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Unique DNA methylation signature in HPV-positive head and neck squamous cell carcinomas

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ABSTRACT

Background: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited.

Methods: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours.

Results: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort.

Conclusions: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13073-017-0419-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus

Hypomethylated DMRs associated with HPV infection show CpG island shore loss of boundary and functional correlation with gene expression. a Heatmap showing the top 50 DMRs associated with HPV status (FDR <0.05). b CpG context of the identified DMRs in the different anatomic sites compared with their distribution in the Illumina HumanMethylation 450 K array (HM450). c Correlation between CpG methylation at the SYCP2 DMR and relative gene expression in the HNSCC cases of the TCGA cohort. HPV(+) cases are indicated by full pink dots, HPV(–) cases by empty blue dots. d Co-methylation plots showing the CpGs in SYCP2 DMR ranked by p value and visualized based on their chromosomal coordinates, relative position to CpG island (green bar) and CpG content (red peak). The average methylation values in the DMR in HPV(+) (pink) or HPV(–) (blue) cases are shown. The correlation plot shows Spearman correlation values among the CpGs in the region
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Fig3: Hypomethylated DMRs associated with HPV infection show CpG island shore loss of boundary and functional correlation with gene expression. a Heatmap showing the top 50 DMRs associated with HPV status (FDR <0.05). b CpG context of the identified DMRs in the different anatomic sites compared with their distribution in the Illumina HumanMethylation 450 K array (HM450). c Correlation between CpG methylation at the SYCP2 DMR and relative gene expression in the HNSCC cases of the TCGA cohort. HPV(+) cases are indicated by full pink dots, HPV(–) cases by empty blue dots. d Co-methylation plots showing the CpGs in SYCP2 DMR ranked by p value and visualized based on their chromosomal coordinates, relative position to CpG island (green bar) and CpG content (red peak). The average methylation values in the DMR in HPV(+) (pink) or HPV(–) (blue) cases are shown. The correlation plot shows Spearman correlation values among the CpGs in the region

Mentions: Since alterations in DNA methylation can affect multiple neighbouring CpG sites, we applied dimension reduction to identify differentially methylated regions (DMRs, at least three CpGs in a 1-kb region), using the DMRcate package (see Methods for details) in the whole dataset (Additional files 4 and 5) and in each stratified organ group (Additional files 6, 7 and 8). Globally, we identified 4371 hypermethylated DMRs and 2044 hypomethylated DMRs (FDR <0.05). However, using more stringent criteria (increasing the differential methylation to Δβmax >20% or Δβmax >30%), we found an increased proportion (from 32% to 65% or 95%, respectively) of hypomethylated regions associated with HPV(+) status (Table 2). Interestingly, we found that average methylation levels of the top 25 hypomethylated and top 25 hypermethylated DMRs (Additional files 4, 5, 6, 7 and 8) were able to separately cluster HPV(+) from HPV(–) HNSCCs for each anatomic site (57/63) (Fig. 3a, Additional file 2: Figure S2D–F). Globally, both hyper- and hypomethylated regions presented an enrichment not only in CpG islands, as previously described, but also in shores (Fig. 3b), thus revealing new genomic regions targeted by DNA methylation changes in HPV(+) HNSCCs.Table 2


Unique DNA methylation signature in HPV-positive head and neck squamous cell carcinomas
Hypomethylated DMRs associated with HPV infection show CpG island shore loss of boundary and functional correlation with gene expression. a Heatmap showing the top 50 DMRs associated with HPV status (FDR <0.05). b CpG context of the identified DMRs in the different anatomic sites compared with their distribution in the Illumina HumanMethylation 450 K array (HM450). c Correlation between CpG methylation at the SYCP2 DMR and relative gene expression in the HNSCC cases of the TCGA cohort. HPV(+) cases are indicated by full pink dots, HPV(–) cases by empty blue dots. d Co-methylation plots showing the CpGs in SYCP2 DMR ranked by p value and visualized based on their chromosomal coordinates, relative position to CpG island (green bar) and CpG content (red peak). The average methylation values in the DMR in HPV(+) (pink) or HPV(–) (blue) cases are shown. The correlation plot shows Spearman correlation values among the CpGs in the region
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC5382363&req=5

