LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer

Abstract

Background and purpose: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation.

Results: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival-OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24-10.38) and progression (HR 4.54, 95% CI 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences.

Conclusions: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Keywords: DNA methylation; HPV; LINE-1; Oropharyngeal squamous cell carcinoma; p53.

Fig. 1

Oncological outcomes according to level of LINE-1 methylation. Overall survival (a) and progression-free survival (b) were calculated through the Kaplan–Meier method. LINE-1 methylation was categorized using a recursive procedure which maximizes the difference in OS

Fig. 2

Oncological outcomes according to HPV16 status and level of LINE-1 methylation. Overall survival (a) and progression-free overall survival (b) were calculated through the Kaplan–Meier method, stratifying patients according to HPV16 status and LINE-1 methylation level

Fig. 3

LINE-1 methylation according to HVP16 status and p53 expression pattern. Mean LINE-1 methylation, and corresponding 95% confidence intervals, was reported for HPV16 status and level of p53 expression. The independent association of HPV16 status and p53 expression with LINE-1 methylation was evaluated through the analysis of variance

Fig. 4

DNA methylation analysis in HPV16-negative OPSCC patients. a Pie chart showing the percentage of differentially methylated CpG in promoter, gene body, and intergenic regions. b Box plot showing the average methylation level (beta-value) of all CpG differentially methylated in not relapsed (NR) vs relapsed (R) HPV16-negative OPSCC patients. c Histogram showing the molecular function where the genes showing differentially methylated promoter are involved

Fig. 5

Integrative analysis of gene expression and DNA methylation in HPV16-negative OPSCC patients. a Leftside: heatmap summarizing expression data for the up-regulated genes in HPV16-negative OPSCC patients (P) who relapsed within 2 years from the end of treatment (P4, P9) with those who did not (P19, P36, P74). Data are shown as normalized expression values in log2 and centered on the median value. Rightside: heatmap showing the methylation values (beta value) in relapsed (P4, P9, P10, P32, P43) vs relapsed-free (P19, P30, P36, P72, P74) HPV16-negative OPSCC patients. b Kaplan–Meier estimates overall survival according to high- and low-methylation levels of the PIK3C2G cg17881542 in MethSurv