Droplet digital PCR of tumor suppressor gene methylation in serial oral rinses of patients with head and neck squamous cell carcinoma

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) currently lacks sensitive approaches to detect cancer-related traits in body fluid.

Methods: Methylation of tumor suppressor genes (TSGs) (PAX5, EDNRB, and DCC) were measured in the oral rinses from 50 HNSCC and 58 control subjects using droplet digital PCR (ddPCR). Diagnostic accuracies in detecting HNSCC and the detection rate of recurrence in the post-treatment monitoring were analyzed.

Results: ddPCR TSG methylation detection in oral rinses for diagnosis of HNSCC had an AUC of 0.892 for PAX5, 0.753 for EDNRB, and 0.729 for DCC. Significant drop of TSG methylation was observed after completion of surgery (p < 0.01). 76.9% of the relapse cases had a pre-emptive rebound of methylation above presurgery levels in at least one of the tested markers before confirmed recurrence.

Conclusions: Utilizing ddPCR for TSG methylation detection in oral rinses shows potential for detection and monitoring of HNSCC.

Keywords: droplet digital PCR; head and neck cancer; head and neck squamous cell carcinoma; liquid biopsy; methylation.

FIGURE 1

Optimization of the droplet digital quantitative methylation specific PCR (ddqMSP) assays. (A) Testing of the ddqMSP assay of Deleted in Colorectal Cancer (DCC) using universally methylated DNA and universally unmethylated DNA, the assay showed specific identification of only methylated signal and unmethylated signal. (B) Quantification performances of the assays were evaluated under low methylation concentration testing, showing the assays could correctly identify the methylated sequence in low concentration conditions. Pearson correlation tests showed close correlation between the droplet digital PCR (ddPCR) results with the expected methylation density of the tested concentrations [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

Methylation density of tumor suppressor gene (TSG) markers in head and neck squamous cell carcinoma (HNSCC) tissue specimens and tissues from recruited control subjects. Paired Box 5 (PAX5) (A), Endothelin Receptor Beta (EDNRB) (B), and Deleted in Colorectal Cancer (DCC) (C). All three markers showed aberrant methylation in tumor, compared with the paired normal tissues (p < 0.001, Mann–Whitney U test) and with the control tissues (p < 0.001, Mann–Whitney U test). Each dot in the three groups represents individual patients. Median methylation level of each group was indicated [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

Comparison of the HNSCC diagnostic accuracy of TSG methylation markers. (A) Diagnostic accuracy of the individual TSG methylation markers. The cut‐off selected in the model was marked by a dot in each ROC curve. Diagnostic accuracy of Paired Box 5 (PAX5) methylation in oral rinse is significantly better compared with Endothelin Receptor Beta (EDNRB) and Deleted in Colorectal Cancer (DCC) (p < 0.004, log‐rank test). The cut‐off for PAX5 methylation was set at methylation density (M%) >0.5%. The cut‐off for EDNRB and DCC methylation was set at EDNRB M% >0.1% and DCC M% >0.08%, respectively. (B) Comparison between the marker combinations and the best‐performed TSG marker (PAX5). In the trial of the combination of the TSG methylation markers, the best performed three marker combination did not show significant improvement compared with PAX5 (p = 0.306, log‐rank test) [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

Change in methylation density of tumor suppressor gene (TSG) markers in oral rinse specimens before and after surgical treatment. The figure showed the paired comparison of the methylation densities of Paired Box 5 (PAX5) (A), Endothelin Receptor Beta (EDNRB) (B), and Deleted in Colorectal Cancer (DCC) (C) in oral rinse collected on presurgery and post 4‐week (first postsurgery timepoint) timepoint. Median level in each group was indicated by the blue mark. Three markers all shown significant drop in the methylation densities after completion of the surgery [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

Longitudinal variation of the oral rinse tumor suppressor gene (TSG) methylation in head and neck squamous cell carcinoma (HNSCC) patients. (A) The methylation densities of TSG markers in serial collected oral rinse samples from non‐relapse patients showed significant drop after completion of the treatment and remained at low levels till final followup time point. (B) Some relapse cases showed close correlation between the variation of TSG methylation in oral rinse and the disease progression and treatment [Color figure can be viewed at wileyonlinelibrary.com]