Neuropeptide receptor genes GHSR and NMUR1 are candidate epigenetic biomarkers and predictors for surgically treated patients with oropharyngeal cancer

Abstract

Pathological staging and histological grading systems are useful, but imperfect, predictors of recurrence in head and neck squamous cell carcinoma (HNSCC). Aberrant promoter methylation is the main type of epigenetic modification that plays a role in the inactivation of tumor suppressor genes. To identify new potential prognostic markers, we investigated the promoter methylation status of five neuropeptide receptor genes. The methylation status of the target genes was compared with clinical characteristics in 278 cases; 72 hypopharyngeal cancers, 54 laryngeal cancers, 75 oropharyngeal cancers, and 77 oral cavity cancers were studied. We found that the NTSR1, NTSR2, GHSR, MLNR, and NMUR1 promoters were methylated in 47.8%, 46.8%, 54.3%, 39.2%, and 43.5% of the samples, respectively. GHSR and NMUR1 promoter methylation independently predicted recurrence in HNSCC. In patients with oropharyngeal cancer (n = 75), GHSR and NMUR1 promoter methylation significantly correlates with survival in surgically treated patients. We classified our patients as having a low, intermediate, or high-risk of death based on three factors: HPV status, and GHSR and NMUR1 promoter methylation. The disease-free survival (DFS) rates were 87.1%, 42.7%, and 17.0%, respectively. Combined data analysis of the methylation status of ten-eleven translocation (TET) family genes indicated a trend toward greater methylation indices as the number of TET methylation events increased. In the current study, we presented the relationship between the methylation status of the GHSR and NMUR1 genes and recurrence in HNSCC, specifically in risk classification of oropharyngeal carcinomas cases with HPV status.

Figure 1

Methylation of the neuropeptide receptor gene promoters in 278 HNSCC samples. (a) Bar graph showing the methylation frequencies of the five genes. (b) Bar graph showing the percentage of tumors that express zero to five methylated target genes. (c) Comparison of the methylation status of the promoters of the five genes in patients with hypopharyngeal, laryngeal, oropharyngeal, or oral cancer. Filled boxes indicate the presence of methylation, and open boxes indicate the absence of methylation (d) Bar graph showing the methylation indices (MIs) according to selected clinical parameters. The mean MI for each parameter was determined by the Student’s t-test.

Figure 2

Correlation between promoter methylation levels of the five neuropeptide receptor genes and TET family genes in cancer tissues. (a) Distribution of promoter methylation in TET family genes and the five neuropeptide receptor genes. Filled boxes indicate the presence of methylation, and open boxes indicate the absence of methylation. (b) Combined analysis of the MIs and methylation status of TET family genes. The number of methylation events is indicated for hypermethylated TET family genes. The mean MIs for the different groups were compared using the Student’s t-test. **P < 0.001. The data are shown as mean ± SD.

Figure 3

Kaplan-Meier survival curves for the 278 patients with HNSCC according to the methylation status of the five target genes. DFS for (a) NTSR1, (b) NTSR2, (c) GHSR, (d) MLNR, and (e) NMUR1 in the case of methylated (red lines) and unmethylated (blue lines) genes. (f) Combined analysis of the five genes. Blueline: patients with 0–3 methylated genes; red line: patients with 4–5 methylated genes. A probability of <0.05 (*P < 0.05) was considered a statistically significant difference.

Figure 4

Risk of recurrence based on gene methylation in tumors with different origins. Odds ratios for recurrence were determined using a Cox proportional hazards model adjusted for age (≥65 vs. <65 years), sex, smoking status, alcohol intake, and tumor stage (I–II vs. III–IV). CI: confidence interval.

Figure 5

Odds ratios for recurrence based on the Cox proportional hazards model. Multivariate Cox regression analyses were performed to assess the correlations between (a) recurrence and patients with T1–2 (n = 135) and T3–4 tumor sizes (n = 143) and between (b) recurrence and patients with HPV-positive (n = 37) and HPV-negative oropharyngeal cancer (n = 38).

Figure 6

Classification of study patients with oropharyngeal carcinoma into the risk of recurrence categories and Kaplan-Meier estimates of DFS according to the categories. (a) Kaplan-Meier estimates of DFS among oropharyngeal cancer patients to classify patients into categories of low-, intermediate-, or high-risk of recurrence, according to GHSR methylation, NMUR1 methylation, and HPV status. (b) Patients with oropharyngeal carcinoma were classified into three categories with respect to the risk of recurrence. Group 1 and 2: low-risk group; Group 4 and 5: intermediate-risk group; Group 3 and 6: high-risk group.