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. 2016 Nov 15;7(46):75379-75393.
doi: 10.18632/oncotarget.12211.

Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

Damian T Rieke  1 , Sebastian Ochsenreither  1   2 , Konrad Klinghammer  2 , Tanguy Y Seiwert  3 , Frederick Klauschen  4 , Inge Tinhofer  5   6 , Ulrich Keilholz  1   2
Affiliations

Methylation of RAD51B, XRCC3 and other homologous recombination genes is associated with expression of immune checkpoints and an inflammatory signature in squamous cell carcinoma of the head and neck, lung and cervix

Damian T Rieke et al. Oncotarget. .

Abstract

Immune checkpoints are emerging treatment targets, but mechanisms underlying checkpoint expression are poorly understood. Since alterations in DNA repair genes have been connected to the efficacy of checkpoint inhibitors, we investigated associations between methylation of DNA repair genes and CTLA4 and CD274 (PD-L1) expression.A list of DNA repair genes (179 genes) was selected from the literature, methylation status and expression of inflammation-associated genes (The Cancer Genome Atlas data) was correlated in head and neck squamous cell carcinoma (HNSCC), cervical and lung squamous cell carcinoma.A significant positive correlation of the methylation status of 15, 3 and 2 genes with checkpoint expression was identified, respectively. RAD51B methylation was identified in all cancer subtypes. In HNSCC and cervical cancer, there was significant enrichment for homologous recombination genes. Methylation of the candidate genes was also associated with expression of other checkpoints, ligands, MHC- and T-cell associated genes as well as an interferon-inflammatory immune gene signature, predictive for the efficacy of PD-1 inhibition in HNSCC.Homologous recombination deficiency might therefore be mediated by DNA repair gene hypermethylation and linked to an immune-evasive phenotype in SCC. The methylation status of these genes could represent a new predictive biomarker for immune checkpoint inhibition.

Keywords: DNA repair; HRD; immune checkpoints; immune therapy; inflamed gene expression signature.

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Conflict of interest statement

CONFLICTS OF INTEREST

DTR: none; SO: Consulting: Novartis, BMS, MSD; KK: none; TYS: Honoraria: Astra Zeneca, Amgen, BMS, Innate, Jounce, SD; FK: none; IT: none; UK: Honoraria: AmGen, Astra Zeneca, BMS, Glycotope, GSK, Merck Serono, Merck/MSD, Pfizer, Research Support: Innate Pharma, Sirtec, Pfizer

