Global Hypomethylation Identifies Loci Targeted for Hypermethylation in Head and Neck Cancer

Abstract

Purpose The human epigenome is profoundly altered in cancers, with a characteristic loss of methylation in repetitive regions and concomitant accumulation of gene-promoter methylation. The degree to which these processes are coordinated is unclear so we investigated both in head and neck squamous cell carcinomas. Experimental Design Global methylation was measured using the luminometric methylation assay (LUMA), and pyrosequencing of LINE-1Hs and AluYb8 repetitive elements in a series of 138 tumors. We also measured methylation of over 27,000 CpG loci with the Illumina HumanMethylation27 microarray (n=91). Results LINE-1 methylation was significantly associated with LUMA and Infinium loci methylation (Spearman’s rho=0.52/rho=0.56, both p<0.001), but not that of AluYb8. Methylation of LINE-1, AluYb8, and Infinium loci differed by tumor site (each Kruskal-Wallis p<0.05). Also, LINE-1 and LUMA methylation were associated with HPV16 E6 serology (each Mann-Whitney p<0.05). Comparing LINE-1 methylation to gene-associated methylation, we identified a distinct subset of CpG loci with significant hypermethylation associated with LINE-1 hypomethylation. An investigation of sequence features for these CpG loci revealed that they were significantly less likely to reside in repetitive elements (GSEA p<0.02), enriched in CpG islands (p<0.001), and were proximal to transcription factor binding sites (p<0.05). We validated the top CpG loci that had significant hypermethylation associated with LINE-1 hypomethylation (at EVI2A, IFRD1, KLHL6, and PTPRCAP) by pyrosequencing independent tumors. Conclusions These data indicate that global hypomethylation and gene-specific methylation processes are associated in a sequence-dependent manner, and that clinical characteristics and exposures leading to HNSCC may be influencing these processes.

Differences in methylation profiles between HPV-positive and HPV-negative oropharynx squamous cell carcinoma

“…Seventeen articles met the inclusion criteria and were eligible for further analysis. [28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] The main reasons for exclusion were duplicates and studies not investigating correlation between HPV status and methylation status in OPSCC.…”

“…28,33,34,44 In other publications studying overall HNSCC, OPSCC subgroup size ranged from 9% to 80% of the total study population. [29][30][31][32][35][36][37][38][39][40][41][42][43] There was only one study that reported the distribution of average age, nicotine and alcohol consumption between HPV-positive and HPV-negative tumors of which promoter methylation was evaluated. 34 The mean age in HPV-positive tumors was 56.9 compared with 58.4 in HPV-negative tumors.…”

“…28,29,32,[34][35][36][37]40 Six used fresh frozen (FF) tissue or both. 31,38,[41][42][43][44] Three studies did not report used material. 30,33,39 The majority of studies used polymerase chain reaction (PCR) to detect HPV 16 DNA.…”

“…Three studies 34,41,43 used PCR and immunohistochemistry (IHC) and Poage et al used multiplex serology. 38 Sartor et al did not describe their method to detect HPV DNA. 40 There was a wide variation in percentage of HPV positivity between studies ranging from 7% to 56%.…”

Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma

“…20,21 MSH4 was recently shown to be hypermethylated in HNSCC. 22 No data on aberrant promoter methylation have been published so far for NEIL1, APEX2 and TREX2. Differential methylation of MLH1 and MSH2 was not observed, maybe due to our detection method or the relatively small number of samples in our screening.…”