. Histologically cancer-negative deep surgical margin samples were prospectively collected from 82 eligible HNSCC surgeries by an imprinting procedure (n = 75) and primary tissue collection (n = 70). Bisulfite-treated DNA from each sample was analyzed by both conventional quantitative methylation-specific PCR (QMSP) and QMSP by droplet digital PCR (ddQMSP) targeting Paired box 5 (PAX5) gene promoter methylation. The association between the presence of PAX5 methylation and locoregional recurrence-free survival (LRFS) was evaluated. PAX5 methylation was found in 68.0% (51 of 75) of tumors in the imprint samples and 71.4% (50 of 70) in the primary tissue samples. Among cases that did not have postoperative radiation (n = 31 in imprint samples, n = 29 in tissue samples), both conventional QMSP and ddQMSP revealed that PAX5 methylation-positive margins was significantly associated with poor LRFS by univariate analysis. In particular, ddQMSP increased detection of the PAX5 marker from 29% to 71% in the nonradiated imprint cases. Also, PAX5 methylated imprint margins were an excellent predictor of poor LRFS [HR, 3.89; 95% confidence interval (CI), 1.19-17.52; P = 0.023] by multivariate analysis. PAX5 methylation appears to be an excellent tumor-specific marker for molecular deep surgical margin analysis of HNSCC. Moreover, the ddQMSP assay displays increased sensitivity for methylation marker detection.
| S-EPMC4633357 | biostudies-literature
Project description:Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in circulating cell‑free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non‑invasive procedure that collects tumor‑derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)‑based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20 patients with HNSCC during follow‑up. The results highlighted: i) The ability of the ddPCR‑based assay to detect very low copies of methylated molecules; ii) the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow‑up with respect to T0; iii) a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.
| S-EPMC10865173 | biostudies-literature
Project description:MotivationDroplet digital PCR (ddPCR) holds great promises for investigating DNA methylation with high sensitivity. Yet, the lack of methods for analyzing ddPCR DNA methylation data has resulted in users processing the data manually at the expense of standardization.ResultsPoDCall is an R package performing automated calling of positive droplets, quantification and normalization of methylation levels in ddPCR experiments. A Shiny application provides users with an intuitive and interactive interface to access PoDCall functionalities.Availability and implementationThe PoDCall R package is freely available on Bioconductor at https://bioconductor.org/packages/PoDCall/. The Shiny application can be executed from the R console using the wrapper function PoDCall::podcallShiny().Supplementary informationSupplementary data are available at Bioinformatics online.
| S-EPMC9825742 | biostudies-literature
Project description:During posttreatment surveillance of head and neck cancer patients, imaging is insufficiently accurate for the early detection of relapsing disease. Free circulating tumor DNA (ctDNA) may serve as a novel biomarker for monitoring tumor burden during posttreatment surveillance of these patients. In this exploratory study, we investigated whether low level ctDNA in plasma of head and neck cancer patients can be detected using Droplet Digital PCR (ddPCR).TP53 mutations were determined in surgically resected primary tumor samples from six patients with high stage (II-IV), moderate to poorly differentiated head and neck squamous cell carcinoma (HNSCC). Subsequently, mutation specific ddPCR assays were designed. Pretreatment plasma samples from these patients were examined on the presence of ctDNA by ddPCR using the mutation-specific assays. The ddPCR results were evaluated alongside clinicopathological data.In all cases, plasma samples were found positive for targeted TP53 mutations in varying degrees (absolute quantification of 2.2-422 mutational copies/ml plasma). Mutations were detected in wild-type TP53 background templates of 7667-156,667 copies/ml plasma, yielding fractional abundances of down to 0.01%.Our results show that detection of tumor specific TP53 mutations in low level ctDNA from HNSCC patients using ddPCR is technically feasible and provide ground for future research on ctDNA quantification for the use of diagnostic biomarkers in the posttreatment surveillance of HNSCC patients.
| S-EPMC5477260 | biostudies-other
Project description:Diet is associated with cancer prognosis, including head and neck cancer (HNC), and has been hypothesized to influence epigenetic state by determining the availability of functional groups involved in the modification of DNA and histone proteins. The goal of this study was to describe the association between pretreatment diet and HNC tumor DNA methylation. Information on usual pretreatment food and nutrient intake was estimated via food frequency questionnaire (FFQ) on 49 HNC cases. Tumor DNA methylation patterns were assessed using the Illumina Goldengate Methylation Cancer Panel. First, a methylation score, the sum of individual hypermethylated tumor suppressor associated CpG sites, was calculated and associated with dietary intake of micronutrients involved in one-carbon metabolism and antioxidant activity, and food groups abundant in these nutrients. Second, gene specific analyses using linear modeling with empirical Bayesian variance estimation were conducted to identify if methylation at individual CpG sites was associated with diet. All models were controlled for age, sex, smoking, alcohol and HPV status. Individuals reporting in the highest quartile of folate, vitamin B12 and vitamin A intake, compared with those in the lowest quartile, showed significantly less tumor suppressor gene methylation, as did patients reporting the highest cruciferous vegetable intake. Gene specific analyses identified differential associations between DNA methylation and vitamin B12 and vitamin A intake when stratifying by HPV status. These preliminary results suggest that intake of folate, vitamin A and vitamin B12 may be associated with the tumor DNA methylation profile in HNC and enhance tumor suppression.
