Genes encoding neuropeptide receptors are epigenetic markers in patients with head and neck cancer: a site-specific analysis

Abstract

Staging and pathological grading systems are useful but imperfect predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). To identify potential prognostic markers, we examined the methylation status of eight neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinomas. The NPFFR1, NPFFR2, HCRTR1, HCRTR2, NPY1R, NPY2R, NPY4R, and NPY5R promoters were methylated in 80.5%, 79.2%, 67.1%, 73.2%, 35.1%, 36.4%, 38.5%, and 35.9% of the samples, respectively. In a multivariate Cox proportional hazards analysis, the odds ratio for recurrence was 2.044 (95% confidence interval [CI], 1.323-3.156; P = 0.001) when the NPY2R promoter was methylated. In patients without lymph node metastasis (n = 100), methylation of NPY2R (compared with methylation of the other seven genes) best correlated with poor disease-free survival (DFS) (odds ratio, 2.492; 95% CI, 1.190-5.215; P = 0.015). In patients with oral cancer (n = 69), methylated NPY1R and NPY2R were independent prognostic factors for poor DFS, both individually and, even more so, in combination (odds ratio, 3.90; 95% CI, 1.523-9.991; P = 0.005). Similar findings were observed for NPY2R and NPY4R in patients with oropharyngeal cancer (n = 162) (odds ratio, 5.663; 95% CI, 1.507-21.28; P = 0.010).

Keywords: GPCR; epigenetic markers; head and neck cancer; metastases; neuropeptide receptors.

Figure 1. Methylation of the neuropeptide receptor gene promoters in 231 head and neck squamous cell carcinoma samples

(A) Bar graph showing the percentage of tumors that express zero to eight of the methylated target genes. (B) Bar graph showing the methylation frequencies of the eight genes. (C) Bar graph showing the methylation indices (MIs) according to selected clinical parameters. The mean MI for each parameter was determined by using Student's t-test.

Figure 2. Kaplan-Meier survival curves for the 231 patients with head and neck squamous cell carcinoma according to the methylation status of the eight target genes

Disease-free survival for (A) NPFFR1, (B) NPFFR2, (C) HCRTR1, (D) HCRTR2, (E) NPY1R, (F) NPY2R, (G) NPY4R and (H) NPY5R. The log-rank test was used to compare the survival times in patients with methylated (red lines) and unmethylated (blue lines) genes.

Figure 3. Risk of recurrence based on gene methylation in tumors with different origins

Odds ratios for recurrence were determined by using a Cox proportional hazards model adjusted for age (≥70 versus <70 years), sex, smoking status, alcohol intake, and stage (I–III versus IV). CI: confidence interval.