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. 2015;10(3):229-36.
doi: 10.1080/15592294.2015.1006506.

DNA methylation profiles and biomarkers of oral squamous cell carcinoma

Yu-Fen Li  1 , Yi-Hsiu Hsiao, Yi-Hui Lai, Yi-Chen Chen, Ying-Ju Chen, Jian-Liang Chou, Michael W Y Chan, Yu-Hsing Lin, Yung-An Tsou, Ming-Hsui Tsai, Chien-Kuo Tai
Affiliations

DNA methylation profiles and biomarkers of oral squamous cell carcinoma

Yu-Fen Li et al. Epigenetics. 2015.

Abstract

Oral squamous cell carcinoma (OSCC) constitutes >90% of oral cancers and is the sixth most common malignancy among males worldwide and the fourth leading cause of death due to cancer among males in Taiwan. However, most patients do not receive a diagnosis of OSCC until the late stages, which have a lower survival rate. The use of molecular marker analysis to identify early-stage OSCC would permit optimal timing for treatments and consequently prolong survival. The aim of this study was to identify biomarkers of OSCC using the Illumina GoldenGate Methylation Cancer Panel, which comprised a total of 1,505 CpG sites covering 807 genes. Samples of buccal mucosa resected from 40 OSCC patients and normal tissue samples obtained from 15 patients (normal mucosa from OSCC patients or from patients undergoing surgery unrelated to OSCC) were analyzed. Fms-related tyrosine kinase 4 (FLT4) methylation exhibited a perfect specificity for detecting OSCC, with an area under the receiver operating characteristic curve of 0.91 for both all-stage and early-stage OSCC. Methylation of 7 genes (ASCL1, FGF3, FLT4, GAS7, KDR, TERT, and TFPI2) constitutes the top-20 panels for detecting OSCC. The top-20 panels for detecting early-stage OSCC contain 8 genes: ADCYAP1, EPHA7, FLT4, GSTM2, KDR, MT1A, NPY, and TFPI2. FLT4 RNA expression and methylation level were validated using RT-PCR and a pyrosequencing methylation assay. The median level of FLT4 expression was 2.14-fold for normal relative to OSCC tissue samples (P < 0.0001). Among the 8 pyrosequenced FLT4 CpG sites, methylation level was much higher in the OSCC samples. In conclusion, methylation statuses of selected genes, and especially FLT4, KDR, and TFPI2, might be of great potential as biomarkers for early detection of buccal OSCC.

Keywords: DNA methylation array; FLT4; KDR; TFPI2; biomarkers; oral squamous cell carcinoma; pyrosequencing.

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Figures

Figure 1.
Figure 1.
Hierarchical clustering heat maps and dendrograms for distinguishing (A) OSCC from normal samples and (B) early-stage OSCC from normal samples. Mean β values from the selected CpG sites were used for hierarchical agglomerative clustering based on the Manhattan distance and complete linkage. Low, medium, and high methylation levels are indicated in green, black, and red, respectively.
Figure 2.
Figure 2.
Box plots of methylation levels of 8 FLT4 CpG sites determined by pyrosequencing methylation assay. (A) Normal samples. (B) OSCC samples for internal validation. (C) OSCC samples for external validation. In each box plot, the whiskers represent the 10th and 90th percentiles, the lower and upper limits of the box indicate the 25th and 75th percentiles. An asterisk mark (*) indicates that the difference between normal and tumor samples reaches statistical significance.
Figure 3.
Figure 3.
Comparison of FLT4 RNA expression between 21 paired normal and OSCC tissue samples. The mRNA level of FLT4 in each normal tissue sample was compared with that of its corresponding OSCC tissue sample, which was assigned a value of 1. Error bars indicate standard deviations.
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