RESUMO
OBJECTIVE: DNA methylation data can be used to derive mitotic indices from complex tissues. Here, we assessed if the DNA methylation-derived mitotic ageing indices are associated with oral squamous cell carcinoma (OSCC) development and recurrence-free survival (RFS). METHODS: DNA methylation-based mitotic indices (MitoticAge, TNSC and hypoSC) were derived using algorithms "MitoticAge" and "epiTOC2" for the discovery [non-malignant (n = 22), premalignant (n = 22) and OSCC (n = 68) tissues] and validation datasets (GSE87053, GSE136704 and TCGA-HNSCC). Differences in mitotic indices between non-malignant, premalignant and OSCC tissues were assessed. Finally, the association between estimated mitotic indices and RFS was evaluated in OSCCs. RESULTS: In the discovery and validation datasets, increased mitotic ageing was observed in OSCC compared to non-malignant and premalignant oral tissues. HPV-positive HNSCCs had higher mitotic index TNSC. Mitotic age index hypoSC was associated with RFS in OSCC (p = 0.011, HR 2.61, 95% CI 1.24-5.48). CONCLUSIONS: DNA methylation-derived mitotic indices are associated with OSCC development and RFS. Thus, DNA methylation-derived mitotic indices may be a valuable research tool to reliably estimate the cumulative number of stem cell divisions in malignant and non-malignant oral tissues. Future research utilizing mitotic indices for predicting clinical outcomes in OSCC is warranted.
RESUMO
BACKGROUND: Gingivobuccal complex oral squamous cell carcinoma (GBC-OSCC) is an aggressive malignancy with high mortality often preceded by premalignant lesions, including leukoplakia. Previous studies have reported genomic drivers in OSCC, but much remains to be elucidated about DNA methylation patterns across different stages of oral carcinogenesis. RESULTS: There is a serious lack of biomarkers and clinical application of biomarkers for early detection and prognosis of gingivobuccal complex cancers. Hence, in search of novel biomarkers, we measured genome-wide DNA methylation in 22 normal oral tissues, 22 leukoplakia, and 74 GBC-OSCC tissue samples. Both leukoplakia and GBC-OSCC had distinct methylation profiles as compared to normal oral tissue samples. Aberrant DNA methylation increases during the different stages of oral carcinogenesis, from premalignant lesions to carcinoma. We identified 846 and 5111 differentially methylated promoters in leukoplakia and GBC-OSCC, respectively, with a sizable fraction shared between the two sets. Further, we identified potential biomarkers from integrative analysis in gingivobuccal complex cancers and validated them in an independent cohort. Integration of genome, epigenome, and transcriptome data revealed candidate genes with gene expression synergistically regulated by copy number and DNA methylation changes. Regularised Cox regression identified 32 genes associated with patient survival. In an independent set of samples, we validated eight genes (FAT1, GLDC, HOXB13, CST7, CYB5A, MLLT11, GHR, LY75) from the integrative analysis and 30 genes from previously published reports. Bisulfite pyrosequencing validated GLDC (P = 0.036), HOXB13 (P < 0.0001) promoter hypermethylation, and FAT1 (P < 0.0001) hypomethylation in GBC-OSCC compared to normal controls. CONCLUSIONS: Our findings identified methylation signatures associated with leukoplakia and gingivobuccal complex cancers. The integrative analysis in GBC-OSCC identified putative biomarkers that enhance existing knowledge of oral carcinogenesis and may potentially help in risk stratification and prognosis of GBC-OSCC.
