A Minimal DNA Methylation Signature in Oral Tongue Squamous Cell Carcinoma Links Altered Methylation with Tumor Attributes

Abstract

Oral tongue squamous cell carcinomas (OTSCC) are a homogenous group of aggressive tumors in the head and neck region that spread early to lymph nodes and have a higher incidence of regional failure. In addition, there is a rising incidence of oral tongue cancer in younger populations. Studies on functional DNA methylation changes linked with altered gene expression are critical for understanding the mechanisms underlying tumor development and metastasis. Such studies also provide important insight into biomarkers linked with viral infection, tumor metastasis, and patient survival in OTSCC. Therefore, we performed genome-wide methylation analysis of tumors (N ¼ 52) and correlated altered methylation with differential gene expression. The minimal tumor-specific DNA 5-methylcytosine signature identified genes near 16 different differentially methylated regions, which were validated using genomic data from The Cancer Genome Atlas cohort. In our cohort, hypermethylation of MIR10B was significantly associated with the differential expression of its target genes NR4A3 and BCL2L11 (P ¼ 0.0125 and P ¼ 0.014, respectively), which was inversely correlated with disease-free survival (P ¼ 9EÀ15 and P ¼ 2EÀ15, respectively) in patients. Finally, differential methylation in FUT3, TRIM5, TSPAN7, MAP3K8, RPS6KA2, SLC9A9, and NPAS3 genes was found to be predictive of certain clinical and epidemiologic parameters.Implications: This study reveals a functional minimal methylation profile in oral tongue tumors with associated risk habits, clinical, and epidemiologic outcomes. In addition, NR4A3 downregulation and correlation with patient survival suggests a potential target for therapeutic intervention in oral tongue tumors. Data from the current study are deposited in the NCBI Geo database (accession number GSE75540).

A minimal set of internal control genes for gene expression studies in head and neck squamous cell carcinoma

“…The gene expression profiling using Illumina HumanHT-12 v4 expression BeadChips (Illumina, San Diego, CA), raw data collection and data processing using the R package lumi 7 and individual batch-correction using ComBat 8 is described previously 9 10 . The raw signal intensities from arrays were exported from GenomeStudio for pre-processing and analyzed using R. Gene-wise expression intensities for tumor and matched control samples from GenomeStudio were transformed and normalized using VST (Variance Stabilizing Transformation) and LOESS methods, respectively, using lumi 7 and top genes with least across-sample variance in expression were selected.…”

“…In the discovery and validation stage, HNSCC tumor: adjacent normal paired samples ( n =63 and n =14 respectively), were used. Previously published whole-genome microarray data 9 10 , RNA-seq data from TCGA 11 and data from individual studies in the literature were used in the discovery stage. For the validation set, 10 HNSCC tumor: normal pairs were from a completely independent cohort (which were not a part of discovery cohort) and 4 were used from the in house discovery cohort.…”

High-risk human papillomavirus in oral cavity squamous cell carcinoma

“…Production of whole genome methylation data using Illumina Infinium Methylation450 BeadChip, chip scanning and the data pre-processing are described previously 27 . Three tumors in HPV +ve group (relatively with high-copy HPV DNA and/or E6/E7 RNA) and 18 tumors in HPV -ve group (HPV DNA –ve and HPV RNA –ve) were considered for further analysis.…”

CAFE MOCHA: An Integrated Platform for Discovering Clinically Relevant Molecular Changes in Cancer—An Example of Distant Metastasis– and Recurrence-Linked Classifiers in Head and Neck Squamous Cell Carcinoma

“…The methylation data was pre-processed, including normalization and batch correction, as described previously (Krishnan et al, 2016). Tumour samples assayed for all the four events (mut, cnv, expr and meth) were considered for the discovery phase.…”