| S-EPMC3427284 | biostudies-literature
Project description:AIMS: To study the homozygous deletion and methylation status of the 5' CpG island of the p16 and p15 genes (9p21) in a set of primary advanced head and neck squamous cell carcinomas (SCC) and to test whether inactivation of these genes by these mechanisms contributes to head and neck SCC development. METHODS: DNA was extracted from fresh tumours. Homozygous deletion was determined by the polymerase chain reaction (PCR) followed by hybridisation with the corresponding probe, radioactively labelled by the random priming method. Methylation status of the CpG island of the 5' region of these genes was assessed by digestion with the appropriate restriction enzymes followed by PCR and subsequent hybridisation with the corresponding probe. The presence of point mutations was determined by PCR-SSCP (single strand conformation polymorphism). RESULTS: The p16 and p15 genes were homozygously deleted in 20% and 10% of the tumours, respectively. No point mutations were found at p16 and p15. The 5' CpG island at the p16 gene was methylated in 20% of the cases. CONCLUSIONS: The tumour suppressor gene p16 is inactivated through homozygous deletion or methylation in a significant proportion of cases of head and neck SCC.
| S-EPMC499991 | biostudies-other
Project description:DNA methylation plays an important role in the etiology and pathogenesis of head and neck squamous cell carcinoma (HNSCC). The current study aimed to identify aberrantly methylated-differentially expressed genes (DEGs) by a comprehensive bioinformatics analysis. In addition, we screened for DEGs affected by DNA methylation modification and further investigated their prognostic values for HNSCC. We included microarray data of DNA methylation (GSE25093 and GSE33202) and gene expression (GSE23036 and GSE58911) from Gene Expression Omnibus. Aberrantly methylated-DEGs were analyzed with R software. The Cancer Genome Atlas (TCGA) RNA sequencing and DNA methylation (Illumina HumanMethylation450) databases were utilized for validation. In total, 27 aberrantly methylated genes accompanied by altered expression were identified. After confirmation by The Cancer Genome Atlas (TCGA) database, 2 hypermethylated-low-expression genes (FAM135B and ZNF610) and 2 hypomethylated-high-expression genes (HOXA9 and DCC) were identified. A receiver operating characteristic (ROC) curve confirmed the diagnostic value of these four methylated genes for HNSCC. Multivariate Cox proportional hazards analysis showed that FAM135B methylation was a favorable independent prognostic biomarker for overall survival of HNSCC patients.
| S-EPMC6140809 | biostudies-other
Project description:BACKGROUND:Alu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology. METHODS:Forty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR. RESULTS:Using ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D. CONCLUSIONS:In our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.
| S-EPMC6303857 | biostudies-other
Project description:Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1) and Erythropoietin (EPO) transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.
| S-EPMC4222945 | biostudies-literature
Project description:Regulatory T cells (Tregs) suppress immune responses in vivo in an antigen-specific manner. Of clinical relevance, Tregs can be isolated and expanded in vitro while maintaining immunoregulatory function. Tregs are classified as CD4+CD25highCD127low FOXP3+ cells. Demethylation of the Treg-specific demethylation region (TSDR) of FOXP3 is found in natural Tregs (nTregs). We report a method for the characterization of the differential methylation pattern of the FOXP3 TSDR in patient-derived and expanded nTregs. Human TSDR sequences from nTregs (unmethylated sequence) and pancreatic (methylated sequence) cells were amplified and cloned into plasmids. A droplet digital TaqMan probe-based qPCR (ddPCR) assay using methylation-specific primers and probes was employed to quantify unmethylated and methylated sequences. The methylation-specific droplet digital PCR (ddMSP) assay was specific and selective for unmethylated DNA in mixtures with methylated DNA in the range of 5000 copies/μL to less than 1 copy/μL (R 2 = 0.99) even in the presence of non-selective gDNAs. CD4+CD25highCD127lowFOXP3+ human nTregs, in the presence of Dynabeads or activators, were expanded for 21 days. There was a decrease in the unmethylated ratio of Tregs after expansion with essentially the same ratio at days 10, 14, and 17. However, the activator expanded group showed a significant decrease in unmethylated targets at day 21. The suppression activity of activator-expanded nTregs at day 21 was decreased compared to cells expanded with Dynabeads. These data suggest that the ddMSP can quantitatively monitor nTreg expansion in vitro. These data also indicate that the assay is sensitive and specific at differentiating nTregs from other cells and may be useful for rapid screening of nTregs in clinical protocols.
| S-EPMC7154152 | biostudies-literature