Unique DNA methylation loci distinguish anatomic site and HPV status in head and neck squamous cell carcinoma

Abstract

Purpose: We have used a genome-wide approach to identify novel differentially methylated CpG dinucleotides that are seen in different anatomic sites of head and neck squamous cell carcinoma (HNSCC), as well as those that might be related to HPV status in the oropharynx.

Experimental design: We conducted genome-wide DNA methylation profiling of primary tumor samples and corresponding adjacent mucosa from 118 HNSCC patients undergoing treatment at Montefiore Medical Center, Bronx, NY, using the Illumina HumanMethylation27 beadchip. For each matched tissue set, we measured differentially methylated CpG loci using a change in methylation level (M-value).

Results: When datasets were individually analyzed by anatomic site of the primary tumor, we identified 293 differentially methylated CpG loci in oral cavity squamous cell carcinoma (SCC), 219 differentially methylated CpG loci in laryngeal SCC, and 460 differentially methylated in oropharyngeal SCC. A subset of these differentially methylated CpG loci was common across all anatomic sites of HNSCC. Stratification by HPV status revealed a significantly higher number of differentially methylated CpG loci in HPV+ patients.

Conclusion: Novel epigenetic biomarkers derived from clinical HNSCC specimens can be used as molecular classifiers of this disease, revealing many new avenues of investigation for this disease.

Figure 1

(A) Dot plots of relative DNA methylation levels (tumor to adjacent normal tissue), measured as a change in M value (ΔM), for CpG loci cg13877915 (ZNF132), cg08668790 (ZNF154) and cg21790626 (ZNF154) for specimen pairs originating from oral cavity (OC), oropharyngeal (OP) and laryngeal (LX) SCC. The median level of differential methylation is indicated by a bar for each patient population. (B) Relative gene expression (tumor to adjacent normal tissue) of corresponding target genes ZNF132 and ZNF154, as obtained by beadchip probe fluorescence measurements in corresponding gene expression microarray datasets, for specimen pairs originating from oral cavity (OC), oropharyngeal (OP) and laryngeal (LX) SCC. The median level of differential expression is indicated by a bar for each patient population.

Figure 2

(A) Dot plot of relative DNA methylation levels (tumor to adjacent mucosa), measured as a change in M value (ΔM) for CpG loci cg08319991 (UCHL1) for specimen pairs originating from oral cavity (OC), oropharyngeal (OP) and laryngeal (LX) SCC. The median level of differential methylation is indicated by a bar for each patient population. Statistical significance (p<0.05) is indicated by an asterisk. (B) Plot of relative gene expression (tumor to adjacent mucosa) of UCHL1 (beadchip probe fluorescence measurements) versus relative DNA methylation (ΔM) for all HNSCC patients. Outliers are indicated by an ‘x’. Correlation between cg08319991 methylation and UCHL1 gene expression is calculated by linear regression analysis.

Figure 3

Dot plot of relative DNA methylation levels (tumor to adjacent mucosa) (ΔM) in oropharyngeal SCC patients for each of the 28 CpG loci whose methylation status best differentiated HPV⁻ from HPV+ oropharyngeal cases. HPV⁺ oropharyngeal cases are indicated by red dots; HPV⁻ oropharyngeal cases are indicated by green dots. Details for each CpG loci include Illumina Target ID, associated gene symbol, as well as the average ΔM for HPV⁻ and HPV⁺ patient sets. Abbreviations: *p<0.05, **p<0.01, ***p<0.001, ns not significant.