Figures

Figure 1
Figure 1. List of DNA repair genes with methylation associated with expression of CTLA4 or CD274 (TCGA)
Included are all genes with correlation coefficients (spearman) exceeding 0.3 in the respective cancer types. Mechanistic groups are listed in the boxes corresponding to the dashed lines. The total number of repair genes in this list and number of all repair genes with available data in the respective types are listed under “Total Number”. Genes that exceed the threshold for CTLA4 and CD274 are included in the candidate gene list for the respective cancer type (indicated by black boxes). All Bonferroni corrected p-values for the candidate genes are < 0.05 (in the lung cancer candidate genes only one of the correlations remained significant after multiple testing correction, respectively).
Figure 2
Figure 2. Candidate gene lists, as established for HNSCC (left box), lung squamous (middle box) and cervical carcinoma (right box) were cross-validated in the respective other cancer types for correlation with CD274 and CTLA4 expression (indicated by color)
Several genes in the HNSCC candidate list showed similar correlation values also in lung and cervical cancer. MLH3, PMS1, RAD51B, XRCC3, RAD54B, BRCA1, SHFM1, FAAP20 and SPRTN exhibited positive correlations in all three cancer types.
Figure 3
Figure 3. The correlation between XRCC3 and RAD51B methylation and mRNA expression of CD274 or CTLA4 is tested in available TCGA datasets (y-axis)
The highest correlations (x-axis) across all four interactions were identified in the three squamous cancer histologies. Positive associations were not identified in all cancer types suggesting a histology-specific methylation signature.
Figure 4
Figure 4. Heatmaps showing clustering and differential methylation of candidate genes from all three squamous histologies
A. Heatmap for HNSCC. Among the genes with no or little differential methylation are all candidate genes from lung and cervical histology that were not identified in HNSCC (CHEK1, OGG1, CHEK1, MSH5). XRCC3, RAD51B and SPRTN that showed similar correlations across all three cancer types cluster closely together. B. No relevant differences are identified between TP53 mutant and TP53 wt samples as surrogate parameter for HPV status. C. Heatmap for lung squamous cell carcinoma. SPRTN, XRCC3 and RAD51B again exhibit differential methylation and cluster closely together. D. Heatmap for cervical carcinoma. Hypomethylation seems more prominent in most genes and RAD51B, SPRTN and XRCC3 again cluster closely together.
Figure 4
Figure 4. Heatmaps showing clustering and differential methylation of candidate genes from all three squamous histologies
A. Heatmap for HNSCC. Among the genes with no or little differential methylation are all candidate genes from lung and cervical histology that were not identified in HNSCC (CHEK1, OGG1, CHEK1, MSH5). XRCC3, RAD51B and SPRTN that showed similar correlations across all three cancer types cluster closely together. B. No relevant differences are identified between TP53 mutant and TP53 wt samples as surrogate parameter for HPV status. C. Heatmap for lung squamous cell carcinoma. SPRTN, XRCC3 and RAD51B again exhibit differential methylation and cluster closely together. D. Heatmap for cervical carcinoma. Hypomethylation seems more prominent in most genes and RAD51B, SPRTN and XRCC3 again cluster closely together.
Figure 5
Figure 5. Bars indicate the total number of homologous recombination (HR) genes across all DNA repair genes (light blue), the total number of HR genes in the respective DNA repair candidate genes (grey), the percentage of HR genes among all repair genes (dark blue) and the percentage of HR genes in all DNA repair candidate genes (black) in the respective cancer types
HNSCC candidate genes show statistically significant enrichment for the homologous recombination pathway (p=0.004, one-sided student's t-test). All genes that exceed the threshold for either CTLA4 or CD274 were included for the analysis in cervical and lung squamous histologies since candidate genes were too few for statistical analysis in both cancer types. All cervical carcinoma genes but only 22 lung squamous cell carcinoma genes were statistically significant after correcting for multiple testing. Cervical carcinoma also shows a statistically significant enrichment for the homologous recombination pathway (p=0.038). Asterisk indicating p<0.05.
Figure 6
Figure 6. Methylation of candidate genes is analyzed for correlation with mRNA expression of a broad range of inflammation-associated genes (including checkpoint molecules, ligands, T-cell markers, interferon gamma, MHC1 and 2, TCGA)
A. Correlations (spearman) of the two top-performing genes from previous analyses, RAD51B and XRCC3 with inflammation-associated genes in HNSCC. Positive correlations are identified in all genes except for CD276 and VTCN1. All Bonferroni corrected p-values < 0.001 with the exception of CD276, VTCN1 and ICOSLG-RAD51B interaction (p>0.5). B. Correlation values (spearman) of XRCC3 and RAD51B in lung squamous cell carcinoma exhibits a similar pattern like HNSCC. C. Correlations (spearman) of XRCC3 and RAD51B in cervical carcinoma. CD276 and VTCN1 are again the only outliers and VTCN1 shows a statistically significant negative correlation with RAD51B (p=0.015) methylation after Bonferroni correction.
Figure 6
Figure 6. Methylation of candidate genes is analyzed for correlation with mRNA expression of a broad range of inflammation-associated genes (including checkpoint molecules, ligands, T-cell markers, interferon gamma, MHC1 and 2, TCGA)
A. Correlations (spearman) of the two top-performing genes from previous analyses, RAD51B and XRCC3 with inflammation-associated genes in HNSCC. Positive correlations are identified in all genes except for CD276 and VTCN1. All Bonferroni corrected p-values < 0.001 with the exception of CD276, VTCN1 and ICOSLG-RAD51B interaction (p>0.5). B. Correlation values (spearman) of XRCC3 and RAD51B in lung squamous cell carcinoma exhibits a similar pattern like HNSCC. C. Correlations (spearman) of XRCC3 and RAD51B in cervical carcinoma. CD276 and VTCN1 are again the only outliers and VTCN1 shows a statistically significant negative correlation with RAD51B (p=0.015) methylation after Bonferroni correction.
Figure 7
Figure 7. Correlation of DNA repair gene methylation with the expression of a 6-gene IFNG signature (consisting of STAT1, HLA-DRA, IFNG, IDO1, CXCL9 and CXCL10) that has been identified as a predictive biomarker for PD-1 inhibitor efficacy in head and neck cancer
A. Methylation of RAD51B and XRCC3 is correlated with the six-gene IFNG-signature expression in HNSCC. B. The ROC-area under the curve for the predictive value of XRCC3 binomial methylation (as defined by methylation > mean) is 0.77. C. The IFNG-signature shows a dose-dependent expression according to the number of hypermethylated candidate genes in HNSCC (methylation signature. A score of 1 indicates one candidate DNA repair gene with methylation > mean).
Figure 7
Figure 7. Correlation of DNA repair gene methylation with the expression of a 6-gene IFNG signature (consisting of STAT1, HLA-DRA, IFNG, IDO1, CXCL9 and CXCL10) that has been identified as a predictive biomarker for PD-1 inhibitor efficacy in head and neck cancer
A. Methylation of RAD51B and XRCC3 is correlated with the six-gene IFNG-signature expression in HNSCC. B. The ROC-area under the curve for the predictive value of XRCC3 binomial methylation (as defined by methylation > mean) is 0.77. C. The IFNG-signature shows a dose-dependent expression according to the number of hypermethylated candidate genes in HNSCC (methylation signature. A score of 1 indicates one candidate DNA repair gene with methylation > mean).
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