Methylation Array Analysis of Preoperative and Postoperative Saliva DNA in Oral Cancer Patients

Abstract

Purpose: To perform methylation array analysis of 807 cancer-associated genes using tissue and saliva of oral squamous cell carcinoma (OSCC) patients with the objective of identifying highly methylated gene loci that hold diagnostic and predictive value as a biomarker. Experimental Design: We did the methylation array on DNA extracted from preoperative saliva, postoperative saliva, and tissue of 13 patients with OSCC, and saliva of 10 normal subjects. We identified sites that were highly methylated in the tissue and preoperative saliva samples but not methylated in the postoperative saliva samples or in normal subjects.Results: High quality DNA was obtained and the methylation array was successfully run on all samples. We identified significant differences in methylation patterns between the preoperative and postoperative saliva from cancer patients. We established a gene classifier consisting of 41 gene loci from 34 genes that showed methylation in preoperative saliva and tissue but were not methylated in postoperative saliva or normal subjects. Gene panels of 4 to 10 genes were constructed from genes in the classifier. The panels had a sensitivity of 62% to 77% and a specificity of 83% to 100% for OSCC. Conclusions: We report methylation array analysis of 807 cancer-associated genes in the saliva of oral cancer patients before and after oral cancer resection. Our methylation biomarker approach shows the proof of principle that methylation array analysis of saliva can produce a set of cancer-related genes that are specific and can be used as a composite biomarker for the early detection of oral cancer. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3603 -11)

DNA methylation profiles and biomarkers of oral squamous cell carcinoma

“…In a recent study, Jithesh et al 39 also employed the same array and suggested that EPHA5, GSTM2, HS3ST2, MME, MT1A, MYOD1, NPY, and PENK are potential biomarkers for detecting oral cancer, as in the present study. The only triple-hit gene among the studies of Viet et al, 10 Jithesh et al, 39 and ourselves was EPHA5. In addition, Nagata et al 40 reported that the aberrant methylation of a combination of ECAD, TMEFF2, RARb, and MGMT from rinse samples can detect OSCC with a sensitivity and specificity of >90% by using noninvasive methods to retrieve samples and a different methylation platform.…”

“…Several methods of determining the occurrence of promoter methylation have been described in the literature, including methylation-specific PCR (MSP), combined bisulfite restriction analysis, bisulfite sequencing assay, pyrosequencing methylation assay, and microarray-based methylation analysis; the overwhelming majority of published data were obtained using the MSP method. Literature reviews [10][11][12] have revealed a wide variation in the reported sensitivity and specificity for this method, ranging from 30% to 90%. Given the limited number of CpG sites detected by MSP and the inconsistent results obtained using this method, we developed a diagnostic tool for OSCC using a high-throughput, quantitative methylation platform-the Illumina GoldenGate Methylation Array-to allow a more comprehensive profiling of DNA methylation.…”

“…ADCYAP1 is associated with hormone activity, 20 GSTM2 belongs to a well-known phase II detoxifying enzyme family, 21 MT1A regulates cell growth and differentiation, 22 TFPI-2 counteracts the activity of several extracellular matrix-associated proteases 23,24 and suppresses tumor invasion and metastasis, [25][26][27] and Viet et al 10 used the Illumina GoldenGate Methylation Array to identify potential biomarkers for oral cancer by comparing the saliva samples of oral cancer patients before and after resection of their tumors. About one half of the identified genes (e.g., ADCYAP1, FGF3, GAS7, and KDR) also exhibited potential in our tissue samples as biomarkers of oral cancer.…”

A Review on Salivary Genomics and Proteomics Biomarkers in Oral Cancer

“…The tumor-specific DNA in saliva could also be used as biomarker for oral cancer [45]. Methylation array of salivary DNA was supported as an effective biomarker for early detection of oral cancer [46]. Hypermethylation on the promoter of DNA in specific gene All the biomarkers were significantly altered in oral cancer and found to be useful as a supportive tool for diagnosis, prognosis and post-operative monitoring.…”

“…An assay was developed that could rapidly quantify the promoter hypermethylation of the gene of interest and could potentially be applied into a clinical setting. [46,48] Salivary IL-8 mRNA and protein EC sensor was developed with multiplexing biomarker detection for salivary diagnostics. IL-8 mRNA and IL-8 protein levels measured by the EC sensors showed significant differences between oral cancer patients and controls.…”