Fig3: Hypomethylated DMRs associated with HPV infection show CpG island shore loss of boundary and functional correlation with gene expression. a Heatmap showing the top 50 DMRs associated with HPV status (FDR <0.05). b CpG context of the identified DMRs in the different anatomic sites compared with their distribution in the Illumina HumanMethylation 450 K array (HM450). c Correlation between CpG methylation at the SYCP2 DMR and relative gene expression in the HNSCC cases of the TCGA cohort. HPV(+) cases are indicated by full pink dots, HPV(–) cases by empty blue dots. d Co-methylation plots showing the CpGs in SYCP2 DMR ranked by p value and visualized based on their chromosomal coordinates, relative position to CpG island (green bar) and CpG content (red peak). The average methylation values in the DMR in HPV(+) (pink) or HPV(–) (blue) cases are shown. The correlation plot shows Spearman correlation values among the CpGs in the region
Mentions: Since alterations in DNA methylation can affect multiple neighbouring CpG sites, we applied dimension reduction to identify differentially methylated regions (DMRs, at least three CpGs in a 1-kb region), using the DMRcate package (see Methods for details) in the whole dataset (Additional files 4 and 5) and in each stratified organ group (Additional files 6, 7 and 8). Globally, we identified 4371 hypermethylated DMRs and 2044 hypomethylated DMRs (FDR <0.05). However, using more stringent criteria (increasing the differential methylation to Δβmax >20% or Δβmax >30%), we found an increased proportion (from 32% to 65% or 95%, respectively) of hypomethylated regions associated with HPV(+) status (Table 2). Interestingly, we found that average methylation levels of the top 25 hypomethylated and top 25 hypermethylated DMRs (Additional files 4, 5, 6, 7 and 8) were able to separately cluster HPV(+) from HPV(–) HNSCCs for each anatomic site (57/63) (Fig. 3a, Additional file 2: Figure S2D–F). Globally, both hyper- and hypomethylated regions presented an enrichment not only in CpG islands, as previously described, but also in shores (Fig. 3b), thus revealing new genomic regions targeted by DNA methylation changes in HPV(+) HNSCCs.Table 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Head and neck squamous cell carcinomas (HNSCCs) represent a heterogeneous group of cancers for which human papilloma virus (HPV) infection is an emerging risk factor. Previous studies showed promoter hypermethylation in HPV(+) oropharyngeal cancers, but only few consistent target genes have been so far described, and the evidence of a functional impact on gene expression is still limited.

Methods: We performed global and stratified pooled analyses of epigenome-wide data in HNSCCs based on the Illumina HumanMethylation450 bead-array data in order to identify tissue-specific components and common viral epigenetic targets in HPV-associated tumours.

Results: We identified novel differentially methylated CpGs and regions associated with viral infection that are independent of the anatomic site. In particular, most hypomethylated regions were characterized by a marked loss of CpG island boundaries, which showed significant correlations with expression of neighbouring genes. Moreover, a subset of only five CpGs in a few hypomethylated regions predicted HPV status with a high level of specificity in different cohorts. Finally, this signature was a better predictor of survival compared with HPV status determined by viral gene expression by RNA sequencing in The Cancer Genome Atlas cohort.

Conclusions: We identified a novel epigenetic signature of HPV infection in HNSCCs which is independent of the anatomic site, is functionally correlated with gene expression and may be leveraged for improved stratification of prognosis in HNSCCs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13073-017-0419-z) contains supplementary material, which is available to authorized users.

No MeSH data available.


Related in: MedlinePlus