Research Article: Intragenic DNA methylation of PITX1 and the adjacent long non-coding RNA C5orf66-AS1 are prognostic biomarkers in patients with head and neck squamous cell carcinomas

Date Published: February 9, 2018

Publisher: Public Library of Science

Author(s): Verena Sailer, Arthur Charpentier, Joern Dietrich, Timo J. Vogt, Alina Franzen, Friedrich Bootz, Dimo Dietrich, Andreas Schroeck, Scott M. Langevin.


Patients with squamous cell cancer of the head and neck region (HNSCC) are at risk for disease recurrence and metastases, even after initial successful therapy. A tissue-based biomarker could be beneficial to guide treatment as well as post-treatment surveillance. Gene methylation status has been recently identified as powerful prognostic biomarker in HNSCC. We therefore evaluated the methylation status of the homeobox gene PITX1 and the adjacent long intergenic non-coding RNA (lincRNA) C5orf66-AS1 in publicly available datasets.

Gene methylation and expression data from 528 patients with HNSCC included in The Cancer Genome Atlas (TCGA, there obtained by using the Infinium HumanMethylation450 BeadChip Kit) were evaluated and methylation and expression levels of PITX1 and lincRNA C5orf66-AS1 was correlated with overall survival and other parameters. Thus, ten beads targeting PITX1 exon 3 and three beads targeting lincRNA C5orf66-AS1 were identified as significant candidates. The mean methylation of these beads was used for further correlation and the median was employed for dichotomization.

Both PITX1 exon 3 and lincRNA C5orf66-AS1 were significantly higher methylated in tumor tissue than in normal adjacent tissue (NAT) (PITX1 exon 3: tumor tissue 58.1%, NAT: 31.7%, p<0.001; lincRNA C5orf66-AS1: tumor tissue: 27.4%, NAT: 18.9%, p<0.001). In a univariate analysis, hypermethylation of both loci was significantly associated with the risk of death (univariate: exon 3: Hazard ratio (HR): 4.97 [1.78–16.71], p = 0.010, lincRNA C5orf66-AS1: Hazard ratio (HR): 12.23 [3.01–49.74], p<0.001). PITX1 exon 3 and lincRNA C5orf66-AS1 methylation was also significantly correlated with tumor localization, T category, human papilloma virus (HPV)-negative and p16-negative tumors and tumor grade. Kaplan-Meier analysis showed, that lincRNA C5orf66-AS1 hypomethylation was significantly associated with overall survival (p = 0.001) in the entire cohort as well in a subgroup of HPV-negative tumors (p = 0.003) and in patients with laryngeal tumors (p = 0.022). Methylation status of PITX1 and even more so of lincRNA C5orf66-AS1 is a promising prognostic biomarker in HNSCC, in particular for HPV-negative patients. Further prospective evaluation is warranted.

Partial Text

Squamous cell carcinoma of the head and neck region (HNSCC) is a common malignancy with 5-year survival rates ranging from 61% for laryngeal cancers to 63% for cancers of the oral cavity and pharynx [1]. For 2017, 63,030 new cases are estimated in the United States and 13,360 patients are estimated to die of cancer-related causes [2].

The present study evaluates for the first time the clinical utility of PITX1 and lincRNA C5orf66-AS1 methylation status as prognostic biomarker in HNSCC. Both parameters added significant information about risk of death in univariate analysis and hyomethylation of lincRNA C5orf66-AS1 is associated with better survival, in particular in patients with negative HPV-status. While overall survival rates have improved in HNSCC, no improvement has been achieved for patients with laryngeal carcinoma, which is not thought to be associated with HPV infection [45–47]. As yet, only few prognostic biomarkers have been investigated in this patient cohort. For example, in silico analysis of expression data deposited in the Gene Expression Omnibus (GEO) identified spectrin, a cytoskleton protein, as a promising new biomarker [48]. Expression of the tyrosine kinase c-MET has been shown to be prognostic in oral squamous cell cancer, which is in general not associated with HPV-infection [49]. Expression of sonic hedgehog pathway related genes Gli-1 and Gli-2 was found to be associated with overall survival in a prospective study in patients with HPV-negative HNSCC [50]. Methylation status of lincRNA C5orf66-AS1 could emerge as a useful prognostic biomarker to identify HPV-negative patients at risk for disease recurrence and metastases, who might benefit from additional therapy like immune checkpoint inhibitors.




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