Prognostic significance of LINE-1 hypomethylation in oropharyngeal squamous cell carcinoma

Abstract

Background: Inclusion of new biomarkers to improve a personalized treatment approach for oropharyngeal squamous cell carcinoma (OPSCC) is urgently needed. Hypomethylation of the Long interspersed nucleotide element-1 (LINE-1) repetitive elements, a widely accepted surrogate of overall genomic DNA methylation content, was found to be associated with a poor prognosis in several cancers. At present, no studies have investigated the influence of LINE-1 methylation levels on OPSCC relapse. The main goal of this study was the evaluation of the prognostic value of LINE-1 methylation status in predicting early tumor relapse in locally advanced OPSCC.

Methods: We retrospectively reviewed a cohort of 77 patients with stage III-IVB OPSCC. Methylation of LINE-1 repetitive sequences was evaluated by real-time quantitative methylation-specific PCR in formalin-fixed paraffin-embedded tissues. The prognostic relevance of LINE-1 methylation was assessed by comparing patients who relapsed within 2 years from the end of treatment (cases) with those who did not (controls). Results were validated in an independent cohort of 33 patients with OPSCC.

Results: With respect to early OPSCC relapse, the mean LINE-1 methylation level was significantly lower in relapsed cases than in control group (p < 0.01). Interestingly, LINE-1 methylation was lower in relapsed cases than in controls in both HPV16-negative and HPV16-positive OPSCC patients, even if statistical significance was reached only for the former group (p = 0.01). LINE-1 methylation levels were also significantly reduced in relapsed cases with respect to the controls in OPSCC current smokers (p = 0.02). Consistently, in HPV16-negative current smokers, OPSCC relapse was significantly associated with decreased levels of LINE-1 methylation (p = 0.02). Using logistic regression model, we found that patients with hypomethylated LINE-1 were associated with a 3.5 higher risk of early relapse than hypermethylated ones (OR = 3.51; 95% CI 1.03-12.00). Adjustment for potential confounders did not substantially change the risk magnitude. Results from the validation cohort confirmed the lower LINE-1 methylation in patients who early relapsed compared to relapse-free patients.

Conclusions: LINE-1 hypomethylation is associated with higher risk of early relapse in stage III-IVB OPSCC. Further validation in a prospective study is needed for its application in daily clinical practice.

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

Fig. 1

LINE-1 methylation according to socio-demographic characteristics, smoking habits, tumor features, and surgical treatment. Black horizontal bars represent the median values of LINE-1 methylation for each group. p values were determined by the non-parametric Kruskal–Wallis test

Fig. 2

LINE-1 methylation in patients who relapsed within 24 months from the end of treatment (cases) and in patients who did not (controls), according to HPV16 status, current tobacco smoking, and risk group. Risk group was defined on HPV16 status and current tobacco smoking as follow: low (HPV16+/non-smokers), intermediate (HPV16−/non-smokers or HPV16+/smokers), high (HPV16−/smokers). Black horizontal bars represent the median values of LINE-1 methylation for each group. p values were determined by the non-parametric Kruskal–Wallis test

Fig. 3

Predicted probability of relapse within 24 months from end of treatment in the discovery set and in the validation set

Fig. 4

Relationships between LINE-1 methylation and age in the discovery and validation sets. The linear regression is represented by a scattered line (discovery set) and a bold line (validation set). Regression line coefficients (b) are also reported for both lines