PITX3 DNA methylation is an independent predictor of overall survival in patients with head and neck squamous cell carcinoma

Abstract

Background: Molecular biomarkers assisting risk-group assignment and subsequent treatment stratification are urgently needed for patients with squamous cell cancer of the head and neck region (HNSCC). Aberrant methylation is a frequent event in cancer and, therefore, a promising source for potential biomarkers. Here, the methylation status of the paired-like homeodomain transcription factor 3 (PITX3) was evaluated in HNSCC.

Methods: Using a quantitative real-time PCR, PITX3 methylation was assessed in a cohort of 326 HNSCC patients treated for localized or locally advanced disease (training cohort). The results were validated with Infinium HumanMethylation450 BeadChip data from a 528 HNSCC patient cohort (validation cohort) generated by The Cancer Genome Atlas (TCGA) Research Network.

Results: PITX3 methylation was significantly higher methylated in tumor compared to normal adjacent tissue (NAT; training cohort: median methylation NAT 32.3%, tumor 71.8%, p < 0.001; validation cohort: median methylation NAT 16.9%, tumor 35.9%, p < 0.001). PITX3 methylation was also significantly correlated with lymph node status both in the training (p = 0.006) and validation (p < 0.001) cohort. PITX3 methylation was significantly higher in HPV-associated (p16-positive) tumors compared to p16-negative tumors (training cohort: 73.7 vs. 66.2%, p = 0.013; validation cohort: 40.0 vs. 33.1%, p = 0.015). Hypermethylation was significantly associated with the risk of death (training cohort: hazard ratio (HR) = 1.80, [95% confidence interval (CI) 1.20-2.69], p = 0.005; validation cohort: HR = 1.43, [95% CI 1.05-1.95], p = 0.022). In multivariate Cox analyses, PITX3 added independent prognostic information. Messenger RNA (mRNA) expression analysis revealed an inverse correlation with PITX3 methylation in the TCGA cohort.

Conclusions: PITX3 DNA methylation is an independent prognostic biomarker for overall survival in patients with HNSCC and might aid in the process of risk stratification for individualized treatment.

Keywords: Biomarker; DNA methylation; HNSCC; HPV; Head and neck squamous cell carcinoma; PITX3; Prognosis.

Fig. 1

Genomic organization and assay position. Genomic organization of the PITX3 gene and locations of the PITX3 qPCR assay [22] and the Infinium HumanMethylation450 BeadChip bead cg20023259. The information was obtained from Ensembl Homo sapiens version GRCh38.p7

Fig. 2

PITX3 DNA methylation in HNSCC specimens. Frequency of PITX3 DNA methylation in the training cohort (a) comprising 326 HNSCC patients and validation cohort (b) including 528 HNSCC patients. Shown are the methylation levels in tumor tissue only. PITX3 DNA methylation in tumor tissue compared to normal adjacent tissue from the training (c) and the validation (d) cohort, respectively. Tumor tissue was methylated significantly higher compared to corresponding normal adjacent tissue. p values refer to Wilcoxon-Mann-Whitney test

Fig. 3

Overall survival in patients stratified according to PITX3 DNA methylation. Kaplan-Meier analysis of overall survival in HNSCC patients stratified according to PITX3 DNA methylation levels in tumor tissue. a Training cohort (all patients). b Training cohort (p16-negative patients). c Validation cohort (all patients). High PITX3 DNA methylation was associated with an adverse prognosis in both cohorts, in particular in p16-negative patients from the training cohort. A subgroup analysis of p16-negative patients from the validation cohort was not performed due to largely missing data